35-39 Randolph St. 
 fcHICAGO. 
 
 f '■ 
 
 
 
 O: ? CORNELL UNIVERSITY. 
 
 u<H THE ! 
 
 fSosroctl p. Matnitv Clbrairg 
 
 
 THE GIFT OF 
 
 
 ROSWELL P. FLOWER 
 
 
 FOR THE USE OF 
 
 
 THE N. Y. STATE VETERINARY COLLEGE. 
 
 
 1897 
 
 
 
Cornell University 
 Library 
 
 The original of this book is in 
 the Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924000912133 
 
/U ' t ?ylT 
 
 Laboratory Manual : 
 
 Elementary Chemical Physiology 
 
 URINE ANALYSIS. 
 
 JOHN H. LONG, M. S., Sc. D., 
 
 PROFESSOR OF CHEMISTRY AND DIRECTOR OF THE CHEMICAL LABORATORIES IN THE SCHOOLS OF 
 MEDICINE AND PHARMACY OF NORTHWESTERN UNIVERSITY. 
 
 WITH NUMEROUS ILLUSTRATIONS. 
 
 ' <r. 
 
 Chicago Mbdical Book Co., 
 
 35-37 Randolph Street, 
 
 1897 
 

 i 
 
 t 
 
 Entered according to act of Congress, in the year 1894. 
 
 By JOHN H. LONG, 
 
 in the office of the Librarian of Congress, at Washington. 
 
 Mo 5 5"<f 
 
 t rf<4( 
 
 1 -'... 
 
PREFACE. 
 
 This little book is an outgrowth of the course given to 
 second year students in the Medical School of Northwest- 
 ern University, and represents in its present form the prac- 
 tical laboratory work as now followed in the course of 
 study required for graduation. 
 
 The time devoted to the study of chemistry and chem- 
 ical physiology in our American medical colleges has been 
 altogether too short, and only within very recent years, 
 with a few notable exceptions, has any attempt been made 
 to impart systematic knowledge in this direction. But now 
 the views of medical teachers are undergoing a rapid 
 change and among the scientific studies recognized as 
 forming a rational groundwork for the study of medicine 
 proper, physiological chemistry is justly regarded as of the 
 first importance. 
 
 It is the belief of the Author that instruction in chemis- 
 try should be given through two years of the course of the 
 medical student. While it may be urged that general 
 chemistry forms no proper part of the curriculum of the 
 medical school it is certainly true that no one of our schools 
 can afford to abandon it until it is generally agreed to make 
 it a requirement for admission. But this is a long way in 
 the future. In his first year work, therefore, the student 
 should be taught the elements of general chemistry both 
 in the classroom and the laboratory, the laboratory work 
 consisting of simple experiments followed by qualitative 
 analysis. Such a course opens the way for the laboratory 
 practice in chemical physiology in the second year, and this 
 should be made practical and simple. 
 
In the set of experiments offered to students the Author 
 has aimed to select only such as illustrate some point of 
 importance, and which, at the same time, can be easily 
 performed. Complicated formulas and reactions which 
 are so largely introduced in some of our recent books on 
 medical chemistry, so called, the Author has thought 
 proper to exclude as not in any degree essential. 
 
 This book consists of two parts, the first dealing with 
 simple experiments in chemical physiology, while the second 
 takes up the subject of urine analysis as properly following 
 and partly illustrating the first. While intended primarily 
 for medical students, .it is believed that it contains not a 
 little matter of interest to the medical practitioner as it dis- 
 cusses fully all the practical processes of urine analysis of 
 value at the present time and in the chapters on chemical 
 physiology presents several features more in detail than is 
 customary in books of this class. 
 
 The Author can make no great claims of originality but 
 would freely acknowledge his indebtedness to such stand- 
 ard works as the Physiologies of Landois and Stirling and 
 Foster, the Physiological Chemistry of Hoppe-Seyler, the 
 Urine Analysis of Neubauer and Vogel and, others. He 
 would extend his thanks to Messrs. E. H. Sargent & Co., 
 of Chicago, to Mr. A. Kruess, of Hamburg and to Messrs. 
 Franz Schmidt and Haensch, of Berlin, for the use of 
 cuts used in illustrations. Finally, he wishes to express 
 his sense of obligation to his assistant, Mr. Chas. H. 
 Miller and also to his colleague, Professor Mark Powers, 
 for valuable aid rendered in the reading of proofs. 
 
 The Author. 
 Chicago, Aug. 15, 1894. 
 
TABLE OF CONTENTS. 
 
 Part I. Elementary Chemical Physiology. 
 
 Chapter I. Introduction 1 
 
 Chapter II. The Carbohydrates 19 
 
 Chapter III . The Fats : 59 
 
 Chapter IV. Proteids or Albumins 67 
 
 Chapter V. The Blood 91 
 
 Chapter VI. Bone Constituents, Saliva, Gastric 
 
 Juice, The Bile Ill 
 
 Chapter VII. Milk, Beef Extracts, Flour and Meal. . 121 
 
 Chapter VIII. Water and Air 134 
 
 Chapter IX. Special Problems 143 
 
 Part II. Urine Analysis. 
 Chapter X. Outline of Tests. Preliminary Tests. .. 177 
 
 Chapter XI. The Tests for Albumins 187 
 
 Chapter XII. The Tests for Sugar 213 
 
 Chapter XIII. The Coloring Matters in Urine. 
 
 Biliary Acids 242 
 
 Chapter XIV. Determination of Uric Acid 254 
 
 Chapter XV. Urea 263 
 
 Chapter XVI. The Determination of Phosphates and 
 
 Chlorides 282 
 
 Chapter XVII. The Sediment from Urine 298 
 
 Chapter XVIII. Unorganized Sediments and Calculi. 321 
 Appendix. Test Solutions and Tables 343 
 
Part I. 
 
 Elementary Chemical Physiology. 
 
Chapter I. 
 
 INTRODUCTION. 
 
 /"""HEMICAL Physiology is in the main concerned with 
 ^— * the study of certain organic compounds existing in the 
 animal body and with the changes which occur in the va- 
 rious food stuffs from the time they enter the stomach until 
 they leave the body, in the form of broken down excreta, 
 by the lungs or through the kidneys. Beginning with the 
 digestive changes in the stomach a series of complex 
 reactions take place which we are able to follow only to a 
 very limited extent. The formation of the important 
 elements in the blood, bile, saliva, the gastric and pancreatic 
 juices, the building up of the solid tissues of the body 
 and the subsequent breaking down of the same, the final 
 appearance of all the elements of these tissues or 
 secretions in the urea, uric acid, phosphates and other 
 products of the urine or in the carbon dioxide from the 
 lungs involve a number of chemical reactions of the 
 highest interest and importance. It is the province of 
 chemical physiology to investigate as far as possible the 
 conditions under which these changes take place, to study 
 the constitution of the food products at the one end of the 
 scale and of the excreta at the other and by means of 
 laboratory experiments to bridge over the space between 
 the two. 
 
 Chemical physiology attempts to duplicate in the test- 
 tube or beaker the changes taking place in the body 
 and to build up from the end products intermediate ones 
 and those at the beginning. Most of the problems sug- 
 
2 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 gested in this direction are as yet far from solution. A 
 study of the urine, expired air and gastric juice has shown 
 the nature of these products under what may be consid- 
 ered normal conditions of the body. Further study has 
 developed certain facts connecting variations in the urine, 
 for instance, with variations in the condition of health in 
 the individual. Study has shown that in health certain 
 bodies are absent from the urine, while in certain diseases 
 they are present, and from this fact there has been 
 developed the very practical department of chemical 
 physiology, known as urine analysis, as an aid to diagnosis. 
 
 Much of the experimental work connected with the 
 investigation of problems in chemical physiology is of so 
 difficult a character as to be quite beyond presentation in 
 an elementary manual. Fortunately, however, other 
 problems, and among them very important ones, are more 
 easily approached, so that they can be worked out in the 
 form of simple laboratory experiments. 
 
 The phenomena of salivary, gastric and pancreatic 
 digestion, of coagulation of blood and of oxidation and 
 reduction of its most important element, of the emulsifi- 
 cation and absorption of fats and of diffusion and osmosis 
 are certainly among the most important and interesting in 
 the whole field of physiology. They have for the student 
 a new meaning when he learns them, not alone from the 
 printed page or the discourse of the lecturer, but also by 
 his own laboratory experiments. Chemical physiology 
 offers a vast field for the research of the specialist, but it 
 offers also abundant simple illustrations for the beginner. 
 
 Chemical physiology is, in a sense, a branch of applied 
 organic chemistry inasmuch as by far the larger number of 
 compounds discussed are compounds of carbon such as 
 are commonly termed organic. It was at one time sup- 
 posed, in fact was maintained as one of the fundamental 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. . 3 
 
 principles of chemical theory, that organic bodies, so-called, 
 are produced only through the agency of the animal or 
 vegetable cell, that is by what were termed vital forces, or 
 by simple transformation of compounds so formed. The 
 building up of a complex carbon compound from the 
 elements of the earth or air was supposed to be a problem 
 which could be solved only by the plant or animal as a 
 function of its growth. 
 
 In 1828 Woehler showed that this view was a false one 
 and that characteristic organic compounds can actually 
 be built up in the chemist's laboratory. This was demon- 
 strated first by the production of a body of the greatest 
 interest to the physiologist, viz.: urea, which under the 
 old view would certainly be one of the last to be con- 
 sidered within the possibility of artificial production. 
 
 The discovery of Woehler marks the beginning of 
 modern scientific organic chemistry, but it proved of 
 scarcely less importance in the development of physiology, 
 as it paved the way for the preparation and study of a 
 great number of compounds, occurring in the animal body, 
 whose relations were imperfectly or not at all understood, 
 or whose existence even in many cases, was not suspected. 
 
 Nature of Organic Compounds. 
 
 Organic compounds contain carbon as their characteris- 
 tic element. Nearly all contain hydrogen. Oxygen is 
 present in a very large number and nitrogen is present in 
 many which are of importance to the physiologist. Sul- 
 phur is present in certain important organic bodies and in 
 a still smaller number there are found phosphorus, chlorine 
 and several other elements. Compounds of carbon are 
 characterized by the fact that when heated alone or with 
 certain oxidizing agents to a high temperature they are 
 decomposed with liberation of carbon dioxide. The 
 
4 • ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 presence of hydrogen is shown under the same conditions 
 by the formation of water. Nitrogen in organic com- 
 pounds may be recognized in several ways but usually 
 most conveniently by conversion into ammonia. Sulphur, 
 phosphorus and chlorine are converted into sulphates, 
 phosphates and chlorides which may be detected by the 
 usual methods of qualitative analysis. 
 
 The proof of the presence of oxygen in an organic 
 compound is often indirect. Some of these reactions may 
 be illustrated by simple experiments, as follows: 
 
 For Carbon. Nearly all organic substances are 
 decomposed when strongly heated. If heated in the air 
 they give off inflammable vapors and often leave a residue 
 rich in carbon which, at a high temperature, burns, form- 
 ing carbon dioxide. 
 
 Solid organic substances when intimately mixed with 
 a large excess of fine granular black oxide of copper and 
 heated to a high temperature in a hard glass tube are 
 decomposed and completely oxidized at the expense of 
 the oxygen of the copper oxide. Carbon present is con- 
 verted into carbon dioxide and hydrogen into water. If 
 the combustion tube is furnished with a delivery tube 
 which dips beneath the surface of clear lime water the 
 ■characteristic reaction for carbonates is given. Organic 
 liquids are characterized by being inflammable or by 
 yielding inflammable vapors. 
 
 For Hydrogen. The presence of hydrogen may be 
 shown by burning the substance in the air in such a man- 
 ner as to condense the products of the combustion by a 
 cold porcelain or metallic surface. The appearance of 
 ■droplets of water is proof of the presence of hydrogen. In 
 the oxidation by copper oxide, if the process is properly 
 •conducted, drops of water condense on the cooler parts of 
 the combustion tube. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 5 
 
 For Nitrogen. Nitrogen in most organic substances 
 can be recognized by heating the latter with soda-lime to a 
 high temperature. The nitrogen is given off as ammonia, 
 which is recognized by the smell or reaction with litmus. 
 
 When a solid nitrogenous substance is heated in a nar- 
 row test-tube with some small fragments of clean metallic 
 sodium, cyanide of the metal is formed. Nitrogenous liq- 
 uids are mixed with dry sand and then with the sodium. 
 The cyanide is dissolved out from the fused mass with 
 distilled water in small amount, to the solution a drop of 
 ferrous sulphate solution and a drop of ferric chloride solu- 
 tion are added and then hydrochloric acid to acid reaction. 
 The appearance of the Prussian-blue color proves the cya- 
 nide and therefore the nitrogen. To recognize nitrogen in 
 nitro-compounds a mixture of soda-lime and sodium thio- 
 sulphate may be used. 
 
 For Sulphur. When organic compounds containing 
 sulphur are fused with a mixture of potassium nitrate and 
 sodium carbonate a sulphate is formed which may be recog- 
 nized in the dissolved mass by the usual tests. 
 
 Organic sulphur compounds, albumin for instance, give 
 up their sulphur as sulphide when heated in a glass tube 
 with metallic sodium. The sulphide can be detected in 
 the usual way. 
 
 Classification of the Physiologically Important 
 Organic Bodies. 
 
 From a strictly scientific point of view organic bodies 
 should be grouped here as elsewhere, that is as products 
 derived from fundamental hydrocarbons. But for many 
 reasons such a classification would be inconvenient or even 
 misleading for our purpose. 
 
 For the physiologist it is in most cases sufficient to con- 
 sider bodies as belonging to, or related to members of, 
 
6 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 three important groups of natural substances, viz. : the car- 
 bohydrates, the fats and the albuminoids. 
 
 Certain compounds appear properly as decomposition 
 products of members of one or the other of these groups. 
 
 By far the greater number of organic substances con- 
 sidered by the physiologist are bodies of natural origin, or, 
 at any rate, difficult of synthesis. While several of the car- 
 bohydrates have been produced artificially, and fats have 
 likewise been built up, no albuminoid has yet been made 
 by an artificial process. The plant alone seems to have 
 the power of building up proteid substances. In the ani- 
 mal body these are converted into new forms or digestion 
 products, but cannot be created outright. Carbohydrates 
 are produced also, in the main, in the vegetable cell, but 
 fats can be built up in the animal body from substances 
 not fat. 
 
 The animal, however, has not the power of producing 
 any organic substance directly from the elements. 
 
 In the following, attention will be paid primarily to 
 groups and derivatives as outlined. 
 
 General Methods of Experimentation. 
 
 It is assumed that the student beginning these experi- 
 ments is reasonably familiar with elementary general 
 chemistry and qualitative analysis. Most of the work fol- 
 lowing is of a qualitative nature, involving the use of very 
 simple methods only, and such apparatus as the student 
 has already used in other work. 
 
 A few volumetric quantitative processes are introduced, 
 the details cf which will be explained in the proper conec- 
 tion. The simple, general principles common to all volu- 
 metric methods may, however, be outlined here. 
 
 Quantitative determinations are made by gravimetric or 
 volumetric methods. In the first the substance sought, or 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 7 
 
 some compound bearing a definite and known relation to 
 it, is precipitated, usually collected and weighed on a filter 
 or in a crucible or other small receptacle. In volumetric 
 methods the procedure is different. Advantage is taken of 
 the fact that many reactions in solution are completed in 
 some definite manner which permits the end-point to be 
 accurately observed. For instance, if to some dilute sul- 
 phuric acid in a beaker, to which a little litmus or phenol- 
 phthalein had been added, a dilute solution of potassium 
 hydroxide be next added a sudden change of color appears 
 as soon as enough alkali is present to neutralize the acid 
 and leave a trace in excess. 
 
 An alkali solution made blue with litmus becomes as 
 suddenly red on addition of an acid. 
 
 If a solution of salt is added to a solution of silver 
 nitrate, drop by drop, a precipitate appears. If the silver 
 solution is contained in a bottle which is violently shaken 
 after each addition of salt, the precipitate formed soon set- 
 tles out so that it is possible to observe the formation of a 
 new precipitate as fresh drops of salt are added. By work- 
 ing carefully the point can be determined where the addi- 
 tion of a drop of salt solution ceases to produce a precipi- 
 tate in the silver bottle, and this indicates the end of the 
 reaction. 
 
 Again, suppose we have a hot dilute solution of oxalic 
 acid containing a small amount of sulphuric acid. To this 
 is added, drop by drop, some solution of potassium per- 
 manganate, the deep color of which is very characteristic. 
 As the drops of permanganate touch the acid solution the 
 color disappears almost instantly, because of decomposition 
 of the compounds, (oxidation of the oxalic acid). The in- 
 stant the last trace of oxalic acid has been decomposed the 
 solution is almost colorless, but the addition of a drop more 
 of permanganate is sufficient to impart a pink color readily 
 
8 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 seen. In this case the end of the reaction is indicated by 
 a sudden change of color. 
 
 We determine in these experiments the relation between 
 acid and alkali, between silver and salt, between oxalic 
 acid and permanganate, in solution and in the same vessel 
 in which we began the test, and besides in a very short time. 
 
 It is evident that if we know the strength, grams 
 per liter, of the potassium hydroxide, of the salt solu- 
 tion and of the permanganate we determine the amount 
 of sulphuric acid in the first, of silver in the second and of 
 oxalic acid in the last of the above experiments by noting 
 simply the volumes of the alkali, salt and permanganate 
 solutions needed to give the characteristic end reaction. 
 This follows because we know that the several compounds 
 react on each other according to the following equations: 
 
 2 K O H + H 2 S 4 = K 2 S 4 + 2 H O H 
 
 112 + 98 = 174 + 36 
 
 Na CI + Ag N O a = Ag CI + Na N 3 
 58.4 -|- 169 = 142.4 + 85 
 
 2 K Mn 4 + 5 C 2 H 2 4 + 3 H 2 S 4 = 
 
 316 + 450 -+- 294 = 
 
 2 Mn S 4 + K 2 S 4 + 8 H 2 O + 10 C 3 
 
 302 -f 174 + 144 + 440 
 
 112 Gm. of potassium hydroxide neutralize 98 Gm. of 
 sulphuric acid; 58.4 Gm. of sodium chloride precipitate 
 169 Gm. of silver nitrate and 316 Gm. of potassium perman- 
 ganate oxidize 450 Gm. of anhydrous oxalic acid. 
 
 The proportions hold good if we substitute here milli- 
 grams, grains, ounces or pounds for grams throughout. 
 
 If now we prepare solutions containing in one liter 112 
 Gm. of KOH, 58.4 Gm. of NaCl and 3. 16 Gm. of K Mn Q 4 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 9 
 
 one cubic centimeter of these solutions will react with 98 
 milligrams of H 2 S0 4 , 169 milligrams of Ag N O s and 4.5 
 milligrams of C 2 H 2 4 . 
 
 Solutions prepared in this manner are called standard 
 or volumetric solutions, because a definite volume of each 
 reacts with a certain weight of something to be measured. 
 A normal solution is usually defined as one which contains 
 a weight in grams equivalent to the molecular weight of the 
 substance in question, dissolved in one liter. Thus, 58.4 
 Gm. of Na CI dissolved in one liter would yield a normal 
 solution. A tenth-normal (/ T ) solution of salt would con- 
 tain 5.84 Gm. in the liter. A twentieth-normal (j^) solution 
 would contain 2.92 Gm. of salt in a liter. A normal solu- 
 tion of sulphuric acid according to this definition would be 
 one with 98 Gm. of H 2 S0 4 in the liter. But many chem- 
 ists take 49 Gm. of H 2 S0 4 (half the molecular weight) as 
 constituting here a normal solution. One cubic centimeter 
 of this would exactly neutralize one cubic centimeter of 
 normal potassium hydroxide (always taken as containing 
 56 Gm. to the liter) and would be equivalent in acidity to 
 one cubic centimeter of a normal hydrochloric acid with 
 36.4 Gm. to the liter. From this standpoint a normal acid 
 solution is one which furnishes in each cubic centimeter as 
 many replaceable hydrogen atoms as are furnished by 
 normal hydrochloric acid with 36.4 Gm. to the liter. 
 
 Many standard solutions are made empirically of such 
 a strength that one cubic centimeter will react with some 
 conveniently small whole number of milligrams of the sub- 
 stance to be measured. A well-known test for sugars 
 depends on the reducing action of these compounds on 
 alkaline copper solutions. Based on this behavior we 
 have a volumetric process in which the standard solution 
 (Fehling's solution) is made of such a strength that one 
 cubic centimeter is completely reduced by 5 milligrams of 
 
10 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 dextrose. This important test will be explained in detail 
 later. Another volumetric solution of frequent use in 
 the physiological laboratory is the mercuric nitrate solution 
 for measurement of urea. Under certain conditions of 
 dilution urea and mercuric nitrate react with each other in 
 a perfectly definite manner, the urea falling as a precipitate 
 with the mercury. The end of the reaction is easily 
 determined and the solution of mercuric nitrate is made of 
 such a strength that one cubic centimeter precipitates 
 exactly 10 milligrams of urea. 
 
 In volumetric analysis indicators are very frequently 
 used to show the end of the reaction. These are usually 
 substances which give a characteristic color change as 
 soon as the principal reaction is completed. Litmus and 
 phenol-phthalein are indicators which show change of 
 reaction from acid to alkaline, or the reverse, and are 
 added, in small quantity, to the solution whose strength is 
 to be measured by a standard acid or alkali solution. 
 
 If to a salt solution whose strength is to be found by 
 silver nitrate, a few drops of neutral potassium chromate 
 solution be added, the chlorine is completely precipitated 
 first, on adding the silver, and then deep red silver chro- 
 mate. The appearance, therefore, of a faint shade of red 
 in making the test indicates the end of the principal 
 reaction. 
 
 In some cases the indicator is not added directly to 
 the solution to be measured, but is put in the form of 
 drops on a glass or porcelain plate ; and with these, from 
 time to time, a drop of the solution to which the standard 
 is being added, is mixed. When enough of the standard 
 has been used a color reaction usually appears in the 
 mixed drops. The end of the reaction in measuring urea 
 by mercuric nitrate or phosphates by uranium solutions is 
 shown in this manner. 
 
ELEMENTARY CHEM/CAL PHYSIOLOGY. 11 
 
 Measuring 1 Apparatus. In volumetric analysis much 
 naturally depends on the accuracy and convenience of the 
 measuring apparatus employed. This apparatus consists 
 of flasks, mixing cylinders, open cylinders, pipettes and 
 burettes. The usual forms of these are shown in the 
 accompanying figures. See next page. 
 
 Flasks are employed of the following dimensions : 1,000 
 Cc. (one liter), 500 Cc, 250 Cc, 100 Cc. and occasionally 
 others. They are made to contain these volumes, and 
 when correctly graduated deliver slightly less of all liquids 
 except mercury. 
 
 Mixing Cylinders are usually employed of the dimen- 
 sions given for flasks, but cannot be used where the great- 
 est accuracy is required. 
 
 Open Cylinders are used for measuring volumes of 
 liquids approximately, and are made to contain 500 Cc, 
 250 Cc, 100 Cc, 50 Cc, 25 Cc, 10 Cc. and 5 Cc, usu- 
 ally. 
 
 Pipettes are made to accurately deliver small volumes of 
 liquids, and those most frequently used have these dimen- 
 sions : 100 Cc, 50 Cc. 25 Cc , 10 Cc. and 5 "Cc. Pipettes 
 are sometimes graduated to resemble burettes. 
 
 Burettes are made to deliver accurately small volumes 
 of liquid down to one-tenth or one-twentieth of a cubic 
 centimeter. They usually hold 50 Cc, sometimes 25 Cc. 
 or 100 Cc. They are frequently closed below with a 
 ground glass stop-cock. Sometimes with a rubber tube 
 compressed by means of a brass clamp, or filled by a little 
 glass ball inside. The ball is placed between the end of 
 the burette and the fine delivery tip, and when the rubber 
 tube is squeezed so as to form a channel on one side, the 
 liquid is allowed to pass. 
 
 These different measuring vessels are readily found in 
 the market graduated with accuracy sufficient for all prac- 
 
12 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 (4M 
 
 A represents a flask; B, a burette; C, an open cylinder; D, a pipette and 
 E a glass-stoppered mixing cylinder. 
 
 FIG. 1. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 13 
 
 tical purposes.* If the user desires to further test their 
 accuracy, this may readily be done by weighing the amount 
 of distilled water at some standard temperature, they hold 
 or deliver. 
 
 Thermometers. While in ordinary qualitative analy- 
 sis the reactions are carred out with little regard to definite 
 temperatures, the room temperature or that of boiling 
 water being sufficiently close for practical purposes, in 
 chemical physiology, for a wide range of investigations, 
 the temperature must be maintained within rather narrow 
 limits. This is necessary because many chemico-physiolog- 
 ical changes are brought about by the activity of living 
 ferments or of enzymes which are destroyed by high tem- 
 perature, and are either destroyed or become inert at low 
 temperature. A certain class of phenomena are best ob- 
 served at a temperature near that of the human body, or 
 a little above, that is about 40° C, while for other reactions 
 a temperature of 65° — 70° C. is found best, while a tem- 
 perature of 75° — 80° C. would be destructive. 
 
 For the ordinary uses of the chemical physiological lab- 
 oratory a thermometer graduated in single degrees with a 
 range — 6° C. to 110° C. is sufficient. Such thermometers 
 can be purchased from chemical dealers and are usually 
 made with accuracy enough for practical purposes. But this 
 is not always the case, and the user should test his instru- 
 ment to ascertain its approximate error. 
 
 First, the error at the zero point should be found by 
 immersing it in clean fresh snow which has been kept an 
 hour or more in a room with a temperature a little above 
 the freezing point, 0° C. This will insure that the snow has 
 the temperature of zero exactly if it has a chance to drain. 
 Snow in this condition packs easily when pressed. The 
 
 * Graduated glassware and other apparatus described in this book may be 
 obtained from Messrs. E. H. Sargent & Co., 106 and 108 Wabash Ave., Chicago. 
 
14 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 mercury of the thermometer left in about half an hour 
 sinks and finally becomes stationary. If the graduation is 
 correct the top of the column should be at the 0° mark. 
 Instead of employing snow, which is seldom found clean 
 enough in cities, and the use of which is limited to the winter 
 season, a better plan is to freeze some pure distilled water 
 in a test-tube by surrounding it with a mixture of crushed 
 ice and salt. When the water begins to freeze it is shaken 
 to produce ice in fine particles. The bulb of the ther- 
 mometer is now immersed in this loose ice, which, as long 
 as any water is present, has accurately the 0° temperature, 
 and stirred about. The level of the top of the mercury col- 
 umn soon becomes constant and can be read off. In many 
 of the cheaper thermometers this level is somewhat above 
 the zero mark. 
 
 The other fixed point on the scale is determined by sus- 
 pending the instrument in the steam of water boiling in a 
 tall metallic vessel. The bulb must not dip in the boiling 
 water, and the top of the mercury column must be a trifle 
 above the neck of the vessel. 
 
 The boiling point is dependent on the atmospheric pres- 
 sure and is by definition 100° C, under a pressure of 
 760 Mm. For temperatures near 100° the boiling point is 
 lowered 1° by a depression of 27 Mm. in the barometric 
 column, from which datum a correction can be made. 
 
 The error of the thermometer at the two fixed points 
 having been determined it remains to determine the errors 
 in the intermediate parts of the scale. The readings at the 
 fixed points may be correct and yet wrong at points 
 between, because of lack of uniformity in the diameter of the 
 capillary tube. To determine the errors accurately between 
 the two fixed points is a matter of some practical difficulty, 
 and will not be explained here in detail. An idea of the 
 amount of the variations in the section of the capillary 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 15 
 
 sufficient for our purpose can be obtained by shaking off 
 a short thread of mercury from the column and allowing it 
 to flow from one point to another, noting its length as 
 measured by degrees on the scale. As the volume of the 
 separated thread is constant a variation in the bore of the 
 capillary is indicated by the lengthening or shortening of 
 the thread as it moves through the tube. Large errors can 
 be detected in this way; small ones we are not concerned 
 with here. 
 
 Thermometers are graduated with the centigrade or 
 Fahrenheit scale in this country. For all scientific meas- 
 urements the former alone is used. The conversion of 
 temperatures on one scale to temperatures on the other is 
 a simple matter and can be done by these formulas. 
 Centigrade temperatures to Fahrenheit: 
 
 F° = 32°+|C° 
 Fahrenheit temperatures to centigrade : 
 C°=4(F°— 32°) 
 
 Separation by Dialysis. For the separation of cer- 
 tain classes of compounds the methods of dialysis are very 
 frequently employed. Many substances in solution pos- 
 sess the property of passing through animal membranes, 
 while other bodies, likewise soluble, do not. Sugar and 
 salt are types of the first class, while glue and albumin are 
 types of the second. The phenomenon depends ultimately 
 on a property common to all bodies in solution, the 
 property of diffusion, which may be illustrated in this way. 
 
 Suppose we partly fill a glass jar with a strong solution 
 of copper sulphate and then bring over this, with as little 
 disturbance as possible, a layer of pure water and leave the 
 jar so charged, several days in a perfectly quiet place at a 
 uniform temperature. It will soon be observed that the 
 blue color of the lower layer begins to diffuse upward, 
 
16 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 notwithstanding the fact that the copper sulphate solution 
 is specifically heavier than water. After a long time the 
 two layers become as one, molecules of water passing 
 down to take the place of the copper sulphate molecules 
 which ascended. 
 
 Something very similar takes place when a layer of 
 water is brought over a layer of solution of salts, acids, 
 alkalies or other substances. Here the change cannot be 
 followed by the eye, however, but must be detected in some 
 other manner. Solid substances in solution show the same 
 tendency to distribute themselves through all available 
 space that is characteristic of gases. The phenomena are 
 the same with miscible liquids, e. g., alcohol over water. 
 We speak, therefore, of diffusion of solids, liquids or gases. 
 
 This fundamentally simple phenomenon is greatly mod- 
 ified if we separate the substances by an animal membrane, 
 as a piece of bladder. The rate of diffusion of many sub- 
 stances is retarded, while that of others is made so slow 
 as to practically prevent passage. Graham, who investi- 
 gated this subject carefully, divided substances roughly 
 into crystalloids and colloids, crystalloids being those bodies 
 which in solution diffuse through animal membranes, and 
 colloids (glue-like substances) those which cannot pass 
 through. It seems to hold true that the failure of bodies to 
 diffuse through membranes depends on the size of their 
 molecules, as the molecules of all those substances called 
 colloids are known to be large, in some cases enormously 
 large. 
 
 In physiological chemical analysis these principles are 
 applied in what was termed above dialysis, or separation by 
 diffusion through membranes. 
 
 This can be carried out by very simple appliances. A 
 dialyser is easily made by stretching a piece of wet parch- 
 ment over a hoop and tying it down close all around. This 
 
ELEMENTARY CHEMfCAL PHYSIOLOGY. 
 
 17 
 
 makes a shallow vessel into which the substance to be in- 
 vestigated is poured. The dialyser is then floated on 
 water in a larger vessel. The water is renewed occasion- 
 ally and so hastens the passage of diffusible substance 
 through the parchment. As an illustration a solution of 
 albumin containing salt can be purified in this manner. 
 The salt diffuses while the albumin fails to. 
 
 Instead of using the common hoop dialyser a parch- 
 ment tube is now frequently employed. The tube is bent, 
 making a narrow double sack into which the liquid to be 
 dialysed is poured. The filled sack is then suspended in 
 pure water which is renewed as before. 
 
 The cut above, Fig. 2, shows the manner of using the 
 first form of dialyser. 
 
 Determination of Specific Gravity. By the specific 
 gravity or density, or specific weight, of a substance we 
 
18 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 understand the ratio of the weight of a given volume of the 
 substance to the weight of the same volume of some other 
 substance considered as a standard. To make our defini- 
 tion exact we must suppose the volumes taken at some 
 normal or standard temperature. The standard substance 
 to which specific gravities of solids and liquids are referred 
 is water and usually at a temperature of 4° C. 
 
 The temperature of the substance compared with water 
 must be accurately known but may be 4°, 15°, 20°, 25° C, 
 or in fact any temperature, but usually one of these. 
 When we say the specific gravity of a liquid is 1.0154 we 
 mean that a volume of it at some definite temperature is 
 that much heavier than the same volume of water at 4° C. 
 If taken at 20° C. we can express the true specific gravity 
 in this manner. 
 
 Sp. gr. 2 4 ° = 1.0154. 
 
 Simple relations exist between the densities of solu- 
 tions and the amounts of dissolved substance, and the de- 
 termination of density is, therefore, usually made in order 
 to find amount of a body in solution or to detect variations 
 in amounts of substances dissolved. The progress of fer- 
 mentation in a sugar solution is readily followed in this 
 manner, and the amount of solid substance in urine is ap- 
 proximately found by making a determination of its density. 
 Special application of these facts will appear later and 
 in the appendix general tables of specific gravities of solu- 
 tions will be given with methods of determination. 
 
Chapter II. 
 
 CARBOHYDRATES. 
 
 The name carbohydrate, as commonly used, is given to 
 a peculiar group of bodies containing carbon, hydrogen and 
 oxygen combined in certain proportions. In this group 
 are included the sugars, the starches, the gums and some 
 allied substances. The molecules of the compounds in 
 this group contain, ordinarily, six, or some multiple of six, 
 atoms of carbon with twice as much hydrogen as oxygen 
 and usually enough to yield five or more molecules of 
 water. In a strictly scientific classification certain mole- 
 cules with five or even four atoms of carbon should prob- 
 ably be included among the carbohydrates. 
 
 The carbohydrates are formed mainly in the vegetable 
 kingdom and are, almost without exception, important food 
 products. A few occur in the animal kingdom, but they 
 are in this case derived from similar substances produced 
 by plants. 
 
 In the experimental study of these bodies it is best to 
 begin with the starches, which are widely distributed in 
 nature and important in the highest degree. 
 
 Starch. 
 
 Ex. I. Prepare starch from the common potato as 
 follows: Grate a potato to a pulp by means of an ordinary 
 tin grater, mix the pulp with water and squeeze it through 
 a piece of coarse unbleached muslin. Moisten the pulp 
 again and repeat this operation several times, collecting 
 the strained liquids in a large beaker. Allow the mixture 
 to settle a half hour or longer and pour off the water, 
 which contains some soluble albuminous substances, some 
 
20 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 cellular floating matter, but very little starch. Most of 
 this will be found in the bottom of the beaker. Add some 
 fresh water, stir up and allow to settle. Now pour the 
 water off again and repeat these operations until the starch 
 appears perfectly clean and white. Transfer this starch to 
 a clean shallow dish and allow what is not intended for im- 
 mediate use to dry spontaneously in an atmosphere free 
 from dust. The dried product will consist of minute glis- 
 tening particles resembling small crystals. 
 
 Corn starch has been made from green corn, by a 
 process quite similar to the above. Ordinary dry corn is 
 used in the United States for the production of starch on 
 the large scale, and to aid in separating the starch from 
 the accompanying materials, it is customary to add a little 
 acid or afkali to the water. Sometimes the meal is allowed 
 to stand with water until a slight fermentation begins, 
 which has the effect of disintegrating the cell walls hold- 
 ing the starch. Potato starch is the common starch of 
 continental Europe. 
 
 Starch consists of minute microscopic, granules varying 
 in size and shape with the root or seed employed. The 
 granules of wheat starch are regular in outline, but vary 
 greatly in size, the smallest having a diameter of less than 
 .005 Mm., while the largest may have a diameter of .04 
 Mm. Corn starch granules have a mean diameter of about 
 .02 Mm., while those from the potato are often .06 Mm., or 
 more in diameter. See Figs. 3 and 4. 
 
 Ex. 2. Examine starch from several sources under 
 the microscope, employing a power of about 300 diameters. 
 Clean a glass slide thoroughly, place in the center of it a 
 small drop of water, and stir into this by means of a 
 needle, or glass rod, a minute quantity of starch. Now 
 drop on a thin, carefully cleaned cover glass, and in such a 
 manner as to exclude air bubbles, and place under the 
 microscope for observation. 
 
ELEMENTARY CHEMICAL P//YS/OLOGY. 
 
 31 
 
 FIG. 3. 
 
 Potato Starch. 300 diameters 
 
 FIG. 4. 
 
 Wheat Starch. 300 diameters. 
 
22 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Ex. 3. Repeat -Experiment 2, using an aqueous solu- 
 tion of iodine instead of water. The starch granules will 
 now appear blue. For the detection of starch in mixtures 
 the use of iodine is often indispensable. 
 
 Starch can be recognized by a number of chemical 
 tests, the best of which are the following : 
 
 Ex. 4. Boil a small amount of starch with water so as 
 to make a thin paste. Allow this to cool, and add a few 
 drops of an aqueous, or dilute alcoholic solution of iodine. 
 A deep blue color is formed which disappears on boiling 
 the mixture. This test is exceedingly delicate and char- 
 acteristic, and serves for the detection of minute traces of 
 iodine as well as starch. 
 
 Ex. 5. Starch is insoluble in cold water, which can 
 be shown by stirring some with water in a beaker, allowing 
 to settle, and pouring the liquid through a paper filter. 
 The filtrate tested with the iodine solution does not give a 
 blue color. 
 
 Action of Acids on Starch. 
 
 When boiled with dilute acids starch is converted into 
 soluble compounds. The nature of these compounds de- 
 pends on the acid used and on the duration of the heating. 
 Two experiments will illustrate these points. 
 
 Ex. 6. Make a paste by boiling about a gram of starch 
 with 100 Cc. of water in a glass flask. Add ten drops of 
 dilute sulphuric acid (1:5) and boil five to ten minutes. 
 Now allow the liquid to cool, remove 5 Cc. with a pipette, 
 dilute this to 25 Cc. with water and add a few drops of 
 iodine solution; a blue violet color results, showing that 
 starch or a starch-like substance is still present. The re- 
 mainder of the acid liquid in the flask is next boiled 
 steadily for one hour, a little water being added from time 
 to time to replace that lost by evaporation. At the end of 
 an hour remove 5 Cc, dilute and test with iodine solution 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 23 
 
 as before. The characteristic starch reaction is now ab- 
 sent, while the liquid has become thin and transparent. 
 Save this for experiments below. 
 
 Ex. 7- Add 5 Cc. of strong sulphuric acid to a gram of 
 starch in a flask holding about 200 Cc. Heat to the boil- 
 ing point and observe that a black mass is soon produced. 
 By prolonged heating this is further decomposed, while 
 fumes of sulphurous oxide escape, leaving finally a color- 
 less liquid. This experiment must be performed in a fume 
 chamber. 
 
 Ex. 8. Add 5 Cc. of strong nitric acid to one gram of 
 starch in a flask holding 200 to 300 Cc, place this on a 
 sand-bath in a fume chamber and apply heat. After a time 
 copious red fumes are given off. Remove the lamp and 
 allow the reaction to continue until the fumes cease to be 
 evolved. Finally, transfer the liquid to a porcelain dish 
 and evaporate to a small volume. On cooling, a crystalline 
 residue remains which consists mainly of oxalic acid. 
 
 These experiments show in a striking manner the be- 
 havior of starch with acids. The action of dilute acids in 
 general resembles that of sulphuric as illustrated in Ex. 6. 
 This reaction is of great practical importance and should 
 be carefully observed by the student. Therefore, with the 
 remainder of the liquid left in Ex. 6 let the following ex- 
 periment be tried: 
 
 Ex. 9. Neutralize the free sulphuric acid by addition 
 of a slight excess of chalk or fine marble dust, heat gently 
 to complete the reaction. Then filter and evaporate the 
 filtrate nearly to dryness on a water-bath. Allow to cool 
 and notice that the residue has a sweet taste. It is, in fact, 
 dextrose and the experiment illustrates the method of man- 
 ufacture on the large scale. Test it by dissolving a little in 
 water and adding a few drops of Fehling's solution, (see 
 appendix). On boiling, a yellowish precipitate appears 
 
24 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 which becomes bright yellow and finally red. Other tests 
 will be given below under the sugars. 
 
 Much of the value of starch depends on the readiness 
 with which it can be converted into sugar. In the animal 
 body this reaction takes place normally and is brought 
 about in two general ways, by the action of saliva and also 
 by the pancreatic juice. Starch, being insoluble in water 
 and very dilute acids, cannot be directly absorbed from the 
 alimentary tract. It must first be brought into soluble 
 form or converted into a soluble compound, and this is 
 just what takes place in digestion. What are com- 
 monly termed digestive processes, are carried on in the 
 body through the action of peculiar complicated chemical 
 compounds called soluble ferments or enzymes. These are 
 unorganized substances and not living cells as in the case 
 of yeast, which may be taken as a type of what are called 
 organized ferments. 
 
 The saliva contains an enzyme known as plyalin, which 
 has the power of converting starch into malt sugar or 
 maltose. A similar active principle is found in malted 
 grain, especially in common malt prepared from barley. 
 The bitter almond contains also an enzyme, called emulsin. 
 The behavior of these bodies will be shown by a few ex- 
 periments below. 
 
 In the pancreas is found a soluble ferment which 
 rapidly converts starch into sugar. The reaction is, in its 
 main features, similar to that produced by saliva, but goes 
 somewhat further. The ptyalin of saliva has been shown 
 to yield maltose as the important product, even after pro- 
 tracted action. But in the case of the pancreatic ferment, 
 while maltose is at first formed, dextrose veiy soon appears 
 and may possibly be looked upon as a normal end product. 
 The action on starch, begun by the saliva, is continued by 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 25 
 
 the amylolytic enzyme of the pancreatic juice and com- 
 pleted, as comparatively recent investigations seem to show, 
 by the secretion of the small intestine. This secretion has 
 only a slight action on starch itself, but rapidly converts 
 maltose into dextrose, which is a point of the highest im- 
 portance, since maltose seems to be only imperfectly 
 assimilable, while of dextrose the reverse is true. 
 
 Action of Saliva on Starch. 
 
 Experimentally the digestive behavior of saliva can be 
 shown without difficulty. 
 
 Ex. 10. After washing out the mouth thoroughly with 
 water, chew a piece of rubber or other neutral insoluble 
 substance to stimulate the secretion of saliva. Collect this 
 in a beaker and continue until about 25 Cc. has been 
 secured. Dilute the more or less turbid liquid with an 
 equal volume of water and allow to stand a short time, and 
 then filter through a 7 or 8 Cm. filter into a small clean 
 flask. 
 
 Ex. II. Make a thin starch paste, about a gram to 200 
 Cc. of water, and observe that it does not respond to the 
 Fehling sugar test referred to above. Mix 10 Cc. of this 
 paste with 5 Cc. of the filtered saliva and warm to a tem- 
 perature not above 40° C. for about fifteen minutes. At the 
 end of the time apply the sugar test again. A yellow or 
 red precipitate will appear now, showing that the starch 
 has been converted, in part at least, into sugar. 
 
 The saliva alone fails to reduce the copper solution, as 
 should be shown by trial. 
 
 Ex. 12. Pour about 5 Cc. of the clear saliva in a test- 
 tube and boil a few minutes; add the starch paste and 
 allow to stand as in the above experiment. On testing 
 with the copper solution no sugar will be found, showing 
 that heat destroys the activity of the ferment. 
 
26 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The digesting power of the saliva is destroyed also, by 
 the addition of a small amount of strong acid or alkali 
 solution, which the student should prove by experiment. 
 
 Our common starchy foods are prepared for use by 
 cooking or baking in some manner. Application of heat 
 produces several interesting changes in starch, some 
 of which will be described below. One important effect is 
 to make it digestible by saliva, which is practically with- 
 out action on raw starch. 
 
 Ex. 13. Stir a small amount of uncooked starch into 
 5 Cc. of saliva, and allow to stand fifteen minutes at 
 35°-40°, and filter. Now apply the Fehling test, and 
 note the absence of precipitated copper suboxide. 
 
 Action of Malt Extract on Starch. 
 
 In the germination of various grains, the enzyme known 
 as diastase is formed, and this is exceedingly active in the 
 conversion of starch. The reaction is largely employed in 
 the arts, especially in the production of the sugar used by 
 the brewer and distiller, and is brought about practically 
 by the use of malt, which is commonly made from sprouted 
 barley. 
 
 The barley is steeped in lukewarm water, and then 
 spread out exposed to the air in a temperate atmosphere until 
 it sprouts. The rootlet fibrils grow very rapidly, and after 
 several days, when they have reached a certain length, the 
 process of growth is checked by thorough drying in hot air 
 kilns. The grain in this condition is rich in the starch 
 converting element, and can be kept for use at any future 
 time. When ground to a coarse meal, and mixed with 
 water, the ferment present goes into solution along with 
 other soluble products formed from the grain. When the 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 27 
 
 solution is evaporated at a low temperature (best in vacuo) 
 it yields the commercial product known as malt extract, 
 which, however, is usually so carelessly made as to possess 
 but little of the diastatic power of the original liquid. 
 
 Like the ptyalin of saliva diastase is destroyed by heat, 
 and evaporation at an}' temperature above 70° C, would 
 result in rendering the product valueless. 
 
 The following experiments show properties of the true 
 malt extract : 
 
 Ex. l4. Mix about 10 Gm. of pale ground malt with 
 25 Cc. of lukewarm water, and allow the mixture to stand 
 a short time, with frequent stirring or shaking. Then filter 
 and add the clear, bright filtrate to a thin starch paste 
 made of 10 Gm. of starch, with 250 Cc. of water. The 
 starch paste must be cool when the malt extract is added. 
 Now, place the mixture on the water-bath and warm to 
 50°-60° C, and maintain at this temperature. Note that 
 the liquid gradually becomes thin and transparent. From 
 time to time remove a few drops by means of a small pipette, 
 and test with iodine solution. At. first a deep blue color 
 appears but this grows weaker, giving place to violet, then to 
 yellowish brown, and finally no color is obtained, indicat- 
 ing completion of the reaction. The starch paste is first 
 converted - into dextrin and finally into maltose. Evap- 
 orate the solution to a very small volume and observe the 
 taste and appearance of the residue. Save it for a later 
 experiment. 
 
 Strong acids or alkalies and high heat destroy the 
 diastase as with ptyalin, and in its behavior with raw starch 
 malt extract resembles saliva. 
 
 Action of Extract of Pancreas on Starch. 
 
 As intimated above the pancreatic fluid contains a fer- 
 ment which converts starch into sugar, maltose at first and 
 by prolonged action into dextrose. 
 
28 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Several methods have been proposed for obtaining an 
 active extract, but all require that the minced gland should 
 remain a number of days in contact with the menstruum. 
 The extracting liquid may be water, alcohol (dilute) or 
 glycerol, and the reaction either acid or alkaline. It has 
 not been found practically possible to separate by any of 
 these processes the amylolytic from the other ferments pres- 
 ent, that is from those which digest proteids, which emulsify 
 fats and which curdle milk. A good extract which shows 
 the characteristic reactions can be made in this manner: 
 
 Ex. 15. Cut a hog's pancreas into small pieces and pass 
 these through a small sausage mill. Take about 10 Gm. of 
 the finely divided matter and cover it with 50 Cc. of dilute 
 alcohol of about 25 per cent strength. Allow the mixture 
 to stand a week, filter it and evaporate the alcohol at a low 
 temperature. Take up the residue with 50 Cc. of water, 
 and use this solution for tests below. 
 
 Ex. 16. Prepare a starch paste with 5 Gm. of starch to 
 100 Cc. of water. Mix 10 Cc. of this paste, after cooling, 
 with 5 Cc. of the pancreatic extract, warm to a temperature 
 of 35°-40° and notice that the paste soon becomes thin 
 and nearly clear. After a time test for sugar. Repeat the 
 experiment, using pancreatic extract which has been boiled 
 before mixing with the starch. The sugar reaction now 
 fails to appear, showing that high temperature destroys the 
 activity of the enzyme, as in the case of saliva. 
 
 Ex. 17. Instead of extracting the pancreas with weak 
 alcohol, glycerol may be used. Take 10 Gm. of minced sub- 
 stance, cover with absolute alcohol in a small dish and 
 allow to stand over night. Then pour off the alcohol and 
 press out the residue between folds of_ filter paper. This 
 treatment removes water. Now put the material in a test- 
 tube and add 10 Cc. of pure glycerol. Cork and allow to 
 stand a week. At the end of the time pour off the liquid 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 39 
 
 and use a little of it for tests on starch paste. Save the 
 rest foT tests on proteids. 
 
 A few drops should be sufficient to give the sugar reac- 
 tions above described. 
 
 Action of Heat on Starch. 
 An important article of commerce is made by heating 
 starch to a temperature of about 250° C, and is called 
 British gum, or dextrin. The same substance is produced 
 at a lower temperature, usually at 125° to 150° by previ- 
 ously moistening the starch with very dilute nitric acid. 
 The acid employed for this purpose need not be stronger 
 than 0.15 of one per cent. The product made in this man- 
 ner is lighter colored than that made in the dry way. 
 
 The dry preparation is illustrated in the following ex- 
 periment : 
 
 Ex. l8. Heat about 10 Gm. of starch in a porcelain 
 dish on a sand-bath to a temperature short of the point 
 where it begins to scorch. It is necessary to stir well all 
 the time, and continue the heat ten minutes after the starch 
 has become uniformly yellowish brown. Then allow the 
 dish to cool, add water and boil thoroughly, which brings 
 part of the product into solution. When sufficiently 
 diluted this solution can be filtered. The filtrate is pre- 
 cipitated by alcohol. 
 
 Dextrin, or British gum, with a small amount of water, 
 constitutes a valuable mucilage or paste which has many 
 uses in the arts. The solution, treated with extract of 
 malt, yields maltose. Dilute acids bring about the same 
 change, leaving dextrose, however, as the final product. 
 
 The Sugars. 
 
 Closely allied to the starches in origin, structure and 
 chemical properties are the sugars, which, for our purposes, 
 
30 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 may be divided into two principal groups, the glucoses and 
 the saccharoses. Representatives of both groups occur 
 widely distributed in nature, in the animal as well as in the 
 vegetable kingdom ; and the members of the second group, 
 the saccharoses, can readily be converted into glucoses by 
 treatment with dilute acids. For our purpose, in the glu- 
 cose group we need consider only dextrose. 
 
 This occurs as grape sugar in many fruits, as diabetic 
 sugar in the urine of persons afflicted with diabetes mel- 
 litus, normally in several animal tissues or secretions, in 
 honey and elsewhere. As intimated above, it is formed as 
 the final product in the complete digestion of starch by 
 the enzymes of the body, and on the commercial scale is 
 produced in immense quantities by the action of dilute 
 sulphuric acid on starch. Illustrations of these processes 
 have been given. We have now to consider some of the 
 important chemical properties of dextrose. 
 
 Reactions of Dextrose. 
 
 For experiments with dextrose, solutions made by dis- 
 solving crystallized commercial grape sugar in water may 
 be used. 
 
 An important property of dextrose is its power of de- 
 composing alkaline solutions of many metallic salts, 
 usually with precipitation of the metal or a low oxide. 
 
 Ex. 19. Add to a dilute solution of dextrose enough 
 copper sulphate solution to impart a very faint greenish 
 blue color and then a considerable excess of strong potas- 
 sium hydroxide solution. This produces no precipitation, 
 but increases the color. On warming the solution a yellow- 
 ish precipitate forms which grows bright red by boiling. 
 This is cuprous oxide, and the test is known as Trommer's 
 test. It is frequently employed to detect the presence of 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 31 
 
 sugar in liquids, especially in urine, but on the whole is not 
 as satisfactory as the next one or Fehling's test. 
 
 Ex. 20. The use of Fehling's solution has already 
 been referred to. Its preparation is given in the appendix. 
 
 Add to a small volume of the sugar solution some 
 Fehling solution, and as long as both liquids are cold and 
 dilute no precipitate forms. On standing, however, in the 
 cold, a precipitate will in time form. Precipitation takes 
 place immediately on heating, the final reaction being the 
 same as in the Trommer test. 
 
 The Fehling solution is in many respects the most val- 
 uable sugar test we have and is used quantitatively as will 
 be described below. 
 
 In the method by the Trommer test if too much copper 
 sulphate is added the color that appears on heating is black 
 instead of red, from the formation of precipitated cupric 
 oxide in place of cuprous, by reduction. 
 
 Ex. 21. Add to a dextrose solution some strong po- 
 tassium hydroxide solution and then a very small amount 
 of bismuth subnitrate. For an ordinary test a few milli- 
 grams will be enough. On boiling, a black precipitate ap- 
 pears which frequently forms a bright mirror on the walls 
 of the test-rube. This precipitate seems to be a mixture 
 of metallic bismuth with some oxide, and shows the strong 
 reducing power of the sugar. 
 
 Ex. 22. Prepare an alkaline solution of mercuric cya- 
 nide as described in the appendix. Heat two or three 
 cubic centimeters to boiling in a test-tube and add a small 
 amount of sugar solution. A reduction of the reagent takes 
 place with deposition of metallic mercury. This solution 
 is sometimes employed in the quantitative determination of 
 sugar, especially in urine. 
 
32 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Among the sugar reactions not depending on the reduc- 
 tion of metallic salts, three may be given as of especial 
 interest. The first of these is the so-called fermentation 
 test and may be briefly explained as follows: 
 
 Under the influence of yeast a number of saccharine 
 substances have the property of splitting up into alcohol 
 and carbon dioxide, when in sufficiently dilute coridition. 
 The practical and theoretical importance of this reaction is 
 so great that it has been made the subject of a large num- 
 ber of investigations, which have established numerous 
 points regarding the kinds of sugars which may be fer- 
 mented, the by-products as well as principal products 
 formed, the best conditions as to temperature and dilution 
 of the fermenting solution, the isolation and peculiarities, 
 of various kinds of yeast and so on, but for our purpose a. 
 few experiments will show such points as may be looked 
 upon as most worthy of attention. 
 
 The yeast ferment differs from the enzymes referred to- 
 above in being a living plant cell, capable of reproduction 
 under proper conditions, but like the enzyme is active only 
 within certain limits of temperature and strength of solu- 
 tion. Strong acids or alkalies destroy both. 
 
 Ex. 23- Rub up a quarter of a cake of compressed 
 yeast with a little water to a thick cream, and then add. 
 about 100 Cc. of water. Allow the mixture to stand in a 
 warm place some hours when it is ready for use. Now pre- 
 pare a sugar solution by diluting 25 to 30 Cc. of commer- 
 cial glucose syrup with an equal volume of water. Pour 
 this solution into a flask holding about 100 Cc. and add 
 about 25 Cc. of the yeast mixture. After mixing thoroughly 
 determine the specific gravity. Close the flask with a good 
 stopper through which a twice bent delivery tube passes, 
 and set aside in a moderately warm place, the temperature 
 of which should not go below 20° C. At the end of 24 
 hours note the escape of gas bubbles from the surface of the: 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 33 
 
 liquid. Now dip the delivery tube in lime water contained 
 in a test-tube or small flask and observe the formation of 
 a precipitate as the gas bubbles pass into the liquid. The 
 precipitate is calcium carbonate. Allow the liquid to stand 
 two days longer, that is about three days in all and observe 
 that it has become much lighter in specific gravity and that 
 it has the odor of alcohol. It is, in fact, a weak solution of 
 alcohol, which may be obtained by distillation. 
 
 Ex.24. Phenyl hydrazine test. A characteristic reac- 
 tion of great practical value is given on the addition of 
 phenyl hydrazine to a solution of dextrose under certain 
 definite conditions. To a dilute dextrose solution add 
 about a gram of phenyl hydrazine hydro-chloride, and two 
 grams of sodium acetate. Heat on a water bath half an 
 hour, and then allow the liquid to cool. There will now 
 be found a beautiful yellow crystalline precipitate of phenyl 
 glucosazone, the nature of which is best seen under the 
 microscope. This test is one of great delicacy, and has 
 been applied to the detection of traces of sugar in urine. 
 
 Another test which serves for the recognition of even 
 minute traces of dextrose and other sugars, is the following, 
 proposed by Molisch . 
 
 Ex. 25. To a small amount of a dilute sugar solution 
 add two drops of a solution of a-naphthol, containing about 
 20 Gm. in 100 Cc. On shaking, the liquid becomes turbid. 
 Now add to it an equal, or slightly greater volume of pure 
 strong sulphuric acid and shake. A deep violet color 
 appears, which gives place to a violet precipitate on addi- 
 tion of water. This reaction has been shown to be due to 
 the combination of the a-naphthol with furfurol produced 
 by the action of sulphuric acid on the sugar present. 
 
 Saccharoses. 
 
 Three sugars deserve our attention in the saccharose 
 group, viz. : Cane sugar, milk sugar and malt sugar. 
 
34 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Cane sugar, or saccharose, occurs widely distributed in 
 the vegetable kingdom, but on a large scale is obtained 
 mainly from several varieties of cane, from the sugar 
 beet, and from maple sap. It has not been produced syn- 
 thetically. 
 
 Several reactions distinguish it from dextrose, as illus- 
 trated in the following experiment : 
 
 Ex. 26. Prepare a dilute solution of pure cane sugar 
 and boil it with Fehling solution in the usual manner. 
 Observe that no reduction of the copper compound takes 
 place. Next boil a similar cane sugar solution with a 
 few drops of dilute hydrochloric or sulphuric acid several 
 minutes, neutralize with sodium carbonate, and then apply 
 the Fehling test. The characteristic red precipitate now 
 appears. In this reaction the cane sugar is broken up by 
 the acid into a molecule of dextrose, and a molecule of 
 laevulose, both reducing sugars. 
 
 Cane sugar solutions do not undergo fermentation 
 directly with yeast, and differ from dextrose solutions also 
 by their behavior with polarized light, which will be ex- 
 plained below. 
 
 Milk sugar resembles cane sugar in some of its chemi- 
 cal properties, but reduces copper solutions. It does not 
 ferment with pure yeast. Dilute acids convert it into 
 galactose and dextrose. A method of preparing milk for 
 sugar tests will be given below. Tests applied directly 
 are unreliable because of the presence of albuminoids. 
 
 The formation of malt sugar has been referred to above. 
 It has few uses in the pure state, but is interesting as 
 being one of the final products of the digestion of starch. 
 Its action on Fehling's solution resembles that of dex- 
 trose. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 35 
 
 The Determination of Sugars. 
 
 Numerous methods have been employed for the deter- 
 mination of sugars in solutions. Two will be described in 
 detail here which depend on different principles and which 
 are generally followed in practice. One of these methods is 
 but a modification of the qualitative test. carried out with 
 the Fehling solution, while the other depends on the be- 
 havior of sugars with polarized light. 
 
 Method with Fehling's Solution. 
 
 Fehling's solution, as described in the appendix, is 
 made arbitrarily of such a strength that one cubic centi- 
 meter is reduced by 5 milligrams of dextrose, on the suppo- 
 sition that the sugar and copper salt react on each other in 
 the proportion of 
 
 5 (CuS 4 . 5 H 2 O) to C 6 H 1S 0„. 
 
 It was formerly held that the reaction was a perfectly 
 definite and simple one, and could be expressed in this 
 manner, but it is now known that the dilution of the solu- 
 tions is a very important factor in determining the amount 
 of copper reduced: The best conditions to be employed 
 in practice have been determined by Soxhlet, who found the 
 reducing power of several sugars to vary as follows, when 
 they were tested in solutions of one per cent strength: 
 
 0.5 Gm. of invert sugar in 1 per cent solution reduces 
 101.2 Cc. of Fehling solution, undiluted. 
 
 0.5 Gm. of invert sugar in 1 per cent solution reduces 
 97.0 Cc. of Fehling solution, diluted with 4 volumes of 
 water. 
 
 0.5 Gm. of dextrose in 1 per cent solution reduces 105.2 
 Cc. of Fehling solution, undiluted. 
 
 0.5 Gm. of dextrose in 1 per cent solution reduces 101. 1 
 Cc. of Fehling solution, diluted with 4 volumes of water. 
 
3(3 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 0.5 Gm. of milk sugar in 1 per cent solution reduces 74 
 Cc. of Fehling solution, undiluted. The reducing power 
 in diluted solutions is the same. 
 
 0.5 Gm. of maltose in 1 per cent solution reduces 64.2 
 Cc. of Fehling solution, undiluted. 
 
 0.5 Gm. of maltose in 1 per cent solution reduces 6*7.5 
 Cc. of Fehling solution diluted with 4 volumes of water. 
 
 The oxidizing power of one cubic centimeter of Fehling 
 solution with each kind of sugar may be tabulated as fol- 
 lows, assuming the sugars to be in solutions of approxi- 
 mately one per cent, strength when acted upon. 
 
 One Cc. of Fehling solution oxidizes: 
 
 Wben When diluted with 
 
 undiluted. 4 vols, of water, 
 
 Dextrose 4.75 Mg. 4.94 Mg. 
 
 Invert sugar 4 94 " 5.15 " 
 
 Milk sugar 6.76 " 6.76 " 
 
 Maltose 7.78 " 7.40 " 
 
 The practical application of the test is best shown by 
 an experiment. 
 
 Ex. 27. Measure out accurately into a flask holding 
 about 250 Cc, 25 Cc. of the copper solution and the same 
 volume of the alkaline tartrate. Heat the mixture, or 
 Fehling solution, on a wire gauze and note that it remains 
 clear. Fill a 50 Cc burette with a dilute dextrose solu- 
 tion and run 10 Cc. into the hot liquid. Boil two minutes, 
 shaking the flask continuously, and allow the mixture to 
 settle. If the supernatant liquid appears yellow it indi- 
 cates that the sugar solution is much too strong and must 
 be diluted with at least an equal volume of water before 
 beginning another test. If, on the other hand, the liquid 
 is still blue, add 2 Cc. more of the sugar solution, boil 
 again for two minutes and allow to settle. If the color is 
 now yellow an approximate value for the amount of 
 sugar in the solution becomes known, but if still blue, the 
 operation of adding solution and boiling must be continued 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 37 
 
 until, after settling, a yellow color appears. Approximate- 
 ly 250 Mg. of dextrose is required to reduce the Fehling 
 solution taken, and this must be contained in the sugar 
 solution added. From this preliminary experiment calcu- 
 late the amount of sugar present in each cubic centimeter. 
 
 Ex. 28. With the data obtained in the above experi- 
 ment as a basis, make now a new sugar solution, having a 
 strength of about one per cent. Measure out 50 Cc. of the 
 Fehling solution, heat to boiling and run the new sugar so- 
 lution from the burette as before, the first addition being 
 about 20 Cc. Boil and note the color after settling and 
 then cautiously continue the addition of sugar solution, a 
 few tenths of a Cc. at a time, boiling after each addition, 
 until the blue color gives place to a yellowish green and 
 then, by the addition of a drop or two, to a pale yellow. 
 
 Under the conditions of this experiment 4. 75 X 50 in- 
 dicates the number of milligrams of sugar in the volume 
 discharged from the burette. By dividing the product, 
 237.5, by this volume the number of milligrams of sugar 
 per cubic centimeter is given. 
 
 If the sugar solution taken for analysis is very dilute the 
 amount oxidized by each Cc. of the Fehling solution ap- 
 proaches five milligrams, as indicated by the table given 
 above. 
 
 The method of employing the test as just outlined is 
 sufficiently exact for the ordinary practical uses. 
 
 Sometimes the final disappearance of the copper from the 
 solution is determined by filtering a few drops through a 
 very small filter and adding a drop of acetic acid and a 
 drop of ferrocyanide solution to the filtrate, when the 
 characteristic color is given if a trace of copper is present. 
 
 With clear aqueous solutions this procedure can hardly 
 be considered necessary as the operator can determine the 
 end of the reaction with very great accuracy after a little 
 
38 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 practice. But in highly colored solutions or with diabetic 
 urine the cuprous oxide precipitate settles very slowly at 
 times, leaving the experimenter in doubt. It is then nec- 
 essary to resort to the ferrocyanide or other test to dis- 
 cover the progress of the reduction. Under the head of 
 urine analysis some modifications of the test will be given. 
 
 The direction is given above to shake the flask in which 
 the mixture of Fehling solution and sugar is boiling. This 
 precaution is necessary to prevent bumping. The neck of 
 the flask is held'by a test-tube holder (a strip of stiff paper 
 is a good substitute) and a continuous rotary motion given 
 to the liquid while it is being heated. 
 
 To determine cane sugar by the Fehling solution it must 
 first be inverted or converted into a mixture of dextrose 
 and lffivulose. If the sugar is in the dry condition the in- 
 version can be accomplished as follows: Weigh out 9.5 
 Gm., dissolve in 700 Cc. of water, add 20 Cc. of normal hy- 
 drochloric acid and heat for 30 minutes on the water bath. 
 Then neutralize with 20 Cc. of normal sodium hydroxide 
 solution and make up to 1000 Cc. on cooling. This gives 
 now a one per cent solution which is employed as given for 
 dextrose using the factor 4.94 instead of 4.75 as the 
 amount of sugar oxidized by each Cc. of the copper solu- 
 tion. On the completion of the experiment take 95 
 parts of cane sugar for each 100 parts of invert sugar found. 
 
 If the cane sugar is in solution it must be diluted to 
 a specific gravity of about 1.005 at 15° C, and then 
 heated with acid, using the proportions given above. 
 
 Malt sugar and milk sugar are determined directly by 
 the Fehling solution, using the proper factors as given in 
 the table. 
 
 If the analyst has under examination a solution con- 
 taining both cane sugar and dextrose the latter can be 
 determined first as outlined above, and then the sum of the 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 39 
 
 two sugars after the inversion of the saccharose. By sub- 
 tracting the amount of Fehling solution required for a 
 given volume of the original from that required for the 
 same volume of the solution inverted, the equivalent of the 
 cane sugar in the original is found, expressed as invert 
 sugar. 
 
 Polarization Method. 
 
 Many substances, solids, liquids and gases, have the 
 power of rotating the plane of polarized light passed 
 through them, the amount, or number of degrees of rota- 
 tion, being proportional to the number of molecules the 
 light passes. 
 
 The observation of this phenomenon in solids and 
 gases is usually a matter of some difficulty, and not, there- 
 fore, practically useful. But in liquids, single substances 
 or solutions, the observation is one readily made, and has 
 become the basis of an important method in the qualita- 
 tive and quantitative examination of organic substances. 
 
 Instruments employed to determine the action of sub- 
 stances on polarized light, are, in general, called polari- 
 scopes, or polarimeters, and are made in a great variety of 
 forms. For the details of construction of these instru- 
 ments the student is advised to consult a special manual 
 or work on general physics. Enough will be given here 
 to render plain the construction and use of one instru- 
 ment which can be used for practically all tests.* 
 
 In an ordinary ray of light the vibrations of the parti- 
 cles of ether producing it take place in all directions and 
 perpendicular to the line of propagation of the light. The 
 projections of these oscillations can be represented as 
 shown in Fig. 5. 
 
 ♦Landolt's work, "The Optical Rotation of Organic Substances," is the 
 standard authority on the subject. 
 
40 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Certain substances have the power of so changing a 
 ray of light passed through, or reflected from them, that 
 the oscillations no longer take place in all directions, but in 
 certain definite directions only. Fig. 6 represents these 
 vibrations as taking place in two directions, in a vertical 
 
 FIG. 5. FIG. 6. 
 
 plane, and in one at right angles to it. Light so modified 
 is spoken of as "plane polarized light," and for most pur- 
 poses is produced by the passage of an ordinary beam of 
 
 g 
 
 FIG. 7. 
 
 light through a specially constructed prism of Iceland spar 
 known as a Nicol's prism. The construction and behavior 
 of this prism can be shown by the accompanying diagrams. 
 
ELEMEN.TARY CHEMICAL PHYSIOLOGY. 
 
 41 
 
 Fig. 7 represents the natural crystal of Iceland spar 
 and a line drawn from d to // represents the principal axis. 
 A plane through b d h f is a principal plane with the 
 angles d b h and h / d, Fig. 8, ='71° nearly. If we suppose 
 a ray of light to fall on the surface abed it will pass 
 
 FIG 8. 
 
 through and emerge as two rays, an ordinary and an extraor- 
 dinary as shown in Fig. 9. 
 
 The light here has suffered double refraction and the 
 ray which is bent the most from its course, o p, is the ordi- 
 
 FIG. 9. 
 
 nary ray and the other, o q, the extraordinary. These rays 
 are found by experiment to be both polarized, one in a 
 plane parallel to the principal plane or section of the 
 crystal b dfh, and the other at right angles to it. It is 
 assumed that the ordinary ray is the one polarized in the 
 direction parallel to the principal section. 
 
42 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 For the purpose before us, it is necessary to eliminate 
 one of these rays, best the ordinary, and this is accomp- 
 lished in the following manner. The four faces a be </and 
 e f g h of the crystal are ground down until the angles h b d 
 and h / d of the principal section are not 71 ° but 68°, 
 which leaves the angle b d h, as shown in Fig. 10,90°. Now 
 
 FIG. 10. 
 
 the crystal is sawed in two halves, the cut passing through 
 rfand h and perpendicular to the plane b d h f. The cut sur- 
 faces are covered with Canada balsam and pressed together 
 again, which gives us a. crystal exactly like the original one 
 in appearance, but with somewhat different physical 
 properties. 
 
 FIG. II. 
 
 The light enters the " Nicol" at o, Fig. 11, suffers 
 double refraction as before, giving the rays o m and o n. 
 The extraordinary ray, o m, passes through the balsam sur- 
 face d h, and emerges in the direction^, while the ordinary 
 ray fails to pass the balsam, but is thrown to one side as 
 it strikes it at a very oblique angle, and emerges from the 
 prism in the direction p. The ray at q is polarized but its 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 43 
 
 intensity is, of course, only one-half of that of the original 
 ray at o. 
 
 Suppose, now, that two of these prisms are placed in 
 positions as shown in the following figure, Fig. 12, with 
 their principal sections in the same plane, that is perpen- 
 dicular to the plane of the paper. 
 
 * w V / \7 \ /» 
 
 T~. — 
 
 FIG. 12. 
 
 A ray of light entering a is polarized and emerges as a 
 ray of half the intensity, the plane of polarization being par- 
 allel to the plane of the paper. This ray now passes into b, 
 the position of whose planes is symmetrical with those of 
 
 a. The light then emerges at n practically as it entered 
 
 b, that is, as an extraordinary plane polarized ray and of 
 nearly half the intensity at m. 
 
 But, on the other hand, if the prism b is turned so that 
 its principal section is in a plane perpendicular to that of a 
 we have the condition shown in Fig. 13. 
 
 a 2 
 
 FIG. 13. 
 
 The extraordinary plane polarized ray leaving a enters 
 b and behaves here as an ordinary ray until it reaches 
 the balsam layer. Here it suffers total reflection and 
 is thrown to one side, no light whatever emerging 
 in the direction n. If the prism b has an interme- 
 diate position with reference to a any ray entering it 
 from a is broken up into two rays, an ordinary and an 
 extraordinary, of which the former is always lost by reflec- 
 
44 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 tion from the balsam layer. If the prism b is placed so 
 that its principal plane makes an angle of 45° with that of a 
 the ordinary and extraordinary rays have equal intensity 
 and the latter emerging finally in the direction n has just 
 half the intensity of the light entering b, or one-fourth the 
 intensity of that entering a. From this it follows that by 
 giving a a fixed position and moving b around a horizontal 
 axis parallel to the plane of the paper the light leaving it 
 in the direction n passes through all degrees of intensity 
 between thftt of the extraordinary ray of a and zero. It 
 would further follow that with the two prisms placed as in 
 Fig. 13, that is so that no light passes both, their principal 
 
 FIG. 14. 
 
 sections being at right angles to each other, a ray of some 
 intensity can still be made to emerge from b after leaving a, 
 provided we place between the two something which is 
 capable of rotating the extraordinary polarized ray before 
 it reaches b. The rotation of the plane of vibration of this 
 ray to the right or left would be equivalent to the rotation 
 of b to the left or right through the same number of degrees. 
 
 Now, there are many substances, solids, liquids and 
 gases, which have the power of twisting or rotating the 
 polarized ray in this manner, and the application of polar- 
 izing apparatus to the study of chemical phenomena 
 depends on this fact. 
 
 For convenience in handling, the Nicol prisms are fas- 
 tened in tubes of brass by the aid of pieces of blackened 
 cork. The mounted prism shows in section as Fig. 14. 
 
 Suppose now we support two of these Nicols on a 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. - 45 
 
 stand, and place between them a glass vessel with plane 
 parallel glass walls. 
 
 Allow a ray of light to pass in the direction indicated. 
 If the glass vessel is filled with clear water, and the Nicol 
 b is placed in position symmetrical with a, the light leav- 
 ing a will pass through the water and through b, as above 
 in Fig. 12. If b is placed as in Fig. 13 no light will pass 
 through ; the water is, therefore, without action. But if 
 instead of with water we fill the glass vessel with a sugar 
 solution, or with a solution of Rochelle salt, we observe 
 now that some light does pass through b, and that to reach 
 the condition of darkness again it will be necessary to 
 rotate the latter prism through a certain number of de- 
 grees. 
 
 •\zz\- 
 
 FIG. 10. 
 
 By trying different solutions in the glass vessel it will 
 be further noticed that the number of degrees through 
 which b must be rotated to secure perfect darkness, varies 
 not only with the nature of the solution, but with its con- 
 centration. 
 
 In this combination of prisms a is called the polarizer, 
 and b the analyzer, because it permits us to detect and 
 measure the phenomena of "rotary" polarization. 
 
 The fact that a number of organic liquids and other 
 organic bodies in solution have the power of rotating the 
 plane of polarized light was discovered by the French 
 physicist, Biot, and at his suggestion several forms of 
 apparatus were constructed with which this phenomenon 
 could be observed. These pieces of apparatus are in gen- 
 eral called polariscopes, or polarimeters, and in their sim- 
 
46 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 plest form consist essentially of a polarizing and an analyz- 
 ing prism, a tube placed between them to hold the liquid 
 under investigation and some arrangement to measure the 
 number of degrees through which the analyzer may be 
 rotated. 
 
 Fig. 16 shows a stand arranged to hold two crystals or 
 two Nicol prisms, with which the general phenomena of 
 
 polarization may be studied, the rotation of one of the 
 prisms, as B, being measured on the graduated circle, C, 
 while Fig. 17 shows one of the first forms employed in the 
 practical examination of liquids. 
 
 The original Mitscherlich apparatus, shown in Fig. VI, 
 contains nothing more than this : a stout brass pillar sup- 
 ports a horizontal arm d, on one end of which is a brass 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 47 
 
 tube containing the polarizing prism, a. At the other end 
 of the arm is a graduated circle fixed in position. In the 
 center of this is an opening in which rotates the tube 
 holding the analyzing prism, b. To this tube a handle, c, is 
 attached, and also a pointer moving over the graduated 
 circle. Between the two prisms a glass or metal tube,/, 
 with ends of plane glass plates perfectly parallel to each 
 
 FIG. 17. 
 
 other is placed. This tube may be 100 to 600 Mm. in 
 length, and holds the liquid to be examined. 
 
 In making an observation the apparatus is directed to- 
 ward a bright light, and the analyzing Nicol turned until 
 the condition of greatest darkness is reached. The tube,/, 
 with the plate at one end in place, is now filled with the 
 liquid to be tested, and the other glass end-plate screwed 
 down by means of the brass cap in such a manner as to ex- 
 
48 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 elude all air bubbles, leaving the tube quite full and clear. 
 It is placed in position, and if it contains a rotating sub- 
 stance it will be found necessary to turn the analyzer 
 through a certain angle, as indicated by the movement of 
 the pointer over the graduated circle, to obtain the darkened 
 field again. The number of degrees read off is called the 
 angle of rotation, and is characteristic of the substance, but 
 depends on the concentration of its solution and the length 
 of the tube. 
 
 This form of instrument cannot be used for exact quan- 
 titative measurements, but only for qualitative indications. 
 More complete instruments have been devised by Wild, 
 Laurent, Jellett, Landolt, and others, some of which can 
 
 HO 
 
 FIG. 18. 
 
 be used for polariscopic observations in general while 
 others are employed only in the examinations of saccharine 
 liquids. A very useful form suitable for all kinds of obser- 
 vations with homogeneous light is the Laurent, as made by 
 several German and French firms. 
 
 The form made by Schmidt & Haensch, of Berlin, is 
 shown in Fig. 19, while the arrangement of the optical 
 parts is illustrated in Fig. 18. 
 
 Fig. 20 shows the lamp by which the yellow light is 
 produced, sodium carbonate or bromide being volatilized 
 in the flame. 
 
 Homogeneous yellow light is supposed to pass from 
 a to g, Fig. 18. It first meets, at a, a thin plate of potas- 
 sium bichromate, held between two glass plates. The ob- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 49 
 
 FIG. 19. 
 
 FIG. 20. 
 
50 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 ject of this is to further purify the sodium light by absorb- 
 ing any foreign rays. The pure yellow light then passes 
 into the polarizing Nicol b, which is mounted in a short 
 brass tube in such a manner that it can be slightly rotated 
 by means of a lever shown at H, of Fig. 19. At c there is 
 a round glass plate over half of which is mounted a thin 
 plate of quartz shown by the shaded part of c. This plate 
 of quartz is ground parallel to the axis of the crystal and is 
 of such a thickness that it produces a difference of half a 
 wave length in the rate of the two polarized rays of yellow 
 light passing through it. Following it is the tube d to hold 
 the liquid to be examined, then the analyzing Nicol e, and 
 finally the achromatic telescope eyepiece made up of / 
 and g. The analyzing Nicol can be rotated around its 
 center and is firmly mounted in the middle of a graduated 
 circle which turns with it. 
 
 The part of this instrument which distinguishes it essen- 
 tially from the simple Mitscherlich and other forms is the 
 small quartz plate at c. When the polarizing prism b is 
 adjusted so that the plane of polarization is exactly paral- 
 lel to the axis of the quartz plate on c (or its vertical edge) 
 the two halves of c appear equally light or dark when 
 viewed through the analyzer; with the latter at right angles 
 to the polarizer there is absolute darkness. But if the adjust- 
 ing lever over b is turned slightly so as to rotate b, and there- 
 fore the plane of polarization, with reference to the vertical 
 line of the quartz plate on c, then there is no condition of 
 absolute darkness for any position of the analyzer, but a 
 condition approaching darkness with the shadow the same 
 on both halves of c when the analyzer is turned to cross the 
 polarizer. The slightest rotation of the analyzer to the 
 one side or the other now causes one-half of c to grow sud- 
 denly dark and the other light. Reversing the direction of 
 the rotation of the analyzer causes a reversion of light and 
 shade on viewing c. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 51 
 
 It is therefore very easy to determine the position of 
 the analyzer for greatest darkness, something which was not 
 possible with the old Mitscherlich instrument. The addi- 
 tion of the small quartz plate is merely a device to enable 
 the observer to find accurately and quickly the zero point 
 of the instrument. In other modern instruments the same 
 end is reached by other means, although not always so 
 perfectly as here. 
 
 The amount of rotation for any substance depends on 
 the length of the column taken, on the concentration and 
 density of the liquid and to a slight extent on temperature. 
 Each substance is characterized by a specific rotation which 
 may be defined as the rotation of a liquid column one deci- 
 meter in length, each cubic centimeter of which is assumed 
 to contain one gram of active ingredient. 
 
 The following designations are commonly employed: 
 
 a=observed angle of rotation. 
 
 /=length of tube in millimeters. 
 
 *r=concentration of liquid, that is number of grams in 
 100 cubic centimeters. 
 
 (/^density of liquid polarized. 
 
 /=percentage strength of liquid, that is the number of 
 grams of active substance in 100 grams of liquid or 
 solution. 
 
 c—p. d. 
 [a]=the specific rotation. 
 
 The specific rotation of an active liquid — oil of turpen- 
 tine, for instance — is expressed as follows: 
 r -. 100 a 
 
 If the liquid under examination is a solution of an ac- 
 tive substance the above formula cannot be used, but the 
 following, as the percentage strength must be taken into 
 
 consideration: , „. 
 
 10 4 « 
 
 [«] = 
 
 l.p.d. 
 
52 ELEMENTARY CHEMICAL PHYSIOLOGY . 
 
 In using this formula it is necessary to know both the 
 percentage strength and the specific gravity, but as p. d 
 is the concentration of the solution, that is the number of 
 grams in 100 Cc, a result equally valuable for many pur- 
 poses is reached by weighing out the active substance in 
 grams and dissolving it to make 100 Cc. of solution. 
 
 In this case the formula to be used is 
 
 10 4 a 
 
 As an illustration of the use of the polariscope deter- 
 mine the specific rotation of .several sugar solutions. 
 
 Ex. 29. Weigh out 10 Gm. of cane sugar and dissolve 
 it in distilled water in a 100 Cc. flask. When solution is 
 •complete, fill to the mark and shake well. 
 
 Close one end of the 200 Mm. polarizing tube with its 
 glass cap and fill with the solution to be tested. Now put 
 on the other glass end so as to exclude all bubbles of air 
 and screw it down. Place the filled tube in its receptacle 
 in the polariscope and direct this toward a bright sodium 
 light in an otherwise dark room. 
 
 If the instrument had been adjusted so that the zero of 
 the scale corresponded to the condition of equal shadows 
 on the circular plate it will be found that with the sugar 
 solution between the polarizer and analyzer, the latter, 
 with the divided circle, must be rotated through a certain 
 number of degrees to reach the same shadow effect. Note 
 the direction and amount of this rotation, and calculate 
 the specific rotation according to the last formula above. 
 
 Ex. 30- Repeat the experiment, using solutions of 
 grape sugar and honey in known amount. The rotation of 
 the latter varies with different samples. 
 
 The following are mean values of the specific rotations 
 of different substances. 
 
 d, in these formulas, indicates sodium light. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY 53 
 
 Saccharose [«]d = 66.5°, ^=10 — 30 
 
 Lactose (C 12 H a2 O n .H, O). . . " = 52.5°, c= 5— 12 
 
 Dextrose (anhydrous) " = 52.1°, c= 2 — 20 
 
 Invert sugar " = — 27.9°, c = 17 — 
 
 Serum albumin " = — 56.° 
 
 Egg albumin " = — 35. 5 C 
 
 - a 
 
 The use of the polariscope in the special examination of 
 proteids will be referred to later. The instrument is em- 
 ployed also in many chemical investigations in lines not 
 related to physiological chemistry. 
 
 In illustration of the methods of calculation the details 
 of some observations will be given. A sample of pure oil 
 of turpentine was polarized in a tube 300 Mm. long and at 
 the temperature of 20° C. The rotation of the plane of 
 polarization was found to be 40.504°. The specific gravity 
 of the oil, accurately determined, was found to be at 
 20°, 0.8650, water at 4° being taken as unity. We have 
 therefore, « d = 40.504 
 
 / = 300. 
 d— 0.865 
 from which follows 
 
 r , _ 100X40.504 
 
 [a n = = lo.fa08 
 
 L 300X 0.865 
 
 In another case 25 Gm. of pure Rochelle salt was dis- 
 solved in distilled water and made up to a volume of 
 exactly 100 Cc. at 20°. The solution was examined in a 
 tube 200 Mm. long and was found to have a rotation of 
 11.058°. 
 
 Here we have 
 
 a D — 11.058° 
 /= 200. 
 c = 25. 
 from which follows 
 
 [«],= 10'X11.05S =22n6O 
 L JD 200X25 
 
54 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Fermentation of Sugars. 
 
 It has been shown above that certain sugars in solution 
 readily undergo fermentation by action of the yeast cell. 
 The products formed are essentially ethyl alcohol and car- 
 bon dioxide. 
 
 Cane sugar does not ferment with ordinary yeast, and 
 strong solutions cannot be fermented at all. In fact, many 
 of the uses of cane sugar depend on its power of pre- 
 venting fermentations or putrefactions. This is illustrated 
 in the "canning" or preservation of fruits, and in the em- 
 ployment of syrups in the pharmacopoeia. 
 
 Ex. 31. Make a strong cane sugar syrup, by boiling or 
 heating together 10 Gm. of sugar and 10 Cc. of water. 
 Allow to cool and add about a gram of crumbled com- 
 pressed yeast, and then set aside for several days. The 
 solution should be found free from any signs of fermentation. 
 
 Conditions of Alcoholic Fermentation. The follow- 
 ing experiments will be of interest in showing some of the 
 conditions on which alcoholic fermentation depends. 
 
 Ex. 32. Prepare a twenty per cent solution of com- 
 mercial dextrose and pour 20 Cc. of it in a test-tube. 
 Add some yeast and allow to stand about two days in a 
 moderately warm place. At the end of this time it should 
 be found in active fermentation, as shown by the escape of 
 gas bubbles and the odor of alcohol. 
 
 Keep for future experiment. 
 
 Ex. 33. Prepare a tube with sugar solution and yeast 
 as in the last experiment. Close it loosely with a plug of 
 absorbent cotton and heat to boiling, allowing steam to 
 escape through the cotton. If the tube is now left to itself 
 for several days it will be found that fermentation has not 
 taken place, showing that heat destroys the characteristic 
 property of the yeast cell. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 55 
 
 Ex. 34- Prepare another tube with sugar and yeast and 
 add 10 Cc. of strong alcohol. Shake the mixture and 
 allow to stand. No fermentation appears as the activity of 
 the yeast cell is destroyed by alcohol. We have good famil- 
 iar illustrations of this in the self-preservation of certain 
 "heavy" wines, as ports, sherries and malagas, while 
 "light" wines, which contain ten to twelve per cent of al- 
 cohol, usually must be kept tightly bottled for preservation. 
 
 Ex. 35. Tests for alcohol. We have many tests by 
 which the presence or formation of alcohol may be shown. 
 The fermentation of a saccharine liquid is followed by a 
 lowering of the specific gravity as already mentioned, and 
 a practical quantitative test is based on this fact. A 
 simple chemical test for the presence of alcohol, which in 
 most cases is sufficient, is the following: Add to the clear 
 liquid to be examined a few small crystals of iodine, warm 
 to about 60° C.,and then add enough sodium hydroxide or 
 carbonate to produce a colorless solution. An excess of 
 the alkali must not be used. In a short time bright yellow 
 crystals of iodoform precipitate, easily recognized by their 
 color and odor. Certain other liquids give the same test. 
 
 Acetic Acid Fermentation. Weak alcoholic solu- 
 tions, if containing small amounts of foreign matter, espe- 
 cially nitrogenous, become sour, if exposed to the air, from 
 formation of acetic acid. This is due to the fact that a 
 small microscopic plant cell, the mycoderma aceti, is gener- 
 ally present in the atmosphere, and in sufficient numbers to 
 oxidize the alcohol to acid. 
 
 Ex. 36. The solution of Ex. 32 becomes sour if al- 
 lowed to stand four or five days in a warm place. When 
 the odor of alcohol has practically disappeared add some 
 precipitated chalk and evaporate to dryness. Extract with 
 a little water, filter and concentrate the filtrate to a small 
 bulk; add equal volumes of alcohol and strong sulphuric 
 acid and apply heat. The characteristic odor of acetic 
 ether is developed, proving the presence of acetic acid as a 
 product of fermentation. 
 
56 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The acetic acid ferment is destroyed by high tempera- 
 ture or strong alcohol, which can be shown by experiments 
 similar to those detailed above. 
 
 Lactic Acid Fermentation. By the action of a pecu- 
 liar ferment solutions of sugar in presence of nitrogenous 
 matters become converted into lactic acid, according to the 
 equation : 
 
 C lf H a2 O,, +H s O=4C 3 H 6 3 
 
 The reaction takes place best in neutral or slightly alka- 
 line solution and is speedily stopped by the accumulation 
 of acid formed. In practice, therefore, it is customary to 
 add some chalk or zinc oxide to the fermenting mass to 
 take up the acid as fast as formed. The fact of the produc- 
 tion of acid is shown by the following experiment: 
 
 Ex. 37. Dissolve 30 Gm. of cane sugar in 150 Cc. of 
 boiling water and add about 30 Mg. of tartaric acid. After 
 this mixture has stood two days, add some putrid cheese, 
 about half a gram, and 40 Cc. of sour milk. Then add 15 
 Gm. of zinc oxide and allow the mixture to stand ten days 
 in a warm place where the temperature is about 45° C, 
 stirring frequently. At the end of this time heat to boiling, 
 filter hot and allow the zinc salt to crystallize out on cool- 
 ing. Dissolve the crystals in a small amount of hot water, 
 and allow them to settle out again. Collect these purer 
 crystals, mix with water and pass hydrogen sulphide into 
 the mixture in excess. Filter off the precipitated zinc sul- 
 phide and shake the aqueous filtrate with ether in two por- 
 tions, using 25 Cc. in all. Allow the ether to evaporate 
 spontaneously, leaving the lactic acid as a thick liquid. 
 
 Butyric Acid is produced from lactic atid by prolonged 
 fermentation. 
 
 If the fermented mass, in the above experiment, contain- 
 ing crystals of calcium or zinc lactate, is allowed to stand 
 some weeks at about the same temperature, bubbles of 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 57 
 
 hydrogen and carbon dioxide will escape, and decompo- 
 sition, according to the following equation takes place: 
 
 2 C 3 H 6 3 =C 4 H 8 2 +2 C 2 +2 H 2 
 
 This action, like the former, is due to the presence of a 
 minute microorganism. 
 
 Glycogen or Animal Starch. 
 
 Closely related to the carbohydrates described above 
 we have a compound produced in the animal body and 
 known as glycogen. This substance is found in small 
 quantities in many of the tissues, but is relatively abun- 
 dant in the liver only, from which organ it is usually pre- 
 pared. It is also found in the common oyster, which is 
 sometimes employed as a starting point in the preparation 
 of the pure substance. 
 
 The laboratory production of glycogen is best carried 
 out as follows: 
 
 Ex. 38. Kill a rat or a rabbit; remove the liver as 
 quickly as possible and without delay cut it into small bits 
 which throw into a vessel of boiling water. The weight of 
 the water should be about five times that of the minced 
 liver. Boil five minutes, then remove from the water and 
 rub up in a mortar with fine clean quartz sand. In this 
 way the fragments of liver become thoroughly disinte- 
 grated. The contents of the mortar, sand as well as liver, 
 are thrown into the boiling water again and kept at 100° C. 
 fifteen minutes. At the end of this time enough dilute 
 acetic acid must be added to impart a faint acid reaction. 
 This coagulates and precipitates some albuminous matters 
 which are separated when the hot mixture is filtered. 
 
 In the opalescent filtrate, which must be collected in 
 a cold beaker, a further precipitation of albuminous mat- 
 ter is effected by adding a few drops of hydrochloric acid 
 and some potassium mercuric iodide as long as a precipi- 
 tate forms. 
 
58 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Filter again and use the dilute aqueous solution of gly- 
 cogen resulting for tests below. 
 
 Ex. 39. Evaporate about half of the liquid above to a 
 small volume and precipitate impure glycogen as an 
 amorphous white powder by addition of strong alcohol. 
 
 Ex. 40. Add a little tincture of iodine to a small por- 
 tion of the solution, and note the red color produced. This 
 color is discharged by heat. Boil some of the solution 
 with dilute hydrochloric acid ten minutes ; neutralize the 
 acid nearly, cool and again test with iodine. No color is 
 now produced, as glycogen has disappeared under the 
 treatment, having been converted into sugar. 
 
 After death the store of glycogen in the liver rapidly 
 disappears, so that tests applied' at the end of a day or 
 two fail to show its presence. 
 
 Ex. 4l. Cut some common beef liver from the market 
 into small bits and extract with boiling water. Boil longer 
 to coagulate the albuminoids, after adding some sodium 
 sulphate. Apply the iodine test for glycogen, which is 
 found absent, and the Fehling test for sugar, which is found 
 present in quantity. 
 
 In Experiment 38, the direction is given to treat imme- 
 diately with boiling water. This is done to destroy a fer- 
 ment-like body in the liver, which speedily converts gly- 
 cogen into sugar. 
 
 Solutions of glycogen in water exert a marked dextro- 
 rotatory action on polarized light, but observers differ as 
 to the exact specific rotation. It is about 
 
 [«]„ = 210°. 
 
Chapter III. 
 
 FATS. 
 
 T^HE bodies commonly called fats are compounds of 
 * glycerol with palmitic, stearic and oleic acids. Butter 
 fat contains in addition glycerol compounds of butyric, 
 caproic and other less important acids. The fats of the 
 vegetable kingdom are in many respects similar to those 
 of the animal kingdom, and like the latter, serve as food 
 for man. 
 
 The important animal fats are butter, lard and the sev- 
 eral kinds of tallow. The important vegetable fats are 
 olive oil, cottonseed oil, sesame oil, peanut oil, cocoa but- 
 ter and others. Pure fats are usually separated from the 
 accompanying animal or vegetable tissue by pressure, or 
 by melting, as illustrated by the manufacture of olive oil 
 and the rendering of lard or tallow. 
 
 Origin of Fats in the Body. The fats found in the 
 animal tissues are obtained from three general sources. 
 First. They are taken directly from the fatty matters of the 
 food, and the amount so absorbed may be considerable, as 
 has been shown by several physiologists who have inves- 
 tigated the question. 
 
 Second. A production of fat goes on in the body when 
 the animal is fed on a diet of albumin alone. The amount 
 of butter which a cow will produce is far in excess of the 
 fat in the feed, and can be accounted for only .on the 
 assumption of decomposition of albuminous matters. Dogs 
 have been starved until their bodies were practically free 
 
60 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 from fat and then fed on muscle and soaps of palmitic and 
 stearic acids. After a time they were killed, and on analy- 
 sis their bodies were found to contain not only palmitin 
 and stearin, but also olein. This must have come from 
 a breaking down of the food proteids. The formation of 
 large quantities of adipocere in cadavers is considered, also, 
 as a proof of the production of fatty acids from albu- 
 minoids. 
 
 Finally, it has been abundantly proven that carbohy- 
 drates give rise directly to fats by molecular changes in 
 the body. Indirectly, the consumption of carbohydrates 
 favors production of fats by taking the place of the al- 
 buminoids in oxidation. The latter are, therefore, left to 
 be broken up into a nitrogenous residue, and a fat-forming 
 residue which is stored up in the tissues. 
 
 Some of the important properties of fats are shown by 
 the following experiments : 
 
 Ex. 42. Note the solubility of small bits of tallow in 
 ether, chloroform, benzine and alcohol, using in each case 
 the same volume of liquid, with equal weights of fat. The 
 solubility in alcohol will be found much less than, in the 
 other menstrua. The common fats are insoluble in water. 
 
 All fats undergo a very remarkable change when heated 
 with solutions of caustic alkalies. This treatment is suffi- 
 cient to split them up into their component parts, glycerol 
 on the one hand and the alkali-metal salt of the fatty acid 
 on the other. Thus stearin, or glyceryl stearate, the com- 
 pound of glycerol with stearic acid, yields, when treated 
 with a strong solution of potassium hydroxide, free 
 glycerol and potassium stearate, as shown by the follow- 
 ing tests: 
 
 Ex. 43. Boil about 25 Gm. of cottonseed oil or tallow 
 with a solution of 10 Gm. of potassium hydroxide in 25 Cc. 
 
ELEMEiYTARY CHEMICAL PHYSIOLOGY. 61 
 
 of water. Stir the mixture thoroughly until it becomes 
 homogeneous, that is, until no oil globules are seen float- 
 ing on top of the aqueous liquid, which may require half 
 an hour. Add water from time to time, to make up for 
 that lost by evaporation. 
 
 The resulting mass is a mixture of glycerol, excess of 
 alkali and soft soap. 
 
 Ex. 44. The presence of fatty acids in the above 
 mixture can be shown by adding to a portion of it enough 
 hydrochloric acid to decompose the soap. Use about half 
 the product of Ex. 43, dilute it with water, and add the 
 acid in slight excess, about 10 Cc. of the strong commercial 
 acid. Warm on the water-bath, which will cause the 
 liberated fatty acids to collect on the surface as a liquid 
 layer as soon as the temperature becomes high enough. 
 Add more water and allow the whole to cool. A semi- 
 solid layer of fatty acids can now be lifted from the surface 
 of the liquid. The hardness of the mixed acids depends 
 on the nature of the fat taken for experiment. Mutton 
 and beef tallows yield very solid acids; with lard the mass 
 is softer, while with some oils the acids do not solidify at 
 all at the ordinary temperature. 
 
 Ex. 45. Dissolve a small portion of the fatty acids in 
 warm alcohol, nearly to saturation. On cooling, the acids 
 separate in crystalline scales. 
 
 Ex. 46- The presence of glycerol as one of the prod- 
 ucts formed by the saponification of fats is best shown as 
 follows: Mix 50 Cc. of cottonseed oil with 25 Gm. of 
 litharge and 100 Cc. of water in a porcelain dish. Place 
 over a Bunsen burner on gauze and stir until all oil 
 globules have disappeared, adding a little water from time 
 to time. The litharge with water acts as lead hydroxide 
 and saponifies the fat, forming an insoluble lead soap, or 
 plaster, and glycerol. When the saponification is com- 
 plete add more water, heat and stir well to dissolve the 
 gl3 T cerol, allow to settle a short time and pour the aqueous 
 solution through a filler. To the residue add water again, 
 
62 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 heat, allow to settle and pour through the same filter. 
 Concentrate the mixed filtrates to a small volume and after 
 cooling observe the sweet taste of the thickish residue. 
 
 Glycerol of commerce was at one time made by this 
 slow process, but at the present time it is made by other 
 reactions from fats, usually by the action of superheated 
 steam. This passed into melted fat effects a separation 
 into glycerol and fatty acid. Sometimes the fat is decom- 
 posed by milk of lime and the glycerol separated by a cur- 
 rent of superheated steam with which it is volatile. 
 
 The inverse process of combining glycerol and fatty 
 acids to form fats is not so easy, but it has been done on 
 an experimental scale, yielding not only the tri-acid com- 
 pounds similar to those existing in nature, but mono- and 
 di-acid compounds as well. 
 
 Fats being insoluble in water, a permanent mixture of 
 the two cannot be obtained directly. A separation always 
 follows after a time. By the action of certain substances, 
 however, the fat can be brought into a very finely divided 
 condition called an emulsion, which has important prop- 
 erties. 
 
 Emulsions. 
 
 Fats can be thrown into this peculiar physical modifi- 
 cation by a variety of additions, which are illustrated in 
 the experiments below. In order that a fatty substance 
 may leave the alimentary canal and enter the circulation it 
 is necessary that it must first be brought into a condition 
 in which it can be absorbed by the lacteals. Emulsifica- 
 tion accomplishes this, which is mainly carried out in the 
 small intestine. 
 
 Ex. 47. Add to 5 Cc. of cottonseed oil half its volume 
 of strong white of egg solution and shake thoroughly. The 
 liquids mix and form a white mass or emulsion. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 63 
 
 Ex. 48. To 5 Cc. of cottonseed oil containing a little 
 free fatty acid add 10 drops of strong sodium carbonate solu- 
 tion and shake. A good stable emulsion is made in this way. 
 
 Ex. 49. In the intestine this reaction is brought about 
 by the action of the pancreatic juice and bile, which can be 
 illustrated in this manner: Pour about 10 Cc. of refined 
 cottonseed oil in a warm mortar and add two or three 
 grams of fresh finely divided pancreas. This can be ob- 
 tained by running the pancreas through a good sausage 
 mill. Rub the mixture thoroughly with a pestle, by which 
 means a white milky mass is obtained. 
 
 To produce an emulsion the presence of free fatty acids 
 seems to be necessary. Now it is probable that the pan- 
 creatic juice contains a product capable of splitting fats 
 into free acid and glycerol, the first of which with the al- 
 kaline juice produces the emulsion. It is certain that the 
 emulsifying power of the juice does not depend on the 
 presence of an enzyme, because emulsification takes place 
 with boiled pancreatic juice as well as with fresh. In its 
 work of splitting fats the pancreatic juice is assisted by the 
 bile, some tests on which will be given later. 
 
 Crystallization of Fats. 
 
 The neutral fats as well as the fatty acids readily 
 assume the crystalline form under certain conditions. 
 Fats in the tissues are undoubtedly free from crystalline 
 structure, and when melted out at a low temperature or 
 separated by pressure the amorphous form is the common 
 one. Melted fats, on standing, generally become crystal- 
 line, which can be shown by melting a little tallow and 
 placing a drop of it on a glass slide to cool. If a cover 
 glass is pressed down on the fat before it becomes hard 
 this will give a flat field in which the crystalline form can 
 be seen under the microscope. 
 
64 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 fig. ai 
 
 Human fat, crystallized from 
 chloroform. 
 
 FIG. 82. 
 
 Dog fat. crystallized from 
 chloroform. 
 
 FIG. 23. 
 
 Cat fat, crystallized from 
 chloroform. 
 
 FIG. 84. 
 
 Beef tallow, crystallized from 
 chloroform. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 65 
 
 Beef tallow, crystallized from 
 chloroform. 
 
 FIG. 86. 
 
 Pure stearin, crystallized from 
 chloroform. 
 
 FIG. 37. 
 
 Mutton tallow, crystallized from 
 chloroform. 
 
 FIG. 88. 
 
 Lard, crystallized from 
 chloroform. 
 
66 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Perfect crystallization can be secured by deposition 
 from a menstruum. 
 
 Ex. 50. Dissolve some mutton or beef tallow in 
 chloroform and with a glass rod put two or three drops of 
 the nearly saturated solution on the center of a glass 
 slide. As the chloroform evaporates a film begins to 
 form on the top of the drop. Now put on a perfectly 
 clean cover glass and allow to stand until crystallization is 
 complete, which may require only a few minutes or some 
 hours, the time necessary depending on the temperature 
 and on the concentration of the solution. Examine the 
 crystals with a microscope. Use a power of 200 to 300 
 diameters. 
 
 Very peculiar crystallizations are shown by certain fats as 
 can be seen by the annexed cuts. Some of these forms have 
 been taken as characteristic and have been considered as suf- 
 ficient for the identification of several fats, even in mixtures. 
 It is true that in general the crystals of mutton tallow 
 or beef tallow are different from those of pure lard, but it 
 is also true that mixtures can be made which yield crystals 
 practically identical with those usually seen in the fields 
 from lard. At various times it has been supposed that 
 adulterations of lard or butter could be demonstrated by 
 these microscopic appearances, but, one by one, the points 
 brought forward as characteristic have been shown to be 
 of little real value. 
 
 From one and the same portion of fat dissolved in chloro- 
 form, for instance, four or five different kinds of crystals 
 may be obtained, and by using other solvents or changing 
 the temperature of crystallization still different forms may 
 be secured. This fact is illustrated in the figures above 
 which are very exact illustrations of the microscopic ap- 
 pearances. They are copies of micropholographs made 
 by the author and show as observed under a magnifying 
 power of about 200 diameters. 
 
Chapter IV. 
 
 PR0TE1DS OR ALBUniNS. 
 
 THE term proteid is applied to a large number of sub- 
 * stances derived from different sources which are very 
 similar in composition, and which in many instances ap- 
 pear to be impure forms of one and the same compound. 
 
 These proteid compounds are found in both the animal 
 and vegetable kingdoms, but never in pure form. Several 
 closely related varieties are often associated, and mixed 
 with these are fats, carbohydrates, mineral salts, etc., in 
 varying proportions. 
 
 Practically, the preparation of a pure albumin is a mat- 
 ter of very great difficulty, and the analyses made by differ- 
 ent experimenters do not show remarkably close agree- 
 ment. It is, therefore, impossible to assign a formula to 
 these bodies, although this has several times been at- 
 tempted. The outside limits assigned for the percentage 
 composition are given below : 
 
 C 50.0 to 55.0 
 
 H 6.8 " 7.3 
 
 O 20.0 " 24.1 
 
 N 15.0 " 18.2 
 
 S 0.3 " 5.0 
 
 As an illustration of the results reported by a single 
 
 observer the analysis of egg albumin by Hammarsten may 
 
 be quoted. He found for the dry substance : 
 
 C 52.25 
 
 H 6.90 
 
 O 23.67 
 
 N 15.25 
 
 S.'.'.V... 193 
 
 100.00 
 
68 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Albumins as a class are recognized by the following re- 
 actions : 
 
 (a.) They give the characteristic test for nitrogenous 
 
 bodies when fused with soda-lime. 
 (£.) They yield precipitates with solutions of salts of 
 
 certain heavy metals. 
 (V.) They give, especially, red precipitates when 
 
 warmed with Millon's reagent. , 
 (d.) They give a characteristic color when heated with 
 
 strong nitric acid, and are then treated with am- 
 monia. 
 (e.~) They give color reactions when mixed with a small 
 
 amount of copper sulphate solution, and then with 
 
 an excess of alkali. 
 
 These reactions are illustrated by the following experi- 
 ments : 
 
 Ex. 51. Mix some common wheat flour with an equal 
 bulk of soda-lime in a test-tube, and heat strongly in the 
 flame of a Bunsen burner. Ammoniacal vapors are given 
 off, which may be recognized by the odor, or by action on 
 litmus paper. The ammonia is produced in the decompo- 
 sition of the proteid matter of the flour by the fixed alkali. 
 This is a characteristic and delicate reaction. 
 
 Prepare a dilute solution of white of egg and use it for 
 tests to follow. Pour the white of one egg into a strong 
 bottle holding about half a liter. Add 100 Cc. of distilled 
 water and some small quartz pebbles, or better, some 
 pieces of clean broken glass, and shake vigorously. After 
 a time add 400 Cc. more and shake again, and allow to 
 stand about fifteen minutes. Strain through clean, un- 
 sized muslin, and use the nearly clear liquid for the tests. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 69 
 
 Ex. 52. Pour some oi the liquid into several test-tubes 
 and add small amounts of solution of mercuric chloride, 
 copper sulphate, lead acetate and silver nitrate. Note 
 that each reagent produces a precipitate. 
 
 Ex. 53. To another portion of the liquid in a test- 
 tube add half its volume of Millon's reagent (for prepara- 
 tion of this see appendix), and heat. A yellowish pre- 
 cipitate which appears at first becomes red on boiling. 
 
 This test shows equally well with other albumins and 
 is very delicate. With very dilute solutions a red color 
 appears without precipitation. 
 
 Ex. 54. Xanthoproteic reaction. To some albumin in 
 a test-tube add strong nitric acid, and boil. A yellow 
 precipitate or color is formed which, on addition of am- 
 monia, becomes deep orange red. 
 
 The colors obtained in this reaction depend on the 
 strength of the albumin solution. With weak solutions a 
 precipitate may not form at all, but the yellow color is 
 characteristic. When only traces of albumin are present 
 the yellow color with the nitric acid may fail to appear, 
 but the addition of ammonia gives the final part of the test 
 with a yellow instead of an orange red color. 
 
 Ex. 55. To some of the albumin solution in a test- 
 tube add enough sodium hydroxide to give a very strong 
 alkaline reaction. Then add two or three drops of a dilute 
 copper sulphate solution. This gives a violet color, which 
 by application of heat becomes much deeper. This is 
 known as the biuret reaction, and serves for the detection of 
 Small traces of albumins in solution. It is absolutely 
 necessarv to employ a considerable excess of the alkali 
 with only a very small amount of copper solution. 
 
 Ex. 56. Make some albumin solution strongly acid 
 with acetic acid, add some sodium sulphate and boil. All 
 proteids except peptones are precipitated in this manner, 
 and the reaction is employed to free solutions from these 
 bodies. The filtrate, after boiling, can be used for other 
 
70 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 tests as the acid and sulphate exert no decomposing action. 
 The method is frequently employed in urine analysis when 
 it is desirable to test for small amounts of sugar in presence 
 of relatively large quantities of albumin. 
 
 Classification of Albumins. 
 
 Investigation has shown that the bodies of this group 
 fall naturally into several classes. At the present time it 
 is usual to consider seven of these classes, the division 
 being based on certain features easily recognized. It must 
 be remembered, however, that this division is somewhat 
 arbitrary and is not universally accepted as here given, 
 according to Hoppe-Seyhr and others. 
 
 n, „„ t xt i.- iL ■ f Egg albumin, 
 
 Class I. Native albumins. . . i c ,. • 
 
 I Serum albumin. 
 
 ' Alkali albumin, 
 
 />, „„ TI t\ ■ j iu ■ Acid albumin, 
 
 Class II. Derived albumins. \ „ . ' 
 
 Casein, 
 
 Syntonin. 
 
 [ Crystallin, 
 | Vitellin, 
 
 Class III. Globulins J P^raglobulin, 
 
 Fibrinogen, 
 ! Myosin, 
 [Globin. 
 
 Class IV. Fibrin. 
 
 Class V. Coagulated proteids. 
 Class VI. Albumoses and peptones. 
 Class VII. Lardacein. 
 
 Native Albumins. 
 
 These are soluble in water, but are precipitated by 
 boiling or by addition of alcohol. 
 
 Their solutions are not precipitated by sodium chloride 
 or sodium carbonate. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 71 
 
 Egg Albumin. Different kinds of eggs furnish albu- 
 mins of slightly different properties, but these variations 
 may be due to accompanying substances rather than to in- 
 herent differences in the albumins themselves. Most of our 
 knowledge of these bodies has been derived from a study 
 of the hen's egg, and this will be taken for the experiments 
 below. Prepare a solution of white of egg as given above 
 and make tests with it as follows: 
 
 Ex. 57- Note that a small portion of the solution is 
 readily coagulated by heat in a test-tube. By the aid of a 
 thermometer find approximately the temperature of coagu- 
 lation, which is not far from 60° C. Some of the proteids 
 present in white of egg begin to coagulate, it is claimed, 
 at even a lower temperature while a temperature of 80° C. 
 may be reached before coagulation is complete. 
 
 Ex. 58. Warm three or four Cc. of strong nitric acid 
 in a test-tube and pour' in about an equal volume of the 
 albumin solution carefully, so that it will float on top and 
 not mix with the acid. A coagulation of the albumin takes 
 place at the juncture of the two liquids and appears as a 
 white ring. This is a valuable and delicate test. 
 
 Ex. 59. To ten Cc. of the egg solution add powdered 
 magnesium sulphate to saturation. A precipitate of globu- 
 lin separates out. Filter and add to the filtrate some 
 strong solution of sodium sulphate. Precipitation of the 
 albumin takes place now, if the solution is. kept at a tem- 
 perature of 40° C. This precipitation seems to be due to 
 the presence of a double sulphate of magnesium and so- 
 dium in the liquid. 
 
 Serum Albumin. Serum albumin occurs in the blood 
 along with several other proteids, but can be obtained 
 nearly pure by the following method: 
 
 Fresh blood from the slaughter house is poured into a 
 shallow dish and whipped with a bunch of twigs or agitated 
 
72 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 vigorously with an egg beater to separate the fibrin. The 
 latter is strained out by means of clean unsized muslin. 
 The liquid passing through the muslin is put into a centri- 
 fugal apparatus and rotated to throw out the corpuscles. 
 If the centrifugal apparatus is not at hand the fibrin-free 
 liquid can be poured into a tall glass jar and allowed to 
 stand until the corpuscles settle. The clarified liquid ob- 
 tained by either method is poured into a large beaker and 
 mixed with four times its volume of saturated solution of 
 ammonium sulphate, or enough to yield a completely satu- 
 rated liquid. This precipitates the albumin and globulin 
 present, and these compounds are then separated by filtra- 
 tion. The moist mass on the filter is dissolved in a small 
 quantity of water and treated again with an excess of am- 
 monium sulphate which gives a purer precipitate. The 
 product is washed on the filter with strong ammonium sul- 
 phate solution, then dissolved in a little water and sub- 
 jected to dialysis, by which means the salts are eliminated 
 while an albumin solution remains on the dialyser. 
 
 During the dialysis globulin separates and at the end 
 of the operation is filtered out leaving a nearly pure serum 
 albumin. This can be further purified by adding a little 
 ammonia until the reaction becomes neutral, dialyzing 
 again and finally filtering. The clear filtrate can be used 
 for tests or further concentrated. To obtain it in dry form 
 evaporate at 40° C. to a small bulk, add strong alcohol in 
 excess to precipitate, filter without delay, press out the 
 alcohol, and displace the remaining alcohol by washing 
 with ether. Dry at a low temperature, powder and pre- 
 serve in a well-stoppered bottle. 
 
 As shown by the method of preparation serum albumin 
 is soluble in water, but insoluble in saturated solution of 
 ammonium sulphate. The tests given for egg albumin are 
 true for serum albumin. These points of difference maybe 
 noted: 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 73 
 
 Ex. 60. Note that a neutral solution of egg albumin is 
 coagulated by ether, while the solution of serum albumin 
 is not. 
 
 Ex. 61. Coagulate a solution of egg albumin by heat 
 and note that the precipitate dissolves but slightly in boil- 
 ing nitric acid. A precipitate of serum albumin dissolves 
 in nitric acid. 
 
 Ex. 62. Precipitate solutions of egg albumin and 
 serum albumin with nitric acid and note that the latter 
 precipitate is much more readily soluble in excess than is 
 the former. 
 
 Derived Albumins. 
 
 These are nearly insoluble in pure water or dilute saline 
 solutions, but are soluble in weak acids or alkalies. Boil- 
 ing does not coagulate the solutions. 
 
 Alkali Albumin. This is most readily prepared by 
 the action of alkali on native albumins. 
 
 Ex. 63. Add strong sodium hydroxide solution to 
 white of egg, with constant stirring, until a thick jelly is 
 formed. Too much alkali must not be added here, but 
 just enough to make the maximum of jelly. This is now 
 cut into small pieces and washed in distilled water several 
 times until the lumps are white throughout. They are 
 then heated with fresh pure water, but very gently, until 
 they go into solution. This is then filtered and the filtrate 
 precipitated by acetic acid, avoiding any excess. The pre- 
 cipitate is washed with pure water. 
 
 Observe that this substance is soluble in dilute acid or 
 alkali solutions. 
 
 Ex- 64. Bring some of the washed alkali albumin into 
 solution with hot water. Add a few drops of phenol 
 phthalein solution, which imparts a red color, and then run 
 in dilute sulphuric until the alkali reaction just disappears. 
 As soon as the neutral point is reached a precipitate falls, 
 but, if the addition of acid be continued it will disappear 
 with formation of acid albumin. 
 
74 ELEMENTARY CHEMICAL PHYSIOLOGY 
 
 The thick jelly-like substance first formed by the action 
 of strong alkali on undiluted white of egg solution is called 
 Lieberkuehn's jelly. It contains an excess of uncombined 
 alkali. A weak solution of alkali albumin can readily be 
 made by warming diluted white of egg solution with a 
 small amount of very weak alkali solution. The tempera- 
 ture should be maintained at 35° C. to 40° C, for about 
 an hour. At the end of this time the solution can be boiled 
 without coagulation. 
 
 Acid Albumin. The bodies called acid albumins are 
 very similar in properties to the alkali albumins and at one 
 time were supposed to be identical with them. But careful 
 experiments have disclosed several points of difference. 
 Acid albumin is most readily made by the action of acid on 
 native albumins. 
 
 Ex. 65. Dilute white of egg with four volumes of 
 of water, take 25 Cc. of the mixture, add 5 Cc. of 0.2 per 
 cent hydrochloric acid and warm it on the water-bath for 
 about two hours to a temperature of 45°. Then carefully 
 neutralize the solution with dilute sodium hydroxide. This 
 precipitates insoluble acid albumin, which can be washed 
 with water by decantation. It is essential that just the 
 right amount of alkali be added here, an excess would 
 redissolve the precipitated acid albumin with formation of 
 alkali albumin. The washed acid albumin can be used for 
 a number of tests. 
 
 Ex. 66- Redissolve a small portion of the precipitate 
 with hydrochloric acid, and observe that the solution is not 
 coagulated by boiling. 
 
 Mix another portion of the precipitate with water and 
 heat to 75° or 80°. This brings about a coagulation sim- 
 ilar to that produced with native albumins. 
 
 Acid albumins are very quickly formed by the action of 
 strong hydrochloric or acetic acid on white of egg. Coagu- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 75 
 
 lated proteids may also be converted into acid albumins by 
 a much longer digestion. 
 
 The striking peculiarity of this acid albumin is that it is 
 insoluble in pure water, but does dissolve in weak acid or 
 alkali. It gives the characteristic yellow color with strong 
 nitric acid, a reddish yellow precipitate with Millon's 
 reagent and the biuret reaction. 
 
 Acid albumin is less soluble than alkali albumin and 
 appears to differ also in specific rotation. 
 
 Casein. Milk contains several proteid substances, the 
 most abundant of which is casein. In normal cow's milk 
 it is present to the extent of about four per cent, while in 
 woman's milk it appears to amount to two and one-half 
 per cent or less. While the determination of casein in 
 cow's milk has been made with great accuracy and is an 
 operation of no special difficulty there seem to be prac- 
 tical difficulties in the way of carrying out this determina- 
 tion in the case of human milk. The first point of uncer- 
 tainty is found in the collection of a normal sample, and 
 from this and other causes it follows that the results given 
 by different chemists for the amount of casein present vary 
 between 0.6 and over 5 per cent. 
 
 Equally great variations have been found in the milk 
 furnished by several animals. 
 
 Our practical knowledge of casein has been in the main 
 derived from a study of the product of cow's milk. 
 
 In most of its reactions casein is closely allied to al- 
 kali albumin and by some has been supposed to be identi- 
 cal with it. But other reactions disclose certain differ- 
 ences which seem to be essential and the weight of evi- 
 dence appears to be against the identity of the two pro- 
 teids. The important properties of casein are made ap- 
 parent by the following experiments. 
 
76 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Ex. 67. To 100 Cc. of fresh milk add 500 or 600 Cc. 
 of distilled water and then enough dilute acetic acid 
 to give a distinct acid reaction. This will throw down a 
 copious white precipitate of casein with fat which is al- 
 lowed to settle and washed with distilled water by decan- 
 tation. The precipitate is then transferred to a filter and 
 allowed to drain and then washed with 50 Cc. of strong 
 alcohol. This removes water and part of the fat present. 
 The precipitate is finally washed with ether to remove the 
 remainder of the fat and allowed to dry in the air. 
 
 After the above treatment the casein is left as a white 
 powder pure enough for tests. A better product is ob- 
 tained by dissolving the precipitate first thrown down in 
 weak soda solution and passing the liquid through several 
 filters. It is then reprecipitated with dilute acetic acid, 
 washed by decantation, collected on a filter and finished as 
 before. 
 
 Ex. 68. Test a small amount of the casein prepared 
 according to the last experiment as to its solubility in 
 water. This will be found to be very slight. It dissolves 
 readily in weak alkali solutions. 
 
 Ex. 69. Mix 5 Cc. of fresh milk in a test-tube with 10 
 Cc. of saturated salt solution. Then add a little more 
 powdered salt, close the test-tube with the thumb and 
 shake vigorously. Casein with fat is precipitated and can 
 be collected on a filter. Boil the opalescent filtrate and 
 note the coagulation of a soluble albumin present. In 
 cow's milk this form of albumin amounts to about 0.5 per 
 cent. 
 
 An excess of magnesium sulphate solution may be used 
 instead of the sodium chloride to precipitate the casein. 
 
 Under the action of rennin, casein undergoes a peculiar 
 modification resulting in the formation of a clot or curd. 
 The nature of this will be explained in the chapter on milk. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 77 
 
 The behavior of milk with rennin serves to distinguish 
 casein from true alkali albumin. With the latter, it is said, 
 no clot can be obtained. 
 
 Pure casein yields no ash on ignition, although it con- 
 tains phosphorus and sulphur in organic combination. 
 Pure alkali albumin yields a small amount of ash when 
 ignited. 
 
 Syntonin. This appears to be an acid albumin, re- 
 sulting from the action of dilute acids on muscle. 
 
 Ex. 70. Free the muscular part of meat from fat as 
 far as possible and run it through a sausage mill several 
 times to bring it to a fine state of subdivision. Wash 
 this chopped mass with distilled water until the washings 
 remain clear. Now, to about 5 Gm. of the moist residue 
 in a small flask, add 50 Cc. of dilute hydrochloric acid, 
 containing ^ per cent of the true acid. Warm the mix- 
 ture slightly (to 35° or 40° C"), and keep at this tem- 
 perature about three hours. Then filter and test the fil- 
 trate. It contains the soluble syntonin. 
 
 Ex. Jl. To a small portion of the filtrate, add weak 
 caustic soda, which produces a precipitate soluble in ex- 
 cess of the alkali. 
 
 Boil another portion of the filtrate. It does not co- 
 agulate. 
 
 Globulins. 
 
 We have here a peculiar class of proteid bodies, in- 
 soluble in pure water and strong saline solutions, but 
 soluble in weak saline solutions. A globulin dissolved 
 in a weak solution of salt is precipitated if the same salt 
 is added to saturation. Magnesium sulphate is most ac- 
 tive in producing this precipitation. The globulins resem- 
 ble the native albumins in being coagulable by heat; they 
 are also insoluble in alcohol. Dilute acids or alkalies 
 dissolve them by forming acid or alkali albumin. 
 
78 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Crystallin. This is the globulin of the crystalline lens, 
 and can be obtained by extracting the latter, in finely di- 
 vided form, with a one per cent salt solution. 
 
 Vitellin. Yolk of egg contains a large amount of this 
 globulin, associated with other substances of which lecithin 
 is, perhaps, the most important. The preparation of pure 
 vitellin is a matter of great difficulty because of the pres- 
 ence of the lecithin which cannot be readily separated. A 
 partial separation can be effected by extracting with ether, 
 leaving a residue which dissolves easily in salt solution. 
 A great excess of sodium chloride does not reprecipitate, 
 as is the case with other globulins. 
 
 Paraglobulin, or serumglobulin occurs with pure albu- 
 min in blood serum, from which it can be separated in 
 nearly pure condition. 
 
 It was shown above, under the head of blood serum, 
 that in the process of dialysis for the final purification of 
 the latter, globulin separates out as the saline liquid loses 
 its salts and becomes aqueous. The precipitation of 
 globulin can be illustrated by the following experiment : 
 
 Ex. 72. Separate fibrin and corpuscles from blood as 
 described above under serum albumin and saturate the re- 
 sulting liquid with magnesium sulphate. This throws down 
 a precipitate of globulin, which can be washed on a filter 
 with a saturated solution of the sulphate without much 
 loss. By mixing the precipitate with a little water, and 
 gradually adding more, it will finally dissolve, but the ad- 
 dition of a very large volume of water produces a precipi- 
 tate again. 
 
 Common salt, added in excess, acts as does the magne- 
 sium sulphate. 
 
 The method illustrated by the above experiment is not 
 sufficient for the preparation of pure globulin. A better 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 79 
 
 product is obtained by dissolving and reprecipitating sev- 
 eral times with magnesium sulphate. Finally the precipi- 
 tate may be dissolved in the right amount of water and 
 dialyzed for the separation of the salts. The globulin re- 
 mains on the dialyzer as an insoluble residue. 
 
 Fibrinogen. In the clotting of blood this substance 
 plays an important part as in the operation it becomes con- 
 verted into the stringy fibrin which encloses the corpuscles 
 forming a thick and jelly-like mass. 
 
 Myosin. This is a globulin formed in muscle after 
 death and is produced from a substance resembling fibrin- 
 ogen existing in the living muscle. The name myosinogen 
 has been given to this primary form. 
 
 The preparation of myosin is illustrated by the next ex- 
 periment: 
 
 Ex. 73- Free muscle (round steak) as far as possible 
 from traces of fat and sinews and then thoroughly disinte- 
 grate it by passing through a sausage mill. Then wash it 
 repeatedly with cold water until the latter is no longer red- 
 dened, and the residue appears white. This is placed in a 
 20 per cent solution of ammonium chloride and allowed to 
 remain about a day, with occasional shaking. Myosin dis- 
 solves in the ammonium chloride and is found in the filtrate 
 when the mixture is filtered. Pour the filtrate into twenty 
 times its volume of distilled water, which causes a precipi- 
 tation of the insoluble myosin. Allow to settle and wash 
 three times by decantation. Collect the precipitate and 
 observe that portions of it dissolve readily in ten per cent 
 solutions of sodium chloride and ammonium chloride or in 
 a 0. 1 per cent solution of hydrochloric acid. The solution 
 in salt is precipitated by the addition of more to saturation. 
 
 Myosin in salt solution coagulates at a low temperature, 
 usually given as 55° to 56° C. By acids it is easily con- 
 verted into syntonin. 
 
80 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Globin is sometimes described as a distinct globulin, 
 formed by the spontaneous decomposition of haemoglobin, 
 but no great amount of study has been given to it by 
 chemists. 
 
 Fibrin. 
 
 Fibrin is the name commonly, given to the substance 
 obtained by whipping blood with a bundle of twigs. So 
 prepared it is very impure, holding white and also some red 
 corpuscles. By other means, however, a much purer 
 product can be obtained. Made in any manner fibrin is a 
 white elastic substance insoluble in water and weak salt 
 solutions. It dissolves gradually in acid or alkali solutions, 
 forming proteids of other groups. A corresponding vege- 
 table product can be easily made by mixing flour with a 
 little water to form a stiff dough. After standing some 
 time this is worked with the hand under running water 
 which washes out the starch, leaving finally the vegetable 
 fibrin or gluten as a white stringy mass. 
 
 Animal fibrin is produced from the proteid referred to 
 above as fibrinogen, occurring in the blood before death. 
 
 Coagulated Proteids. 
 
 We have here a class of substances produced by action 
 of heat on most of the proteid bodies just described. 
 When a proteid assumes the coagulated form it becomes 
 insoluble in water, dilute acids or alkalies, and can be dis- 
 solved by strong acids only through complete modification 
 and partial destruction. Alcohol has also the property of 
 coagulating many proteid substances. 
 
 We have a familiar illustration 'of a coagulated pro- 
 teid in boiled white of egg. This, and similar substances, 
 undergo a peculiar change called digestion when taken into 
 the stomach where they are acted on by the gastric juice, 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 81 
 
 or in the intestines under the action of the pancreatic 
 juice. This digestion will be considered in a few experi- 
 ments later, as it is one of the important reactions taking 
 place in the animal body. 
 
 Albumoses and Peptones. 
 
 Under the action of the gastric juice and the secretion 
 of the pancreas just referred to albuminous substances in 
 general surfer a profound modification whereby they be- 
 come soluble and ready for absorption as preliminary to 
 nutrition. The gastric juice is a weak acid liquid of rather 
 complex composition, but containing at least two sub- 
 stances of importance which merit our attention here. 
 
 The first of these is a peculiar enzyme or soluble fer- 
 ment called pepsin and the second is hydrochloric acid. 
 This acid is present in all normal gastric juice and in man 
 it amounts to about 0.25 per cent of the total weight 
 of the juice. The other substances present have as a mat- 
 ter of course some important function, but the manner of 
 behavior of the acid and pepsin have been made the sub- 
 ject of many investigations. The essential work of these 
 two compounds is to convert other proteids into the forms 
 known as albumoses and peptones, of which the latter are 
 extremely soluble and to some degree diffusible. Between 
 the ordinary coagulated proteids taken into the stomach 
 as food and the final peptone or completely digested mat- 
 ter there are possibly a number of intermediate stages. 
 The albumoses represent one of these and can be distin- 
 guished from the end product by several fairly definite re- 
 actions. It appears, further, that at the beginning of the 
 process of digestion with pepsin and hydrochloric acid that 
 a body similar to or identical with acid albumin is formed, 
 to be soon followed by albumose. 
 
82 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 In studying the digestion of starches it was shown that 
 the pancreatic fluid contains an enzyme very active in this 
 direction, and further that in the emulsification of fats, as a 
 step in digestion, the pancreatic juice plays, likewise, a 
 prominent part. The secretion of the pancreas is active 
 finally in a third direction, viz. : in the digestion of proteids 
 when it continues and completes the work begun by the 
 gastric juice. Of the^actual nature of the pancreatic enzymes 
 which bring about this result little is known, but the end 
 seems to be reached in several stages as with the gastric 
 secretion. The first step is, apparently, the production of 
 an alkali albumin, followed by what is termed hemialbu- 
 mose as distinguished from antialbumose, formed in gas- 
 tric digestion. Hemialbumose is the forerunner of hemi- 
 peptone, the product corresponding to antipeptone pro- 
 duced by pepsin. 
 
 There is not, however, a complete parallelism between 
 the actions of the two digestive fluids. The work of the 
 gastric juice ends with the production of peptone, while 
 that of the pancreatic secretion includes the formation of 
 several decomposition products, as leucine, tyrosine and 
 others. These changes can be represented as follows 
 diagrammatically (according to Kuehne): 
 
 Antialbumose. -1 Antipeptone ■< ¥? P*P" C 
 
 Albumin. 
 
 ( Leucine. 1 By Pan- 
 Hemialbumose. -j Hemipeptone 4 Tyrosine 1 creatic di- 
 
 ( etc. ( gestion. 
 
 Digestion of Proteids. 
 
 The changes referred to above can, in part, be repre- 
 sented by simple experiments, of which a number will here 
 follow. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 83 
 
 Peptic Digestion. An active peptic ferment fluid can 
 be readily prepared as follows: 
 
 Separate the mucous membrane of the hog's stomach 
 from the outer coatings, and cut it into very small bits or 
 disintegrate thoroughly in a sausage mill. Place the 
 chopped mass in a flask or bottle and cover it with about 
 twice its weight of good, strong glycerol. Allow to stand 
 a week or ten days, with frequent shaking, and then pour 
 off the glycerol, which is now a peptic extract. 
 
 For the experiments given below a good solution may 
 be prepared by treating 5 Gm. of the minced membrane 
 with 8 to 10 Cc. of glycerol. 
 
 Ex. 74- Boil an egg until it is hard, take out the 
 white portion and rub it through a clean wire sieve with 
 fine meshes, by means of a spatula. Add about 5 Gm. of 
 this egg to 100 Cc. of 0.2 per cent hydrochloric acid in a 
 flask, and then add 2 Cc. of the glycerol extract. Keep the 
 flask at a temperature of 40° C, with frequent shaking. 
 In time the egg albumin will dissolve, forming an opales- 
 cent liquid. Unless the flask is very frequently shaken 
 the solution of the albumin will be slow. Use the solu- 
 tion for experiment 76. 
 
 Ex. 75. To 2 Cc. of the glycerol extract in a test-tube 
 add a little water and boil a few minutes. Now add this 
 boiled liquid to albumin and 0.2 per cent hydrochloric 
 acid as in the last experiment and note that- under the 
 same conditions digestion does not take place, the heating 
 having destroyed the active enzyme. 
 
 Ex. 76. Test for Albumose and Peptone. About 
 half an hour after the beginning of the digestion explained 
 in the above experiments pour off about half the liquid, 
 neutralize it exactly with ammonia and then saturate with 
 ammonium sulphate. This precipitates albumose but not 
 peptone. Filter off the precipitate and apply the biuret 
 test to a portion of the filtrate, using only a very small trace 
 
84 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 of copper sulphate. A pink color should be observed. 
 Concentrate the remainder of the filtrate from the albumose 
 precipitate by evaporation at a low temperature, pour it in 
 a test-tube and add some strong alcohol. This gives a 
 precipitate of peptone, which dissolves by adding water. 
 
 The half of the liquid of Ex. 74, not used in the last 
 test, if allowed to stand long enough will give tests for 
 peptones only, the albumose being entirely converted. It 
 will afford the student good practice to carry out experi- 
 ments on the digestion of albumin, using some of the pre- 
 pared pepsins on the market. These products are made 
 by several processes from the hog's stomach and vary 
 greatly in their digestive power. 
 
 Comparisons between different commercial pepsins must 
 be made by some arbitrary method, but carefully con 
 ducted as to temperature, amount of liquid employed, 
 character and condition of the albumin taken for experi- 
 ment, and so on. A process frequently followed is here 
 given: 
 
 Process of the United States Pharmacopoeia for 
 Valuation of Pepsin. 1890. 
 
 "Prepare, first, the following three solutions: 
 
 A. To 294 Cc. of water add 6 Cc. of diluted hydro- 
 chloric acid, ten per cent. 
 
 B. In 100 Cc of solution A dissolve 0.067 Gm. of the 
 pepsin to be tested. 
 
 C. To 95 Cc. of solution A, brought to a temperature 
 of 40° C, add 5 Cc. of solution B. 
 
 The resulting 100 Cc. of liquid will contain 0.2 Cc. 
 (0.21 Gm.) of absolute hydrochloric acid and 0.00335 Gm. 
 of the pepsin to be tested. 
 
 Immerse and keep a fresh hen's egg during fifteen 
 minutes in boiling water; then remove it and place it in 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 85 
 
 cold water. When it is cold, separate the white coagulated 
 albumin and rub it through a clean sieve, having thirty 
 meshes to the linear inch. Reject the first portion passing 
 through the sieve. Weigh off 10 Gm. of the second, 
 cleaner portion, place it in a flask of the capacity of about 
 200 Cc, then add one-half of the solution C and shake 
 well, so as to distribute the coherent albumin evenly 
 throughout the liquid. Then add the second half of the 
 solution C, and shake again, guarding against loss. Place 
 the flask in a water-bath, or thermostat, keep at a temper- 
 ature of 38° to 40° C, for six hours, and shake it gently 
 every fifteen minutes. At the end of this time the albumin 
 should have disappeared, leaving at most only a few thin 
 insoluble flakes. (Trustworthy results, particularly in 
 comparative trials, will be obtained only if the temperature 
 be strictly maintained between the prescribed limits, and 
 if the contents of the flasks be agitated uniformly and 
 in equal intervals of time.) 
 
 The relative proteolytic power of pepsin, stronger or 
 weaker than that described above, may be determined by 
 ascertaining, through repeated trials, how much of solution 
 B made up to 100 Cc. with solution A will be required 
 exactly to dissolve 10 Gm. of coagulated and disintegrated 
 albumin under the conditions given above." 
 
 The pharmacopoeia of the United States requires that 
 pepsin must be able to digest 3,000 times its weight of 
 coagulated albumin and the above process serves to de- 
 termine whether or not a given sample comes up to the 
 accepted standard. Another method depending on the 
 same general principles may be described as follows: 
 
 General Process. 
 
 Prepare a weak acid and pepsin solution as described 
 above and determine by two or three trials the weight of 
 
86 ELEMENTARY CHEMTCAL PHYSIOLOGY. 
 
 albumin it will digest. Or, most conveniently, to five por- 
 tions of 100 Cc. each of the acid-pepsin mixture add 6, 8, 
 10, 12 and 14 Gm. of the prepared egg albumin and digest 
 in flasks holding about 200 Cc. The flasks must be im- 
 mersed in a large water-bath, the temperature of which is 
 kept uniform by frequent stirring, and must be agitated at 
 regular intervals and to the same extent through five hours. 
 This length of time is sufficient to show approximately 
 the digesting value of the pepsin. 
 
 Now weigh out accurately an amount of the prepared 
 egg albumin about one-third greater than the largest 
 amount dissolved in the flask experiments above, and treat 
 with 100 Cc. of the acid-pepsin mixture in the same man- 
 ner. At the end Of six hours apart of this albumin will be 
 still undissolved, and must be filtered off, dried and 
 weighed. The weight subtracted from that taken, gives 
 the amount which has been actually digested. 
 
 Some precautions are necessary in the filtration and 
 weighing. In order to obtain a liquid which can be filtered 
 readily, it has been recommended to add to it an accurately 
 weighed amount (about 3 Gm.) of finely divided, well 
 washed and dried asbestos, such as is used in the Gooch 
 funnels, with 100 Cc. of water, and shake thoroughly. The 
 mixture is then poured on a large weighed Gooch funnel, 
 washed thoroughly with distilled water, and finally with 
 some alcohol. It is then dried at 110° C. to a constant 
 weight and accurately weighed. From the gross weight of 
 the contents subtract the weight of the asbestos. The 
 remainder represents the weight of the dry albumin. Mul- 
 tiply this by 7.5, to obtain the moist equivalent, and sub- 
 tract this from the weight originally taken, to find that 
 actually digested. 
 
 In the digestion experiments given above, it has been 
 directed to use weak hydrochloric acid with the active 
 enzyme. This is necessary as can be shown by a trial. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 87 
 
 Ex. 77- Pour about 50 Cc. of 0.2 per cent hydro- 
 chloric acid into a flask, add about half a Cc. of the glycerol 
 extract of pepsin and a gram of finely divided hard boiled 
 white of egg. In a similar flask take 50 Cc. of distilled 
 water with the same amounts of pepsin extract and albu- 
 min as before. Place both flasks in water at a temperature of 
 40° C, and keep them there about an hour. In the flask to 
 which the hydrochloric acid had been added, the digestion 
 will be found far advanced, or complete, while in the other 
 no change will be observed. 
 
 Test the liquid of the first flask for peptones. 
 
 Lactic acid is frequently found in the gastric juice in 
 small amount, but probably as a product rather than as a 
 factor in peptic digestion. Hydrochloric acid is always 
 normally present, but under pathological conditions the 
 amount may be very much diminished, or nearly absent. 
 It becomes, at times, a question of importance to deter- 
 mine the actual amount of this acid present in the juice as 
 collected by a tube. Several tests suffice for the recogni- 
 tion of hydrochloric acid in presence of lactic or other 
 organic acid, some account of which will be given in a sub- 
 sequent chapter. 
 
 Pancreatic Digestion of Proteids. Under the head 
 of digestion of starch directions were given for the prep- 
 aration of a pancreatic extract which serves, also, very 
 well for the digestion of proteids. It was shown that the 
 active ferment of the gastric juice acts only in an acid 
 medium ; the ferment of the pancreas acts in a solution 
 which is alkaline in reaction. The mode of action of the 
 pancreatic enzyme can be shown by the following experi- 
 ment 
 
 Ex. 78- Pour 25 Cc. of a 1 per cent solution of sodium 
 carbonate (crystallized salt) into each of several small flasks 
 or test-tubes. Add to each half a Cc. of the glycerol extract 
 
88 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 of pancreas and about a gram of finely divided hard boiled 
 white of egg. (The white of egg can be easily prepared ac- 
 cording to the methods given above under the head of 
 pepsin testing.) Make one of the tubes slightly acid by 
 the addition of dilute hydrochloric acid. Now place all of 
 them in water kept at 40° C. At the end of half an hour 
 remove one of the alkaline tubes, and note that it still con- 
 tains unaltered coagulated albumin. Test the liquid for 
 albumoses and peptones as given above, Ex. 76. After 
 another half hour, test a second tube (after filtration). 
 It will be observed that as the coagulated proteid disap- 
 pears, peptones become more abundant. 
 
 Allow one of the alkaline tubes to remain several hours 
 at a temperature of 40° C. In time it develops a disagree- 
 able odor, due to the presence of indol formed. The tube 
 containing the hydrochloric acid kept several hours at 
 40° C. does not show the effects of digestions-indicating that 
 an acid medium does not suffice for the converting activity 
 of the pancreatic ferment. 
 
 To readily recognize the final products of the pancreatic 
 digestion of proteids it is necessary to start with larger 
 quantities of materials than are given in the above experi- 
 ment. By the following method enough of the products 
 for demonstration in a class can be secured: 
 
 Ex. 79- Mix 25 Gm. of fresh minced pancreas with an 
 equal weight of fresh fibrin and 250 Cc. of thymolized 
 water, in a flask. Place the flask in a thermostat and keep 
 it at a temperature of 40° C- six hours. At the end of this 
 time pour off about one-third of the mixture, boil it and 
 filter warm. Concentrate the filtrate to a small bulk and 
 place drops on glass slides for further evaporation prelimi- 
 nary to microscopic examination. Leucine, one of the pro- 
 ducts, crystallizes in spherical bunches of minute short 
 needles, while tyrosine, another, crystallizes in long needles 
 often bunched together. Tyrosine, even in small quantity 
 gives a rose red color when boiled with Millon's reagent. 
 
 The larger portion of the digesting mixture is allowed 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 89 
 
 to remain six hours longer in the thermostat at 40° C, for 
 the production of indol the presence of which is shown by the 
 characteristic odor of the fluid and also by chemical tests. 
 
 A splinter of pine wood moistened with hydrochloric acid 
 turns red when dipped in a small portion of the liquid 
 previously acidified with strong hydrochloric acid. 
 
 A red color is also imparted by the action of a small 
 amount of nitrous acid on indol. To obtain this test use 
 25 Cc. of the liquid, neutralize the alkali by addition of 
 dilute sulphuric acid, and leave a very little acid in excess. 
 Then add a small amount of a solution of sodium or potas- 
 sium nitrite, which brings out the reaction. 
 
 In the above experiments leucine and tyrosine appear 
 to be true products of pancreatic digestion, while indol is 
 formed by a subsequent putrefactive process. Leucine 
 and tyrosine are very common and frequent decomposition 
 products formed from proteids, or allied compounds, by a 
 variety of reactions. In a following chapter a process will 
 be given by which they may be obtained through the action 
 of weak acids on horn shavings. 
 
 Diffusion of Peptones. Peptones are soluble in water, 
 and diffusible, but their rate of diffusion is so slow that it can- 
 not be readily observed in a simple laboratory experiment. 
 It has commonly been held that this diffusion plays a very 
 important part in the absorption of the digestive products 
 from the intestines, but the phenomenon is of so complex a 
 nature that the part played in it by diffusion or osmosis is 
 one which cannot be clearly defined. 
 
 Lardacein or Amyloid Substance. 
 
 This is a pathological product closely related to the 
 proteids, found in several organs of the body. 
 
 Most authorities agree that it cannot be digested by the 
 peptic or pancreatic enzymes in which respect it differs from 
 
90 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 the common proteids, but it resembles them in yielding 
 acid albumin with strong acids, and alkali albumin on 
 treatment with alkali hydroxides. 
 
 Lardacein is usually separated from finely divided 
 tissue containing it, by washing out everything soluble in 
 water and dilute alcohol and then digesting with pepsin 
 and hydrochloric acid. The lardacein is left, with small 
 amounts of other substances, as an insoluble residue. 
 
 It gives the color reaction with Millon's solution and is 
 specially characterized by its behavior with several reagents, 
 giving a red or brown color with iodine and a violet to pure 
 blue with iodine and sulphuric acid. Hence the name, 
 from fancied resemblance to amylum. It gives color reac- 
 tion with several of the aniline colors also. 
 
Chapter V. 
 
 THE BLOOD. 
 
 TttE blood of man, normally, is an opaque viscid fluid, 
 red in color and alkaline in reaction. It is not and 
 cannot be constant in composition, because of the several 
 functions it is called upon to perform. The blood carries 
 nutrient matter to the tissues and must vary in density and 
 complexity according to the nature and amount of the 
 nutritive supply. It carries away waste matter from the 
 tissues and delivers it at the principal points of elimination. 
 The character of the blood must depend on the rapidity of 
 this elimination. 
 
 Blood contains a characteristic coloring matter which 
 has the peculiar property of combining with oxygen and 
 other gases with variations in color. Oxidized blood is 
 bright red, while reduced blood or blood combined with 
 other gases than oxygen is much darker in hue. 
 
 The general composition of blood may be represented by 
 this scheme. 
 
 f f Serum 
 
 I Liquor sanguinis -j 
 Blood -j [Fibrin ] 
 
 j~ Clot (on 
 Corpuscles j exposure to air). 
 
 On coagulation the corpuscles become entangled with 
 the stringy fibrin, forming what is commonly termed the 
 clot. 
 
92 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The percentage composition of average normal human 
 blood has been determined by several chemists. The fol- 
 lowing results (Becquerel and Rodier) are frequently quoted. 
 
 IN 1,000 PARTS BY WEIGHT. 
 
 Water 779 
 
 Solids 22 1 
 
 Fibrin 3.2 
 
 Haemoglobin 134.5 
 
 Albumin : 76.0 
 
 Cholesterin, fat and lecithin 1.6 
 
 Salts and extractive matters 6.S 
 
 General Tests for Blood. 
 
 Blood may be recognized by its appearance under the 
 microscope, in which case the corpuscles are characteristic, 
 by the absorption binds of its solution when viewed through 
 a spectroscope and finally by certain chemical tests. Some 
 of these will be given here by way of illustration,. Use 
 blood diluted with about twenty volumes of water for the 
 tests. 
 
 Ex. 8o. Heat the solution of blood until it is near the 
 boiling temperature and note that the red color is largely 
 destroyed and that a brownish precipitate forms which con- 
 tains albumin and decomposed coloring matter. Add now 
 a small amount of sodium hydroxide solution and observe 
 that the precipitate disappears while the blood solution 
 becomes red again by reflected light, but greenish by trans- 
 mitted light, 
 
 Ex. 8i. Guaiacum Test. - To a little blood solution 
 in a test-tube add some fresh tincture of guaiacum and then 
 a few drops of an ethereal solution of hydrogen peroxide. 
 Shake the mixture and observe that the precipitated resin 
 has assumed' a blue color, more or less marked. . In this 
 test turpentine oil which has been exposed to the air, or 
 which has been shaken with air in a bottle, can be used in- 
 stead of the solution of peroxide. Hydrogen peroxide is 
 developed by the action of oxygen on turpentine. -' 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 93 
 
 This test depends on the oxidation of the resin by the 
 peroxide in presence of blood and has practical applica- 
 tions. 
 
 Hsemin Crystals. When acted on by acids or strong 
 alkalies the haemoglobin of blood is broken up into globin 
 and a characteristic compound called hmmatin. Hsmatin 
 in turn is decomposed by hydrochloric acid yielding hcemin 
 which appears in crystalline form. From the name of their 
 discoverer, these crystals are called Teichmann's crystals. 
 Their appearance constitutes one of the best tests we have 
 for blood, and can be illustrated by the following: 
 
 Ex. 82. Evaporate a drop of blood on a slide, add two 
 or three drops of glacial acetic acid and boil. Put on a 
 cover glass and allow to cool. Minute (microscopic) plates 
 or prisms separate out. If old blood, a stain for instance, 
 is examined, it is necessary to add a small crystal of sodium 
 chloride to the acetic acid, by which means sufficient hydro- 
 chloric acid is liberated for the test. 
 
 The crystals have a dark brown color and are very 
 characteristic. 
 
 Reaction of Blood. The reaction of normal fresh 
 blood is alkaline, which can be shown as follows: 
 
 Ex, 83. Prepare some small smooth plaster of Paris 
 surfaces by pouring the well-known plastic mixture of 
 plaster of Paris and water on glass plates and allowing it to 
 harden several hours at least. The prepared plates are re- 
 moved from the glass and soaked in a neutral solution of 
 litmus and are then allowed to dry. The test proper can 
 now be made by putting a few drops of the blood on the 
 smooth plaster surface and allowing it to remain there five 
 minutes. It is then washed off with pure water when it 
 will be found that the part of the plate which had been 
 covered by the blood has become blue from the action of 
 the alkali of the blood on the neutral litmus. 
 
94 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The Coagulation of Blood. Some of the simpler phe- 
 nomena connected with the coagulation of blood may be 
 readily shown by experiment. 
 
 Ex. 84. Have ready two test-tubes. Pour into the 
 first one Cc. of a cold saturated solution of sodium sul- 
 phate, the other is left clean and dry. Decapitate a rat 
 and allow two Cc. of the escaping blood to flow into the tube 
 containing the sodium sulpha.te. The rest of the blood is 
 collected in the dry tube. In a very few minutes coagula- 
 tion takes place in the latter tube, while it is prevented by 
 the sodium sulphate in the former. 
 
 Allow both tubes to stand at rest a day or more. In 
 the salted tube it will be noticed that most of the corpuscles 
 have settled to the bottom, leaving a clear and lighter col- 
 ored liquid, while in the other tube the coagulum has 
 begun to shrink into a smaller mass from which droplets of 
 yellowish serum ooze. The corpuscles in this case remain 
 with the fibrin. 
 
 Ex. 85. Collect a quantity of slaughter house blood by 
 running two volumes of the latter into one volume of sat- 
 urated solution of sodium sulphate. Shake the mixture 
 and allow it to stand at a low temperature several days. 
 Coagulation does not occur, but a gradual precipitation of 
 the corpuscles is observed, leaving a yellowish liquid 
 known as salted plasma, which may be poured off and used 
 for various experiments. 
 
 Ex. 86. Pour a few Cc. of the salted plasma into a test- 
 tube and dilute it with several times its volume of 
 water. On slight warming of the mixture coagulation fol- 
 lows. The effect of the sodium sulphate is to prevent 
 coagulation. In this case dilution favors it. 
 
 Haemoglobin. 
 
 This is a complex proteid-like body which can be ob- 
 tained in crystalline form, but which is not diffusible. It 
 makes up the larger part of the solid matter in the red cor- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 95 
 
 puscles, and is distinguished from the true proteids by con- 
 taining a small amount of iron. 
 
 In the experiments given above the haemoglobin re- 
 mains with the corpuscles, and as they settle out the serum 
 is left as a yellowish, clear liquid. If from any cause the 
 corpuscles become disintegrated the haemoglobin separates, 
 allowing the residue to settle, and imparts now a perma- 
 nent red color to the serum. This disintegration of the 
 corpuscles can be effected in several ways, by addition of 
 water to blood, by repeated freezing and thawing, by treat- 
 ment with certain salts, and by shaking with ether. 
 
 In normal arterial blood haemoglobin exists in, the ox- 
 idized form known as oxyhemoglobin; in venous blood it 
 exists in a partly reduced condition. The most charac- 
 teristic property of haemoglobin, and the one on which its 
 value in the blood mainly depends is its power of com- 
 bining with certain gases to form loose compounds easily 
 broken up. Oxyhemoglobin is such a compound, and by 
 means of it oxygen is conveyed through the blood from 
 the lungs to the remote tissues. The oxygen there being 
 given up as required, the blood returns by the venous sys- 
 tem, darker in color, and containing "reduced" haemoglo- 
 bin, or haemoglobin proper. 
 
 The action of oxidizing and reducing agents on blood 
 can be illustrated by a few simple experiments. 
 
 Ex. 87. Shake about 10 Cc. of defibrinated blood with 
 a few drops of ammonium sulphide solution or with 
 Stokes' reagent. (This is a solution of ferrous sulphate, to 
 which a small amount of tartaric acid has been added, and 
 then ammonia enough to give an alkaline reaction). 
 Warm gently, and observe that the bright color of arterial 
 blood gives place to the darker purple of venous. On 
 shaking the mixture now with air the bright red color 
 returns. For the success of this experiment where Stokes' 
 reagent is employed it should be freshly prepared before 
 use. Various other substances behave in a similar manner. 
 
96 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Ex. 88. Generate some hydrogen gas in the usual 
 manner, and allow it to bubble through defibrinated blood. 
 A change of color follows after a time, due to the mechan- 
 ical loss of oxygen. The same result may be accomplished 
 by exhausting the oxygen of the blood by means of an air 
 pump. Exposure to the air restores the color in a short 
 time, as before. 
 
 The above experiments show that oxygen can easily be 
 removed from normal blood, and as easily restored. 
 Haemoglobin enters into another combination, however, 
 which is far more stable than that with oxygen, and 
 which, when once formed, cannot be broken up by agita- 
 tion of its solution with air. This is the compound known 
 as carbon monoxide haemoglobin, the formation of which 
 can be shown as follows : 
 
 Ex. 89. Pour about 10 Cc. of defibrinated blood into a 
 test-tube, and allow a current of common illuminating gas 
 to bubble through the liquid a few seconds. Close the 
 test-tube with the thumb and shake thoroughly, then 
 allow the gas to bubble through again. This should pro- 
 duce a change of color, the blood becoming a peculiar 
 cherry red. Now try to restore the bright arterial red by 
 shaking the tube in contact with air. The darker color 
 persists, as carbon monoxide forms a very stable compound 
 with the haemoglobin. 
 
 This reaction is very interesting, as illustrating the 
 action of carbon monoxide in cases of illuminating gas 
 poisoning. Illuminating gases contain from eight to thirty 
 per cent of this compound, and when inhaled in sufficient 
 quantity produce death, because of a fixation of the 
 haemoglobin, or oxygen-carrying constituent of the blood. 
 Blood, exposed to illuminating gas long enough, loses the 
 power of conveying oxygen to the tissues from the lungs, 
 and this transfer of oxygen being necessary to the main- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 97 
 
 tenance of life, death must follow. Blood containing car- 
 bon monoxide can be recognized readily by the spectro- 
 scope and also by chemical tests, of which the following is 
 one of the most reliable : 
 
 Ex. 90. Add some strong solution of sodium hydroxide 
 to ordinary blood. This gives a brownish green precipitate 
 at first and then a red solution. Treat blood saturated 
 with carbon monoxide in the same manner. This gives a 
 red precipitate and finally a red solution. 
 
 Since the oxygen carrying power, and therefore the 
 practical value of blood, depends largely on the amount of 
 haemoglobin it may contain various methods have been 
 suggested for the rapid and accurate determination of the 
 amount of this substance present. These methods are 
 both physical and chemical. Many analyses have been 
 made of haemoglobin which show, in the mean, that it con- 
 tains 0.45 per cent of iron. A determination of iron gives, 
 therefore, a measure of the haemoglobin in- a given sample 
 of blood, and this method has been very frequently em- 
 ployed. 
 
 Methods depending on the comparison of the color of 
 the blood with the color of a solution of haemoglobin of 
 known strength are very commonly applied at the present 
 time and yield results sufficiently accurate for all practical 
 purposes. Colors can be compared only in dilute solu- 
 tions and for the purpose tubes similar to those used for the 
 Nessler test in water analysis may be employed. If we 
 have blood which may be considered normal in every way 
 this may be taken as the standard and the sample in ques- 
 tion compared with it. 5 Cc. of the normal blood, defibri- 
 nated, should be diluted with water to make 100 Cc. The 
 blood to be tested is then to be diluted with water until it 
 gives the same color as the standard when viewed under 
 
98 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 similar conditions. If observed in shallow cells a dilution 
 with 5 Cc. of blood to the 100 Cc. is proper, but if the 
 samples are to be compared in Nessler tubes or deepj:ells 
 a dilution of 1 Cc. of blood with 99 of water will be nec- 
 essary. Much better results than can be obtained by 
 the simple Nessler tubes are given by the use of certain 
 special instruments, such as the hcemoglobinometet of Gow- 
 ers and the colorimeter, for general purposes, of Wolff. 
 
 In the Gowers instrument, which is shown in the cut 
 below, a one per cent solution of normal blood is taken as 
 
 FIG. 29. 
 
 the standard and is poured into the ungraduated tube up 
 to the mark D. Then twenty cubic millimeters of the blood 
 under test is poured into the graduated tube and diluted 
 with pure distilled water and shaken until the tint reached 
 corresponds to that of the standard when viewed horizont- 
 ally, the tubes having exactly the same diameter. The 
 right hand tube shown in the figure is divided into 100 
 parts, or degrees, and a blood which can be diluted to 100° 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 99 
 
 before the color of the standard is reached is normal or of 
 full strength. If the shade of the standard is reached by 
 diluting the twenty cubic millimeters of blood to 50° its 
 strength in haemoglobin can be only fifty per cent of the 
 normal amount. 
 
 Instead of using normal blood for comparison glycerol 
 jelly, colored with picrocarmine to the proper tint, may be 
 employed and used continuously. This is done to avoid 
 the preparation of a normal blood solution, which must be 
 made fresh from day to day, as it does not keep well. 
 
 The instrument does not give scientifically accurate 
 results, but is convenient for clinical purposes and is suffi- 
 ciently exact. 
 
 The cut shows a small pipette for measuring the blood, 
 a bottle for distilled water and a puncturing needle. 
 
 Somewhat more accurate results can be secured by use 
 of the Fleischl instrument or hcemometer, the characteristic 
 features of which are shown in the next cut. 
 
 The standard of comparison here is a wedge of colored 
 glass which can be moved under the transparent bottom of 
 the observation tube and give the effect of change of shade 
 by change in the thickness of the glass. 
 
 On the platform M is a cylindrical cell G divided into 
 two equal parts by a vertical partition. One-half is over 
 the movable wedge and is filled with distilled water. In 
 the other half the diluted blood for examination is poured. 
 By means of a screw, R, the wedge is now moved until the 
 colors on the two sides of the partition appear the same to 
 the eye held vertically over the cylinder. Beneath the 
 cylinder is a white reflecting surface, S, by means of which 
 light can be thrown from a lamp, or candle, upward through 
 the blood and colored glass. The wedge is graduated into 
 degrees, empirically, which indicate, usually, percentages 
 of the normal amount of haemoglobin in blood. 
 
100 
 
 ELEMENTARY. CHEMICAL PHYSIOLOGY. 
 
 In making the test a definite, measured small quantity 
 of blood is taken and this diluted with water up to a mark 
 on the cylinder. The pure water in the other half of the 
 cylinder is poured in to the same level. 
 
 In order to obtain uniform and fairly accurate results 
 here it is necessary to measure the blood very carefully to 
 begin with and add water then to the proper level in the 
 two compartments. The practical value of the instrument 
 
 FIG. 30. 
 
 depends largely on the accuracy with which the normal 
 blood colors have been duplicated in coloring the glass 
 used. The instruments made by Kruess, of Hamburg, and 
 Reichert, of Vienna, have been generally satisfactory. 
 
 Ex. 91. Let the student carry out practical measure- 
 ments with the Gowers and Fleischl instruments, taking the 
 blood for the purpose from his own finger by means of the 
 shielded needle furnished with each apparatus. The 
 amount of blood diluted must be accurately measured by 
 the small pipette likewise furnished with the instruments. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 101 
 
 Crystals of oxyhemoglobin can be obtained by the fol- 
 lowing method : 
 
 Ex. 92. Mix 15 Cc. of defibrinated blood with 1 Cc. 
 of strong ether in a test-tube. Shake thoroughly and allow 
 to stand a day or more, the tube being stoppered. On 
 carefully decanting the contents of the tube minute crystals 
 will be found in the bottom which may be recognized by 
 transferring to a slide and examining this with the micro- 
 scope. These crystals differ in form according to the 
 source of the blood; from human blood they are rhombic 
 prisms. 
 
 A test can also be made by mixing a drop of blood 
 (defibrinated) with a drop of water on a slide. A cover 
 glass is put on the mixture, which is allowed to stand ten 
 or fifteen minutes; with a microscope crystals can usually 
 be seen at the end of this time. 
 
 The Use of the Spectroscope. 
 
 Results of great value in the examination of blood are 
 obtained by the use of the spectroscope, with the general 
 construction of which the student is assumed to be familiar. 
 
 When sunlight or the bright light of a lamp is focussed 
 on the slit of a spectroscope the ordinary continuous spec- 
 trum is observed, the bright colors 'being interspersed, in 
 the case of the solar spectrum, with the dark Fraunhofer 
 lines. If, before reaching the slit, the light is made to pass 
 through a dilute colored solution contained in a vessel with 
 thin, clear glass walls, preferably parallel, a very different 
 appearance is noted. The continuous spectrum is broken 
 and perhaps wholly obscured by dark bands due to the 
 absorption of parts of the white light by the coloring mat- 
 ters in the solution. The position and extent of these 
 bands vary with the nature of the substance and the 
 strength of its solution. Under a definite and constant 
 degree of dilution the bands are characteristic for different 
 
102 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 substances. The spectra of oxyhsemoglobin, reduced 
 haemoglobin and carbon monoxide haemoglobin have been 
 accurately studied and constitute excellent tests for blood. 
 The positions of these absorption bands is most con- 
 veniently represented with reference to the Fraunhofer 
 lines which correspond to colors of perfectly definite wave 
 length. 
 
 FIG. 31. 
 
 Spectroscopes to be used for anything more than rough 
 qualitative tests must be furnished with some appliance by 
 which the exact position of the bands in the spectrum can 
 be determined. This is usually accomplished by the addi- 
 tion of a third tube to the two essential tubes, the third one 
 containing a fine photographed scale and focussing lens so 
 adjusted that lamp light can be thrown through the scale 
 slit on the prism and from this reflected to the eye of the 
 observer. The scale tube is then moved until one of its 
 numbers is made to coincide with one of the Fraunhofer 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 103 
 
 lines. Usually the line 50 of the scale is made to coincide 
 with the D line (sodium). The position of other prominent 
 lines is then determined with reference to the scale. The 
 value of the scale divisions being found, once for all, the 
 exact boundaries of any absorption band may be recorded 
 by reference to the scale. The absorption spectrum and 
 reflected image of the scale reach the eye at the same time. 
 The cut opposite represents a spectroscope with absorption 
 cell arranged for such observations. 
 
 Ex. 93. Let the student observe the absorption spec- 
 trum of oxyhemoglobin under the following conditions: 
 Measure accurately 5 cubic centimeters of blood and dilute 
 it with 120 cubic centimeters of water. Mark this mixture 
 "Solution No. 1." Dilute 50 Cc. of No. 1 with 50 Cc. of 
 water and mark the mixture "Solution No. 2." Dilute 50 
 Cc. of No. 2 with 50 Cc. of water and mark the mixture 
 "Solution No. J." Dilute 50 Cc. of No. 3 with 50 Cc. of 
 water and mark the mixture "Solution No. 4." Dilute 50 
 Cc. of No. 4 with 50 Cc. of water and mark the mixture 
 " Solution No. 5." Dilute 50 Cc. of No. 5 with 50 Cc. of 
 water and mark the mixture "Solution No. 6." Finally, 
 dilute 50 Cc. of No. 6 with 50 Cc. of water and mark the 
 mixture "Solution No. 7." 
 
 We have now dilutions beginning with 1 in 25 and end- 
 ing with 1 in 1,600. The last solution is almost colorless. 
 
 Take seven test-tubes of thin colorless glass and as uni- 
 form as possible in diameter. Number them 1 to 1 and 
 two-thirds fill each one with the dilute blood solution cor- 
 responding to its number. Place each tube before the nar- 
 row slit of the spectroscope and adjust the flame of an oil 
 or gas lamp so that its light may pass through the solution 
 into the slit. Pull out the draw tube until the light is prop- 
 erly focussed and observe that the bright field is tra- 
 versed by two black bands which cut out portions of the 
 yellow and green. With strong blood solutions all light 
 except the red is shut out, but with solutions of the dilu- 
 tions 2 to 7 the field is obscured only by the two bands. In 
 solution No. 2 they are very dark and well defined. With 
 
104 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 increasing dilution they grow fainter and are scarcely visi- 
 ble in solution No. 7. In all the solutions examined note 
 the position of these bands with reference to the character- 
 istic colors. 
 
 The two bands are always found in solutions of oxy- 
 hemoglobin, and although altered in depth of shade they 
 are not altered in position by dilution. Other red liquids 
 may give dark bands, but blood or a solution of oxyhaemor 
 globin is the only liquid which gives two bands exactly in 
 the position of these. We have, therefore, here a valuable 
 means for the identification of blood and one which is very 
 frequently applied in medico-legal investigations. 
 
 The two bands lie between the Fraunhofer lines D and 
 E. The one near E is somewhat wider than the other, and 
 between the two a greenish yellow part of the spectrum is 
 distinctly seen. 
 
 As explained some pages back several reducing agents 
 modify the oxyhemoglobin in a very marked manner, 
 which is readily shown by a change in the colflr of the 
 diluted blood itself. The alteration as observed in the 
 spectroscope is very striking and characteristic. 
 
 When treated with Stokes' solution dilute blood be- 
 comes purple in color and shows in the spectroscope one 
 dark broad band filling three-fourths of the space between 
 D and E, instead of two bands. If ammonium sulphide is 
 used instead of the Stokes' solution the same broad band 
 appears, and in addition a single narrow black band, the 
 center of which is to the left of D. 
 
 Finally, diluted blood saturated with carbon monoxide 
 shows two dark bands, differing in position, however, from 
 those of oxyhemoglobin. The space between them is nar- 
 rower and they are both moved toward the blue of the 
 spectrum. The fainter of these bands reaches nearly to 
 the Fraunhofer line b, while the heavier one does not reach 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 105 
 
 D in the other direction. These appearances can be best 
 noted by the student with solutions treated as in the follow- 
 ing experiments: 
 
 Ex. 94. To a dilute solution of blood, about 1 part to 
 50 of water, add a few drops of strong ammonium sulphide 
 solution and warm gently in a test-tube until the change of 
 color noted above is reached. Now place the tube before 
 the slit of the spectroscope and observe the bands referred 
 to, especially the narrow one in the red. 
 
 Hydrogen sulphide gives practically the same result. 
 
 Ex. 95. Repeat the above experiment, using Stokes' 
 solution instead of the sulphide. A single broad band 
 appears now; if the liquid is shaken briskly the air acts on 
 the reduced coloring matter with oxidizing effect, as shown 
 by a division of the band, but only temporarily. On stand- 
 ing a short time the single broad band, not very sharply 
 defined, returns. 
 
 Ex. 96. Into diluted blood, as before, pass a stream of 
 common illuminating gas until the liquid is saturated, which 
 requires but a few minutes. On placing the tube in front 
 of the spectroscope the two dark bands described will be 
 seen and farther from the yellow than is the case with oxy- 
 hemoglobin. 
 
 These bands do not change in extent or position, by 
 agitation of the liquid with air, as follows with reduced 
 haemoglobin. 
 
 Methaemoglobin. This is derived from oxyhemoglo- 
 bin by action of certain oxidizing agents. It is said to 
 be produced in the living body by excessive doses of potas- 
 sium chlorate, and appears also, it is said, in several dis- 
 eases. 
 
 The formation of this body can be illustrated by experi- 
 ment : 
 
 Ex. 97. To diluted blood in the test-tube, (1 to 50), 
 add some small crystals of potassium chlorate, and warm 
 
106 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 107 
 
 very gently. To a like amount of blood in another tube 
 add a few drops of a 1 per cent solution of potassium 
 permanganate and warm. Both solutions should darken, 
 and when examined by the spectroscope should show a 
 number of characteristic bands, especially one in the red. 
 Ammonium sulphide gives with this the spectrum of haemo- 
 globin as shown before. 
 
 The several spectra referred to are shown in the cut 
 on the preceding page. 
 
 The clinical importance of variations in the amount of 
 oxyhemoglobin is so great that besides the approximate 
 methods of measurement given above, other very elaborate 
 and accurate ones have been devised, especially by Vier- 
 ordt, which give results of scientific exactness. 
 
 The method of Vierordt is carried out by the aid of 
 an instrument known as a spectrophotometer, illustrated 
 in a following chapter. 
 
 The principle involved is this : Diluted blood, in a 
 layer of definite thickness, absorbs a certain fraction of the 
 light passing through it, and this fraction depends on the 
 amount of haemoglobin present, but is not the same for 
 all parts of the spectrum. If the amount of absorption 
 in a given spectral region is determined once for all with 
 solutions of known strength, that is if the relation between 
 concentration and absorption is found by direct experi- 
 ment, it will be possible to find the haemoglobin strength 
 of an unknown solution by simply determining its absorp- 
 tion power for the same part of the spectrum. 
 
 Vierordt and others have shown how these tests may be 
 made with great accuracy; and as the method is applied 
 to many physiological investigations besides the' investiga- 
 tion of blood it will be explained in some detail in a fol- 
 lowing chapter to which the student is referred. 
 
108 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The Number of Blood Corpuscles. 
 
 In the normal blood of man there are about 5,000,000 
 red corpuscles in the cubic millimeter. In the blood of 
 men the number is somewhat greater than with women. 
 
 In several diseases the number may suffer a very great 
 decrease, and as a means of diagnosis the determination of 
 the condition of the blood in this respect becomes often of 
 the highest importance. Usually, a change in the total 
 amount of haemoglobin may be taken as proportional to a 
 change in the number of corpuscles, but this does not 
 always hold true, and it is, therefore, necessary to make 
 independent determinations. 
 
 A simple clinical method is carried out essentially in 
 this manner. A small known volume of blood, accurately 
 measured by a fine pipette, is diluted to a definite larger 
 volume by addition of water. Then in a definite small 
 fraction of this diluted mixture the corpuscles are counted 
 under the microscope. With properly constructed meas- 
 uring appliances it is possible to dilute 5 cubic millimeters 
 of blood with 995 of water, or even 1 of blood with 999 of 
 water. In order to effect an accurate count under the 
 microscope it has been found most convenient to place a 
 drop of the diluted blood in a shallow cell, having a depth 
 of exactly one-fifth or one tenth millimeter. The bottom 
 of this cell is ruled in squares a tenth or twentieth of a 
 millimeter on each side. When the cell is filled with the 
 diluted blood and covered with an ordinary cover glass the 
 number of corpuscles between the rulings can be readily 
 counted under the. microscope. Counting instruments have 
 been devised by Gowers, Abbe, and others. The Gowers 
 instrument is shown in the annexed illustration. 
 
 Iji using this instrument 995 cubic millimeters of sodium 
 sulphate solution (with a specific gravity of 1.015) are 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 109 
 
 measured by means of the pipette A, and discharged into 
 the mixing vessel D. 5 cubic millimeters of blood are drawn 
 up by the pipette B, and mixed with the sulphate solution 
 by means of a small glass stirrer. A drop of the mixture 
 is put in the center of the glass slide, in the shallow cell 
 the small squares in which are ruled in tenths of a milli- 
 
 FIG. 33. 
 
 meter. The drop is covered with a cover glass which is 
 pressed down. As the cell is one-fifth of a millimeter 
 deep the volume of blood in the space bounded by the 
 slide, cover and ruling is ^ T of a cubic millimeter. There- 
 fore, 500 times the number of corpuscles counted in each 
 square gives the number in one cubic millimeter of the 
 diluted blood, and 200 times this product the number in 
 the original blood. It is best to count the number in 15 
 or 20 squares and take the average ; this multiplied by 
 
110 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 100,000, gives the number per cubic millimeter in the un- 
 diluted blood. Normally the average number of corpuscles 
 in each square is 50. 
 
 In the instrument made by Reichert, of Vienna, the 
 Thoma-Zeiss, the cell is one-tenth of a millimeter deep, 
 
 t-^s^teR-gST: -" ~ ~ -Cv 
 
 ' ■ 1 
 
 
 --==; 
 
 ■■I ii i ., — ...... 
 
 .._■■■. «. .... . - . — 
 
 
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 — z 
 
 ^K 
 
 ^p ■ 
 
 Cz\ -^7. i _^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 >m : 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 - =->&L \M ' 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ^^^ 
 
 
 
 
 ^-- =- 
 
 j:~z;:. - ■ 
 
 
 
 
 
 
 ' 
 
 -=- — •"■ 
 
 -— -^=— ~ - --— — " ■ ' 
 
 
 , [ °° 1 % 1^. It. ;■_. ;J_ .J.S±£. 
 
 ••• ." •• •.. .••* »•• - .* '. >;•% 
 
 • ■* °° " » o ■ „ a • . 
 
 _ * ■ JJ_ • " _£ • a o a a o_ •£ » Q 
 
 - .... a, - ••,..,o... 
 
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 c La£>> ■■■■ ° ,' ° " ■> .£_ . £ ,£ c «° 
 
 FIG. 34. 
 
 and the space bounded by the slide, cover and lines ¥T Vtt °f 
 a cubic millimeter. The blood is drawn up in a mixing 
 pipette which dilutes it 100 or 200 times. The reading 
 with the dilute blood for each square must be multiplied 
 by 40,000 or 80,000 to get the number of corpuscles in each 
 cubic millimeter of the original. Fig. 34 shows the eel' 
 and the rulings and the measuring pipette. 
 
Chapter VI. 
 
 BONE CONSTITUENTS.— SALIVA.— GASTRIC JUICE — 
 THE BILE. 
 
 A FEW simple tests may be readily carried out by the 
 student to show the general composition of bones. 
 Roughly speaking, bones consist of one part of organic mat- 
 ter to two of mineral matter. The organic substance is 
 termed ossein. The mineral matters present show, in the 
 mean, about the following composition : 
 
 Calcium phosphate 85.0 per cent. 
 
 Magnesium phosphate 1.5 
 
 Calcium carbonate 11.0 " 
 
 Calcium fluoride and chloride 2.5 " 
 
 These mineral constituents are insoluble in water, but 
 readily soluble in dilute hydrochloric acid, by the aid of 
 which they may be separated from the ossein. 
 
 Ex. 98. Clean a long, slender bone (best, a rib), and 
 immerse it in dilute hydrochloric acid of about ten per 
 cent strength. Let it remain several days. At the end 
 of this time remove the bone from the acid and observe 
 that it has lost its rigidity, and has become very flexible. 
 It may be even possible to tie it in a knot. Wash the 
 elastic mass several times in fresh water to remove all 
 the hydrochloric acid, and then boil it with a small amount 
 of pure water. By heating it long enough the ossein be- 
 comes converted into gelatin which solidifies, on cooling, 
 to a jelly. 
 
 By boiling the bone ossein under pressure the forma- 
 tion of the gelatin is very much hastened. 
 
112 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Ex. 99. Make a dilute aqueous solution of gelatin, 
 and add to it an aqueous solution of tannic acid. This 
 gives a white flocculent precipitate which is characteristic 
 as it is produced even in extremely dilute solutions. 
 
 Commercially, gelatin occurs in shreds, or sheets, which 
 are used for many purposes. It is very similar to albumin 
 in chemical composition, although different in many im- 
 portant properties. It fails to give many of the reactions 
 characteristic of albumins as a class, and does not appear 
 to be able to take the place of albumins as a food. 
 
 In the laboratory gelatin is employed in the prepara- 
 tion of the well-known nutrient jellies used in the cultiva- 
 tion of bacteria. 
 
 In the next to the last experiment the mineral matters 
 were left in the hydrochloric acid solution. Filter this, if 
 it is not clear, and use it for tests. 
 
 Ex. 100. To a few cubic centimeters of the solution 
 add some ammonium molybdate solution. In a short time 
 a yellow precipitate appears, indicating presence of a 
 phosphate. 
 
 Ex. 101. To a few cubic centimeters of the solution 
 add solution of sodium acetate until a distinct odor of 
 acetic acid persists. Then add some solution of ammon- 
 ium oxalate which produces a white precipitate of calcium 
 oxalate. 
 
 Ex. 102. To another portion of the hydrochloric acid 
 solution add ammonia until a gocd alkaline reaction is ob- 
 tained. A white precipitate of calcium and magnesium 
 phosphates settles out. Filter and to the filtrate add some 
 ammonium oxalate solution. A further precipitate appears. 
 This is calcium oxalate and proves that the original bone 
 contains calcium in excess of that necessary to combine 
 with phosphoric acid. The calcium combined with car- 
 bonic, hydrofluoric and hydrochloric acids appears here. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 113 
 
 Saliva. 
 
 In our experiments on starches an important property 
 of saliva has been shown. A few other reactions remain to 
 be given. Collect about twenty-five cubic centimeters of 
 saliva (after thoroughly rinsing out the mouth) and filter it 
 to obtain a clear solution, with which make the following, 
 tests. 
 
 Ex. 103. To a few Cc. of the clear saliva in a test-tube 
 add several drops of a dilute solution of ferric chloride. 
 This gives a more or less marked red color from the forma- 
 tion of ferric sulphocyanate. A very strong reaction must 
 not be expected. Make a comparative test by adding a 
 like amount of ferric chloride to dilute solutions of potas- 
 sium sulphocyanate. 
 
 The addition of solution of mercuric chloride discharges 
 the color. 
 
 A similar color is given by ferric salts and solutions of 
 meconic acid, extracts of opium, for instance, and the re- 
 action may therefore have medico-legal importance. Sul- 
 phocyanates are present in normal saliva and may, there- 
 fore, possibly be sometimes found in the liquids of the 
 stomach in traces sufficient to give a test with the ferric 
 salt when laudanum or other opium extract is looked for. 
 Ferric sulphocyanate like ferric meconate is red, but danger 
 of confounding the two may be avoided by noting that the 
 color of the former is destroyed by mercuric chloride,while 
 that of the latter is not affected. 
 
 Ex. 104. Test the reaction of saliva with neutral lit- 
 jnus paper. It will be found slightly alkaline. Now add 
 two or three drops of dilute acetic acid and note that a 
 stringy precipitate of mucin separates. Filter off this pre- 
 cipitate and test the filtrate for proteids by boiling with 
 Millon's reagent or by the xanthoproteic reaction. 
 
114- ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Mixed human saliva has a specific gravity of 1,002 to 
 1,006, and contains in the mean 99.5 per cent of water and 
 0.5 per cent of solids. The potassium sulphocyanate 
 amounts to about 0.005 per cent. 
 
 Gastric Juice. 
 
 Chemical Experiments. Simple laboratory tests of 
 the gastric juice cannot be readily made for want of a con- 
 venient source of the material. But this secretion has in 
 the last three years become an object of accurate clinical 
 investigation, since methods have been devised for collect- 
 ing it without great annoyance to the patient. Investigations 
 are usually instituted to determine the amount and char- 
 acter of the free acids and the peptic activity of the fluid. 
 The latter test is carried out as already given. The acid 
 tests will be explained here. 
 
 For investigation, gastric juice is collected at the time 
 when the stomach is as free as possible from food, prefera- 
 bly before breakfast and by aid of the gastric sound and 
 stomach pump or by a siphon tube. The sound for this 
 purpose consists of an elastic rubber tube small and firm 
 enough to be pushed through the oesophagus into the stom- 
 ach. Thelower end is closed and round but furnished with a 
 number of very fine openings. By attaching the outer end 
 of the tube to a stomach pump and exhausting, it may be 
 partly filled with liquid. This is filtered and is then ready 
 for the several tests. 
 
 Total Acidity. Measure accurately 10 Cc. of the fil- 
 tered gastric juice into a beaker, add a few drops of phenol- 
 phthalein indicator and then from a burette add slowly -fa 
 normal- sodium hydroxide solution until a pink color just 
 persists" on shaking. Each Cc. of the alkali solution used 
 corresponds to 0.00182 Gm. of free hydrochloric acid, but 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 115 
 
 it will not do to consider this as an estimation of hydro- 
 chloric acid as other acids are generally present in some 
 amount, among them lactic. 
 
 Onscubic centimeter of the 3 1 s -' nQrma l sodium hydroxide 
 solution neutralizes 0.0045 Gm. of lactic acid, or nearly 
 two and one-half times the weight of the hydrochloric acid 
 neutralized. The acidity must, therefore, be indicated in 
 terms of alkali used and not expressed as acid found. 
 
 Hydrochloric acid is certainly the most abundant of the 
 gastric acids and near the end of a digestive process it is 
 said to be norjnally the only one present. But at the begin- 
 ning of -digestion lactic acid may be also present. Patho- 
 logically, lactic and other organic acids may be at times very 
 much increased and their detection is a matter of no little 
 clinical importance. A number of tests will here be given. 
 
 Free Hydrochloric Acid. That this acid is present in 
 the uncombined condition has been frequently shown by 
 making an accurate determination of all the bases and also 
 of the total hydrochloric acid. The latter is in excess of 
 the amount which could unite with the bases and must 
 therefore be partly free. For the clinical detection of the 
 acid the following methods are in favor. 
 
 " Emerald Green " Test. An aqueous solution of 
 this substance, when added in small amount to weak 
 hydrochloric acid, is turned yellowish green or yellowish 
 brown. Organic acids do not give this test. The green 
 used for this test must be the pure product of the Bayer 
 laboratory, Elberfeld, having the empirical formula, 
 C„ H 33 N 2 HS0 4 . 
 
 " Congo Red " Test. This substance in aqueous 
 solution is turned blue by very dilute hydrochloric acid. 
 Organic acids do not give the test. 
 
116 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The reaction is most conveniently carried out by means 
 of test papers made by dipping filter paper in a solution of 
 the coloring matter, and then drying. 
 
 "Methyl-Violet" Test. A dilute violet colored aque- 
 ous solution of this substance, when mixed with weak 
 hydrochloric acid, turns blue. The reaction with gastric 
 juice is faint, but when care is observed characteristic. 
 Organic acids, even when present in quantity, do not give 
 the test, which was first successfully used for the detection 
 of traces of mineral acids in vinegar. 
 
 "00 Tropaeolin" Test. When a dilute alcoholic or 
 aqueous solution of this color is added to weak hydro- 
 chloric or other free acid the color changes from yellow 
 to reddish violet. This is a sensitive test, especially for 
 hydrochloric acid. 
 
 " Phloroglucin and Vanillin" Test. A reagent is 
 made by dissolving 2 Gm. of phloroglucin and 1 Gm. of 
 vanillin in 100 Cc. of alcohol. To make the test, mix 5 
 Cc. of this solution with an equal volume of the gastric 
 filtrate, and concentrate in a glass or porcelain vessel on the 
 water-bath. As the liquid becomes concentrated it turns red. 
 
 Tests for Lactic Acid. Prepare a dilute solution of 
 phenol by dissolving 1 Gm. of the pure crystallized product 
 in 75 Cc. of water. To this add 5 drops of a strong solu- 
 tion of ferric chloride, which produces a deep blue color. 
 Fiye. Cc, of this mixture suffices for a test. Add to it a few 
 drops of the liquid containing lactic acid, and note the 
 change from blue to yellow. 
 
 A weak, almost colorless solution of ferric chloride 
 alone serves also as a test substance, as its color becomes 
 much deeper by addition of a trace of lactic acid. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 117 
 
 This reaction is not influenced by the presence of small 
 amounts of hydrochloric acid, as can be readily shown by 
 adding some to the liquid to be tested. 
 
 The Bile. 
 
 In the bile are found a number of characteristic pig- 
 ments and acids which can be recognized without much 
 difficulty and which serve, therefore, as indicators of the 
 presence of the secretion. 
 
 Two acids, glycocholic and taurocholic, are found in 
 human bile in combination as alkali salts; they are found 
 also in bile of many animals. Investigations seem to show 
 that in human bile the glycocholic acid is more abundant 
 than the other. 
 
 Two important pigments are found in human and other 
 bile normally, and these are known as bilirubin and bili- 
 verdin. Pathologically, it is likely that other pigments are 
 present in small amount. Several modified forms of the 
 two normal pigments have been described, which differ 
 mainly in the amount of water of crystallization. The 
 formula of bilirubin is C 16 H 18 N s 3 , while that of bili- 
 verdin is C 16 H 18 N 2 4 . 
 
 In human bile bilirubin is probably in excess, while in ox 
 bile the biliverdin seems to predominate. These pigments 
 appear to be derived from the haemoglobin of the blood, 
 bilirubin being in fact identical in composition with haema- 
 toidin, which is an iron-free derivative of haemoglobin. 
 
 Tests for the Bile Acids. Several color tests are 
 known here, the so-called Pcttenkofer test being perhaps 
 the most characteristic. The student can use ox bile for 
 this and other reactions given below. 
 
 Ex. 105. Add a little cane sugar or some strong syrup 
 to bile in a test-tube. Then pour in an equal volume of 
 
118 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 strong sulphuric acid in such a manner as to mix the 
 liquids as little as possible. The acid may be allowed to 
 trickle down the side of the test-tube and collect beneath 
 the lighter bile. At the junction of the two liquids a dark 
 purple band appears. On shaking the tube the liquids 
 mix and become colored throughout. A modified form of 
 the test is sometimes carried out in this manner; Mix 
 a little syrup with bile and shake the tube until a layer of 
 froth forms. Pour in a few drops of strong sulphuric acid. 
 As it passes through the froth it imparts a purple color 
 to it. 
 
 This reaction depends on the production of furfurol 
 (C 4 H 3 O C H O) by the destruction of the sugar when 
 the sulphuric acid is added. Furfurol in turn combines 
 with cholalic acid, formed b} 7 the action of the sulphuric 
 acid on the bile acids, giving the color. Several substances 
 give a very similar color, and the test, therefore, must 
 be employed with caution. With very dilute solutions the 
 reaction does not appear and this is the case when the test 
 is directly applied to urine. In order to avoid all uncer- 
 tainty, either from dilution of the solution to be examined, 
 or from possible presence of interfering substances it is 
 necessary to apply a method for the separation of the bile 
 acids, and on these perform finally the Pettenkofer test. 
 Under " Urine Analysis," later, such a method will he 
 explained in detail. 
 
 Another modification of the test consists in diluting the 
 colored solution and observing its absorption bands in the 
 spectroscope. The number and position of the bands 
 given by cholalic acid are said to be quite characteristic. 
 
 Ex. 106. The Pigment Tests. These, as usually 
 carried out, depend on the oxidation of the pigments by 
 means of nitric acid. Gmelin's test is performed in this 
 manner. To a few Cc. of strong nitric acid in a' test-tube 
 add a little bile, without mixing much. At the junction of 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 119 
 
 the two liquids a series of colored rings appear, green, 
 blue, violet, red and yellow below. Try the test, also, by 
 placing some bile in a flat-bottomed porcelain dish. When 
 a drop of strong nitric acid is put in the middle of the bile 
 a play of colors is observed as in the test-tube. The oxi- 
 dation is greatest, with yellow color, near the acid, and 
 least, with green color, near the bile. 
 
 This reaction is exceedingly delicate and is applied 
 chiefly to the detection of bile in urine. 
 
 Other constituents of the bile are shown by the follow- 
 ing experiment: 
 
 Ex. 107. To 5 Cc. of bile add an equal volume of 
 water and some alcohol. This produces a precipitate of 
 mucin. Filter this off and divide the filtrate into two por- 
 tions ; to one add some hydrochloric acid which causes 
 precipitation of glycocholic acid, to the other portion add 
 solution of lead acetate, which throws down lead glyco- 
 cholate. Remove this by filtration, and to the filtrate, add 
 solution of basic lead acetate, which gives a further precipi- 
 tation of lead taurocholate. 
 
 Action on Fats. In Chapter III. it was shown that 
 emulsions of fats are produced in various ways, especially 
 by action of the pancreatic juice and by the bile. This 
 latter reaction can now be illustrated by experiment. 
 
 Ex. 108. In a slightly warmed mortar pour about 5 
 Cc. of bile, and add to it 1 Cc. of cottonseed oil. Rub the 
 two thoroughly together for several minutes, and then add 
 another small portion of the fatty oil. An emulsion forms 
 slowly, and becomes more persistent as the working with 
 the pestle is prolonged. The amount of oil which can be 
 brought into the form of a stable emulsion with the 5 Cc.of 
 bile depends largely on the character of the oil. The 
 presence of a small amount of free fatty acid in the cotton- 
 seed oil aids materially in producing the emulsion. 
 
120 ELEMENTARY CHEMICAL PHYSIOLOGY 
 
 The free fatty acids have the power of decomposing the 
 bile salts with liberation of their acids. The soaps formed 
 assist in increasing and holding the emulsion. 
 
 It is also worthy of note "that animal membranes 
 moistened with bile permit the passage of fatty oils, while if 
 they are moistened with water only the oil cannot pass 
 through. This behavior is of the highest importance in 
 aiding the absorption of fatty substances from the intestine 
 in the process of digestion. 
 
Chapter VII. 
 
 MILK BEEF EXTRACTS FLOUR AND MEAL. 
 
 T^ROM its very great importance milk has been made the 
 ■*■ subject of almost countless investigations from nearly 
 every standpoint. Most of the literature is naturally con- 
 cerned with cow's milk as a commercial article having 
 market value. Besides this, however, we have a valuable 
 scientific literature of milk which discusses its secretion as 
 a physiological process and variations in its composition 
 depending on age, race, nutrition, etc., of the animal fur- 
 nishing it. 
 
 For many reasons our knowledge of human milk is far 
 less complete than is our knowledge of the milk of several 
 animals. The difficulty of collecting normal human milk 
 is naturally very great, as any stimulus applied to cause its 
 flow for collection must necessarily be an abnormal one. 
 The wide disagreement between many of the published 
 analyses of human milk may probably be in part accounted 
 for from this fact. Other reasons will be pointed out below. 
 
 The average composition of cow's milk as determined 
 by thousands of analyses is given in the following figures : 
 
 Water 87.4 per cent 
 
 Fat 3.5 " 
 
 Sugar 4.5 
 
 Albumins 3.9 
 
 Salts 7 
 
 The solids amount to 12.6 per cent. 
 In considering milk furnished by individual cows in 
 nearly 400 instances the following variations depending on 
 
122 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 age, race, season, food, etc., were noticed by a well-inown 
 German authority : 
 
 The- solids between 8.50 and 16.03 per cent, the fat 
 between 2,04 and 6.17, the sugarbetween 2.00 and 6.10, the 
 albuminoids between 1.98 and 6.61, and the salts between 
 0.34 and CL9S per cent. 
 
 The following are some analyserwhicfa have been, given 
 of human milk. (Landois and Stirling.') 
 
 Water 87.24 to 90.58 percent. 
 
 Fat 2.67 " 4.30 
 
 Sugar 3.15 " 6.09 
 
 Albumins 2.91 " 3.92 
 
 Salts 14 " .28 
 
 Recent analyses by Palm, made by methods which seem 
 to be reliable and free from errors of older methods, give 
 the following results as the mean of twenty complete an- 
 alyses of nurse's milk : 
 
 Water 87.81 per cent. 
 
 Fat ....: 4.06 
 
 Sugar 5.26 
 
 Albumins 2.36 
 
 Salts 51 
 
 Human milk is poorer than cow's milk in albumins, but 
 richer in sugar. 
 
 Some simple experiments may readily be made to show 
 the presence of the three important constituents in milk. 
 
 The Test for Fat. 
 
 Ex. 109- Pour about 20 Cc. of milk in a porcelain 
 dish, add an equal volume of clean, dry quartz sand and 
 evaporate, with frequent stirring, about an hour on the 
 water-bath. Then loosen the dry mass as well as possible 
 by means of a spatula, or glass rod, and pour over it 25 
 Cc. of light benzine. Stir up well and cover with a sheet 
 of paper and allow to stand 15 minutes. Then pour the 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 133 
 
 liquid through a small, dry filter into a small, dry beaker, 
 and place this in hot water to volatilize the benzine. 
 
 A residue of fat will be left. Do not attempt to evaporate 
 the benzine over a flame, or on a water-bath under which a 
 lamp is burning. Heat the water, then extinguish the flame 
 and immerse the vessel containing the benzine in the hot 
 water. 
 
 In the analysis of milk the determination of the 
 amount of fat is the most important operation, and many 
 quick and accurate methods have been devised by which 
 this may be accomplished. The processes by drying and 
 extraction by ether, carbon bisulphide or benzine, as illus- 
 trated above, can be made very exact, but then they con- 
 sume a great deal of time. It has been found possible to 
 mix the milk with certain reagents which cause the fat to 
 separate in a pure layer, and if the separation takes place 
 in a narrow, graduated tube, the volume of this fat layer 
 may be read off accurately. The specific gravity of the 
 fat being known, the weight of a given observed volume is 
 obtained, and from this the percentage amount. Several 
 forms of apparatus are now in use by means of which this 
 may be easily done, so that it is possible to make many 
 tests in a day, or in an hour even. 
 
 The fat residue is a mixture of the glycerides of oleic, 
 palmitic, stearic, butyric and other acids. The above 
 method can be made quantitative by weighing the milk, 
 drying carefully, extracting completely and drying and 
 weighing the fatty residue. 
 
 The Test for Sugar. 
 
 Ex. 110. Measure out about 10 Cc. of milk, and dilute 
 it with water to make 200 Cc. Add to this 5 Cc. of a 
 copper sulphate solution such as is used in making the 
 Fehling solution, (69.3 Gm. per liter) and then enough 
 potassium or sodium hydroxide solution to produce a 
 
124 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 voluminous precipitate containing copper with all the 
 proteids and fat. For this purpose about 3.5 Cc. of a 1 
 per cent sodium hydroxide solution will be required. 
 Allow the precipitate to subside, pour or filter off some of 
 the supernatant liquid, and boil it with Fehling solution. 
 The characteristic red precipitate forms, showing presence 
 of sugar. 
 
 The sugar appearing in the above test is known as 
 milk sugar ojr lactose. Like cane sugar it suffers inversion 
 when heated with weak acids, the product formed in this 
 way being known as galactose. 
 
 The principle illustrated by the above experiment is 
 readily made the basis of a quantitative process of value. 
 
 It has usually been assumed that the precipitation of 
 proteids and fat is complete by this reaction, but there is 
 good reason for believing .that certain modified albumins 
 are not thrown down in this way, but are left with the fil- 
 trate to slightly impair the accuracy of the sugar determi- 
 nation. The precipitate has frequently been used for the 
 determination of proteids after drying and dissolving out 
 the fats, but the results are probably a little low, for the 
 reason just mentioned, especially in the case of human 
 milk. 
 
 Proteid Test. 
 
 The presence of a proteid in milk can readily be shown 
 as. follows: 
 
 Ex. III. Mix equal volumes of milk and Millon's rea- 
 gent in a test-tube, and boil. The bulky red precipitate 
 which forms, proves the presence of the body in question. 
 
 The total amount of proteid present in milk can be 
 most accurately determined by finding the nitrogen by the 
 methods of organic analysis and multiplying this by 6.25, 
 on the supposition that proteids in the mean contain 16 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 125 
 
 per cent of nitrogen. That milk contains casein as its chief 
 nitrogenous constituent has already been shown in the 
 chapter on proteids. It was also shown that a simple al- 
 bumin is likewise present, which can be coagulated after 
 separation of the casein. Peptone appears to be present 
 in small amount as a rule and this escapes precipitation 
 by the usual methods. 
 
 Action of Rennet on Milk. 
 
 The mucous membrane of the stomachs of most animals, 
 and especially that of the young calf, contains an enzyme 
 known as the"milk curdling ferment," the "rennet ferment" 
 or rennin. 
 
 A crude extract of the stomach mucous membrane from 
 the calf is commonly called "rennet" and has long been in 
 use for the curdling of milk in the production of cheese. 
 This curdling consists essentially in the coagulation or pre- 
 cipitation of the casein, which it will be recalled, is not 
 readily thrown down by the usual methods. 
 
 An active rennet can be readily obtained by digesting 
 the fourth stomach of the calf with glycerol or brine. If a 
 brine extract is precipitated by alcohol in excess a white 
 powder separates, which, when collected and dried, has 
 very active properties. Several powders of this descrip- 
 tion are now in the market. Let the student try the follow- 
 ing experiment with such a product: 
 
 Ex. 112. Warm some fresh milk to a temperature of 
 38° to 40° C. in a test-tube or small beaker, then add 
 about half a gram of commercial "rennin," and after stir- 
 ring it in well keep for fifteen minutes at a temperature not 
 above 40°. Then as the milk cools it assumes the consis- 
 tence of a firm jelly. It is essential in this experiment that 
 the temperature be kept within the proper limits, as the 
 enzyme is not active at low temperature and it is, like 
 others, destroyed by high temperature. 
 
126 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The spontaneous coagulation of milk is due to the ac- 
 tion of lactic acid produced by the conversion of milk sug- 
 ar under the influence of a true ferment which enters the 
 milk from the air usually. Boiling destroys this ferment. 
 Milk when heated to 100° for an hour in bottles loosely 
 stoppered with cotton plugs may be kept sweet almost in- 
 definitely provided the plugs of cotton are not removed. 
 
 Milk which has been boiled and allowed to cool does 
 not coagulate readily with rennet. A perfectly satisfactory 
 explanation of this fact has not been given. 
 
 The spontaneous coagulation of boiled milk is usually 
 very slow, as the necessary lactic acid ferment cannot be 
 supplied by all atmospheres. 
 
 The Action of Pancreatic Extract on Milk. 
 
 The behavior of milk with extract of pancreas is some- 
 what complicated because of the complex nature of milk 
 itself. The three important constituents of milk, the sugar, 
 the fat, and the proteid bodies all suffer some change 
 under the influence of the several pancreatic enzymes. 
 
 The most interesting of these changes, however, is that 
 produced in the proteids, and is commonly called peptoni- 
 zation. 
 
 At the present time the digestion, or peptonization of 
 milk, is a very common practice in the preparation of food 
 for the sick room, and can be illustrated by the following 
 experiment : 
 
 Ex. 113. Dilute about 10 Cc. of milk with an equal 
 volume of water, and add half a gram of sodium bicarbon- 
 ate. Next add a few drops of a liquid extract of pancreas, 
 or a very small amount (10 to 20 Mg.) of one of the con- 
 centrated " pancreatin" powders on the market. Shake 
 the mixture and keep it at a temperature of 40° on the 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 127 
 
 water-bath half an hour. At the end of this time filter and 
 apply the peptone test — potassium hydroxide and dilute 
 copper sulphate — and observe the pink color. 
 
 The following formula will serve for the practical diges- 
 tion of milk in quantity : 
 
 Dissolve 1 Gm. of sodium bicarbonate in 100 Cc. of 
 water, and add from a third to a half gram of pancreatin 
 powder. Warm the mixture slightly, not above 40°, and add 
 500 Cc. of milk, warmed to about 40°. Keep the mixture 
 at this temperature about half an hour. As the action goes 
 on the color changes from white to grayish yellow, and a 
 bitter taste appears which becomes stronger the longer the 
 digestion is continued. As this bitter taste is unpleasant, 
 it is always necessary to stop the reaction before it is fully 
 completed which can be done either by boiling the milk, or 
 by cooling it quickly by placing it on ice. If it is to be 
 used immediately it is not necessary to boil or cool. 
 
 The pancreatin, or pancreatic extract, used for this pur- 
 pose, must be from beef, not from the hog pancreas. An 
 extract from the latter source is very active,in the conver- 
 sion of starch into sugar, but is deficient in proteid con- 
 verting power. Some manufacturers prepare products from 
 both sources. 
 
 Extract of Meat. 
 
 By the term extract of meat several different products 
 may be meant. When lean meat is superficially broiled, 
 minced fine and squeezed out in a meat press, a juice is 
 obtained which holds, besides meat salts and meat bases, 
 a certain amount of soluble albumin. Such a product has 
 considerable direct nutritive value. If, however, this juice 
 is thoroughly boiled and filtered practically all the albu- 
 min is coagulated and lost, and a product so made would 
 have little nutritive value. 
 
128 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 It was at one time supposed that a concentrated "ex- 
 tract" of meat could be secured by boiling meat until com- 
 pletely disintegrated, filtering and evaporating the filtrate to 
 a paste, and that in this manner the most valuable part, or 
 essence of the beef was secured. This view was afterward 
 shown to be fallacious, and the opinion that the product 
 so prepared contained only the meat salts and organic 
 bases, is therefore merely a condiment and stimulant, 
 and not a true food in any sense, took its place. 
 
 The error here seems to be as great as in the other 
 case. It has lately been found possible to effect a separa- 
 tion of the derived proteid products known as albumoses 
 and peptones, described in a former chapter, and a study 
 of meat extract shows that these bodies are often pres- 
 ent in considerable proportion. The amount present de- 
 pends on the time of boiling,, and on some other factors. 
 
 It seems that by prolonged contact with hot water a 
 partial digestion of the meat takes place so that from the 
 coagulated albumin, albumose, and finally peptone is 
 formed. In the boiling operation any fat present would 
 separate so that it could be skimmed off. Among the 
 meat bases present there may be mentioned carnin, krea- 
 tin and sarkin. Some gelatin is always present too. 
 
 The production of extract of meat was first undertaken 
 in order to utilize the flesh of cattle killed on the plains 
 of South America for the hides. It is now made else- 
 where in great quantities. Two samples recently analyzed 
 by the author showed the following constituents : 
 
 Solid matter 80.4 per cent. 80.7 per cent. 
 
 Water and volatile... 19.6 " 19.3 
 
 Ash 20.2 " 24.0 
 
 Soluble albumin trace. " trace. 
 
 Insoluble albumin. .. .0.0 " 0.0 
 
 Albumose 12.7 " 4.3 
 
 Peptone 8.4 ■'■' 8.9 
 
 Other N bodies 39.1 " 43.5 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 129 
 
 From its composition it is apparent that alone it can- 
 not be used as food, but as a condiment and addition to 
 other food. From this standpoint it has great value. 
 
 Several of the organic basic bodies present are active 
 stimulants, and the potassium phosphate and other salts 
 of the same metal are not without marked physiological 
 properties. 
 
 A few simple experiments will show important char- 
 acteristics of the commercial extracts as everywhere found 
 on the market. 
 
 Ex. Il4- Heat a little of the solid extract on a bit of 
 porcelain until it is reduced to a char. Extract this with 
 dilute nitric acid, filter and divide the filtrate into two 
 portions. In one, test for phosphates by the addition of 
 ammonium molybdate, and in the other, for potassium salts 
 by the flame test. Both tests should show good reactions. 
 
 Ex. 115. Add 200 Cc. of water to 10 Gm. of commer- 
 cial extract, warm gently and observe that a nearly clear 
 solution is obtained, showing absence of fat, coagulated 
 albumin, etc. To the solution add very carefully a solu- 
 tion of basic acetate of lead as long as a precipitate forms, 
 but not much in excess. This can be determined by wait- 
 ing after each addition until the precipitate settles enough 
 to leave a moderately clear supernatant liquid. A few 
 drops of the lead solution added to this will show whether 
 more is needed or not. When precipitation is complete 
 the light colored mass of lead salts, organic and inorganic, 
 is filtered off, and through the filtrate enough hydrogen 
 sulphide is passed to throw down the excess of lead con- 
 tained there. The black sulphide is removed by filtration, 
 and the filtrate shaken thoroughly to remove as much as 
 possible of the gas. It is then evaporated at a low temper- 
 ature on the water-bath to a volume of about 8 Cc, and 
 allowed to stand then two or three days in a cool place. 
 Kreatin separates as a crystalline mass. Pour the liquid 
 and crystals on a filter, and wash with strong alcohol, in 
 which kreatin is but slightly soluble. 
 
130 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Ex. Il6. Dissolve the kreatin crystals of the last ex- 
 periment in a small amount of pure hydrochloric acid, and 
 evaporate the solution to dryness on the water-bath. By 
 this action kreatin is converted into kreatinin. Dissolve 
 this residue in a small volume of water, and divide the 
 solution into two parts. To one add a solution of zinc 
 chloride, which produces a white crystalline precipitate, 
 the character of which is best seen under the microscope. 
 With the other try Weyl's reaction. Add a few drops of 
 dilute solution of sodium nitroprusside, and then, a drop 
 at a time, dilute sodium hydroxide solution. This gives 
 a ruby red color which soon fades to yellow. Add now 
 enough acetic acid to change the reaction, and warm. 
 The color becomes green, and finally bluish. 
 
 Kreatinin is interesting as occurring normally in urine 
 to the amount of about 1 Gra. daily. It is probably derived 
 there by dehydration from the kreatin found normally in 
 muscle, blood and brain, as they differ in composition sim- 
 ply by one molecule of water. 
 
 The average composition of the commercial extract, 
 corresponding to the analyses given, is about : 
 
 Water 20 parts. 
 
 Salts 30 " 
 
 Organic substances 60 " 
 
 It is made on the large scale by boiling lean meat until 
 everything soluble has passed into solution. The liquid is 
 filtered and concentrated to the above composition^ after 
 removing fat by skimming, best in vacuum pans. 
 
 The water in which meat is boiled in the canning 
 establishments is now generally used also in the produc- 
 tion of extract, as it becomes concentrated after a time. 
 When extract is made as the principal product the muscu- 
 lar residue remaining after long boiling has little value 
 except as a cattle food, for which purpose it is sometimes 
 employed. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 131 
 
 Fresh beef is sometimes digested with pepsin and 
 hydrochloric acid, or with pancreatic extract and soda and 
 the product neutralized. Such products are sold as "fluid 
 beef " or " peptonized meat," and under other names. 
 Still other commercial articles seem to be prepared by 
 simply heating meat with water and dilute hydrochloric 
 acid under pressure. A preparation of this description 
 contains proteids in a finely divided condition, but cannot 
 be called digested. There are great differences in the value 
 of these products as articles of food. In Chapter IX. 
 further details concerning the examination of these prod- 
 ucts will be given. 
 
 Flour and Meal. 
 
 The average composition of wheat flour may be shown 
 
 by the following analyses : 
 
 Fine flour. Coarse flour. 
 
 Water 18.34 12.65 
 
 Proteids 10 08 11.82 
 
 Fat 0.94 1.36 
 
 Sugar and gum 5.41 5.95 
 
 Starch 69.44 66.28 
 
 Fiber 0.31 0.98 
 
 Ash 0.48 0.96 
 
 100.00 100.00 
 
 Corn meal contains usually less water and more fat than 
 wheat flour. 
 
 Ex. 117. Boil a small amount of wheat flour with 
 Millon's reagent. The red color produced shows presence 
 of proteids. 
 
 Ex. Il8. Moisten about 25 Gm. of flour with water 
 and work it into a dough. Then hold this under a fine, 
 slow stream of water and by kneading between the fingers, 
 slowly work out a portion of the mass as a thin milky 
 
182 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 liquid. This is largely starch. After some time an elastic 
 residue is left insoluble in water. This is gluten, and is the 
 chief nitrogenous element of the flour. 
 
 Gluten corresponds to fibrin of the animal proteids and 
 is accompanied by a vegetable albumin and vegetable 
 casein, besides other related products in small amounts. 
 
 Ex. 119. To about 5 Gm. of flour add 10 Cc. of water, 
 shake thoroughly and allow to stand until a nearly clear 
 liquid appears above a white sediment. Filter the liquid 
 and test for sugar by the Fehling solution. 
 
 Boil some of the residue with water and add iodine 
 solution as a test for starch. 
 
 Ex. 120. To about 5 Gm. of fine corn meal in a test- 
 tube add 10 Cc. of ether. Close the tube with the thumb 
 and shake thoroughly. Then cork and allow to stand half 
 an hour. Shake again and pour the mixture on a small 
 filter, collect the ethereal filtrate in a shallow dish and 
 evaporate it by immersion in warm water. A small amount 
 of fat will remain. 
 
 Rice flour is characterized by containing a large amount 
 of starch with only a small amount of fat, and much less 
 albuminous matter than is found in corn or wheat. Peas 
 and beans are rich in albuminoids with lower carbohydrates 
 than corn or wheat. 
 
 Action of Yeast on' Flour. 
 
 The following experiment is intended to illustrate the 
 work done by yeast in leavening dough: 
 
 Ex. 121. Crumble two or three grams of compressed 
 yeast into 15 Cc. of lukewarm water and shake or stir the 
 mixture until the yeast is uniformly distributed. Then 
 stir in enough flour to make a thick cream and allow to 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 133 
 
 stand over night at room temperature. In this time fer- 
 mentation of the small amount of sugar in the flour begins 
 and the "sponge" swells up by the escape of bubbles of 
 gas. At this stage mix in uniformly and thoroughly 
 enough flour to make a stiff dough, using for the purpose 
 perhaps 25 Gm. Put the dough in an evaporating dish, 
 keep it for an hour or more at a temperature of 30° to 35° 
 C. and observe that it increases very greatly in size, from 
 the continued action of the yeast in liberating bubbles of 
 carbon dioxide. If a good hot air oven is at hand the ex- 
 periment is completed by baking the leavened mass. 
 
 Fresh brewer's yeast, if obtainable, is preferable to the 
 compressed yeast for this experiment as its action is 
 quicker. 
 
 Yeast is sold also in the form of perfectly dry cakes 
 which keep almost indefinitely and give most excellent re- 
 sults. 
 
Chapter VIII. 
 
 WATER AND AIR. 
 
 ""THE sanitary examination of water is a matter of some 
 *■ difficulty and can be carried out properly only by 
 skilled chemists. But certain tests are so commonly used 
 and certain terms so frequently employed that it will not 
 be out of place here to illustrate by a few experiments the 
 nature of the tests and meaning of the terms. 
 
 Absolutely pure water is nowhere known as a natural 
 substance, but can be prepared only by elaborate methods 
 of distillation. 
 
 Natural waters secured from springs, wells, rivers or 
 lakes hold in solution or suspension a variety of substances 
 taken from the air or from the soil through which they 
 have passed. Many of these substances are harmless. In 
 fact, they may be even beneficial in a drinking water, and 
 with them we are not concerned here. Certain other sub- 
 stances, which in themselves are harmless, are generally 
 looked upon with suspicion in drinking waters because 
 they usually enter water accompanied by other substances 
 of really dangerous character, or because they are products 
 of decomposition of possibly dangerous substances. Thus, 
 common salt is found in small amount in nearly all waters, 
 but in the water of ordinary wells and rivers does not exist 
 in more than traces because soils are not strongly impreg- 
 nated with salt. If in a shallow well water we find more 
 than a trace of salt we are led to look for the source from 
 which it has come. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 135 
 
 The urine and solid excreta of common house sewage 
 are the worst offenders in the contamination of well water 
 where they are allowed to soak into the soil through de- 
 fective drains or improperly constructed vaults. 
 
 Most of the organic products in the sewage may be 
 speedily oxidized by the soil. Others may pass on and 
 enter the water, and with them the indestructible salt as an 
 indicator of past contamination, as a reminder of the pos- 
 sible presence of something else for which we have less 
 characteristic tests. 
 
 The test for salt is a very simple one, and we make it 
 in our search for possible past contamination. 
 
 We test for ammonia, for nitrates, and for nitrites, not 
 because the minute quantities of these bodies found in 
 waters are harmful, but because they are usually produced 
 by the decomposition of nitrogenous organic matters and 
 may be accompanied by germs of disease from the same 
 source, from faecal matter, for instance. 
 
 Ex. 122. The Test for Chlorides. A test is often 
 made in this way. Measure out 200 Cc. of the water, add 
 to it a few drops of a solution of pure neutral potassium 
 chromate, and then from a burette run in, with constant 
 stirring, solution of tenth normal silver nitrate until a faint 
 reddish precipitate of silver chromate appears. Each cubic 
 centimeter of the silver solution precipitates 3.54 Mg. of 
 chlorine from common salt or other chloride, and when 
 the last trace of chlorine is combined, the silver begins to 
 precipitate the chromate with production of a red color. 
 The chromate acts here as an indicator, as it shows just 
 when the chlorine is all combined by beginning to pre- 
 cipitate itself. 
 
 In making this test it is well to take two similar beak- 
 ers, place them side by side on white paper, pour equal 
 amounts of water in each, add to each the same number of 
 drops of the indicator, and then with one make the actual 
 test by adding, the silver solution. Note the amount used 
 
136 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 to give a light shade and then discharge it by adding a 
 drop of salt solution. Now, with this opalescent or tur- 
 bid liquid for comparison add silver nitrate to the second 
 beaker until the light yellowish red shade just appears. 
 This reading is usually somewhat more accurate than the 
 first. 
 
 The amount of chlorine in most uncontaminated waters 
 is less than 20 milligrams in a liter. The preparation of 
 the standard silver nitrate solution, and of other solutions 
 to follow, is given in the appendix. 
 
 Ex. 123. The Test for Ammonia. Solutions of 
 ammonia or ammonium salts possess -the -peculiar prop- 
 erty of giving a yellowish brown color with what is known 
 as Nessler's reagent (a solution of mercuric potassium 
 iodide, made strongly alkaline with sodium or potassium 
 hydroxide). With more than traces of ammonia a pre- 
 cipitate is formed. 
 
 To make the test measure out 50 Cc. of the water in 
 a large test-tube, or tall narrow beaker, and add to it 2 
 Cc. of the Nessler solution. By placing the beaker on a 
 sheet of white paper and looking down through it, the 
 depth of color can be observed. A few parts of ammonia 
 in one hundred millions can be readily seen and measured. 
 
 Chemists usually make this test by measuring out 500 
 Cc. of the water, which is made alkaline by the- addition of 
 a few drops of strong, pure solution of sodium carbonate, 
 and then distilled from a large, clean glass retort with Lie- 
 big' s condenser attached. The distillate is collected in 
 portions of 50 Cc. each in a number of thin cylinders of 
 colorless glass, and to each is added 2 Cc. of the Nessler 
 reagent. Four portions are - usuaU,y«;eaaugh,*as the am- 
 monia distills over easily and soon. The colors are dupli- 
 cated by adding to pure distilled water in similar tubes 
 small amounts of standard ammonia solution and then the 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 137 
 
 Nessler solution until like shades are obtained. In this 
 manner it is possible to make a quantitative test. 
 
 In waters containing relatively large quantities of am- 
 monia much less than 500 Cc. must be taken. Whatever 
 the volume is which may be decided on by an approximate 
 preliminary experiment, it should be diluted to 500 Cc. 
 with pure ammonia-free water and then distilled. 
 
 A large amount of ammonia is generally an indication 
 of contamination, but not always. Deep well waters often 
 contain relatively great quantities of ammonia, while at the 
 same time they may be organically pure. 
 
 Chemists apply the Xtxrafree ammonia to that distilled 
 as just explained. If to the residue in the retort after the 
 distillation of 200 or 250 Cc. a strong oxidizing mixture of 
 potassium or sodium hydroxide and potassium perman- 
 ganate be added and heat again applied, a new portion of 
 ammonia may be liberated and collected with the con- 
 densed steam as before. To this the term albuminoid am- 
 monia is applied, because albuminous and other nitrogenous 
 matters are broken up by this treatment with liberation of 
 ammonia. The ammonia collected in the distillate during 
 this operation did not therefore exist already formed in the 
 water in the "free" or saline condition, but potentially. 
 That is, its elements were present in complex nitrogenous 
 bodies which, possibly, by putrefactive or other process of 
 disintegration would yield it. The presence of these com- 
 plex nitrogenous compounds which yield ammonia is a 
 suggestion of the possible presence of worse matters, hence 
 the value of the test. 
 
 Waters furnishing more than 1.5 to 2 parts of albumi- 
 noid ammonia in ten millions are usually condemned or 
 looked upon as suspicious. 
 
 The Oxidation Tests. Pure waters absorb free oxy- 
 
138 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 gen from the atmosphere but have no tendency to decom- 
 pose compounds to secure it. On the other hand waters 
 containing organic matters or certain inorganic contamina- 
 tions have the power of decomposing oxygen salts to 
 secure the oxygen they desire, and the amount of oxygen 
 so taken up becomes a measure of the impurity of the 
 water, Potassium permanganate is a salt, which, under 
 certain conditions, gives up its oxygen to waters contain- 
 ing organic bodies in solution and is frequently employed 
 in water analysis for this purpose. An experiment will 
 show one way in which it is used. 
 
 Ex. 124. Measure out about 100 Cc. of pure, carefully 
 distilled water, pour it into a clean beaker in which water has 
 just been boiled and add 5 Cc. of pure dilute sulphuric acid 
 (1 to .3). Place the beaker on wire gauze and heat to boil- 
 ing. ,Now add 5 drops of a dilute permanganate solution 
 (300 milligrams, to the liter) from a burette or dropping 
 tube and boil five minutes. The pink color persists. 
 
 Repeat the experiment using 100 Cc. of common 
 hydrant water to which a trace of egg albumin or urea has 
 been .added, and after running in the permanganate boil 
 again. The color fades out and more may be . added. 
 Finally, after sufficient has been added the pink color 
 remains. The number of drops or cubic centimeters used 
 is a measure of the contamination of the water, although 
 often, as in this experiment, a very rough one. 
 
 The above experiment is intended to show only the 
 principle involved in the test. As used by chemists prac- 
 tically, it has been modified in several ways in order to 
 make it convenient and reliable. 
 
 Because we are seldom in condition to tell the exact 
 nature of the organic bodies present in a contaminated 
 water, and are therefore unacquainted with their molecular 
 weights or powers of combination we cannot express the 
 results obtained by our tests in terms of organic matter 
 present, but only as oxygen consumed. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 139 
 
 In presence of organic matter (to absorb oxygen) and 
 sulphuric acid, permanganate is decomposed according to 
 the following equation: 
 
 3 H 3 S0 4 + K 2 Mn 2 O g = 
 
 K 2 S0 4 + 2 Mn S0 4 + 3 H s O + 5 O. 
 
 That is, 315 parts of permanganate liberate 80 parts of 
 oxygen. In a solution containing 315 milligrams of per- 
 manganate to the liter, each cubic centimeter will liberate 
 0.08 Mg. of oxygen. 
 
 The test is usually made by adding at once what a pre- 
 liminary experiment shows to be an excess of permangan- 
 ate, boiling five minutes and then determining the actual 
 excess of permanganate by running into the hot liquid a solu- 
 tion of oxalic acid of such strength that one cubic centi- 
 meter will exactly reduce the same volume of perman- 
 ganate. 
 
 The Tests for Nitrites and Nitrates. Nitrogenous 
 matters undergoing oxidation in water and soil usually give 
 rise, in time, to nitrites and finally to nitrates. These com- 
 pounds are therefore looked for in a water as evidence of 
 past contamination. In most instances nitrites, as a less 
 advanced stage of oxidation than nitrates, suggest compar- 
 atively recent contamination. The tests are especially 
 interesting in the examination of well and spring waters. 
 
 Chemists are acquainted with a number of methods for 
 the detection of traces of nitrogen in the form of nitrites 
 and nitrates, but at the present time certain color reactions 
 are, because of their simplicity, mainly in favor. These are 
 illustrated by the following tests. 
 
 A reagent for nitrites is prepared by dissolving 0.5 Gm. 
 of sulphanilic acid in 150 Cc. of acetic acid of 25 per cent 
 strength, and mixing this with- a solution of 0.1 Gm. of 
 
140 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 pure naphthylamine in 200 Cc. of dilute acetic acid. This 
 mixture keeps very well for a time in the dark. 
 
 Ex. 125. To about 50 Cc. of water in a clear beaker 
 add 2 Cc. of the above solution. If the water is quite free 
 from nitrites the reagent imparts no color to it. One hun- 
 dredth of a milligram of nitrogen as nitrite in the water 
 gives a faint pink color at the end of five minutes; with 
 larger quantities the color may become deep rose red. 
 
 A fairly accurate quantitative measurement may be 
 made by comparing the colors obtained with those pro- 
 duced in pure distilled water containing known amounts of 
 added potassium or sodium nitrite when treated with the 
 same reagent. The tests are best made in clear glasscyl- 
 inders with flat bottoms. All waters allowed to stand in the 
 air a short time show the reaction from absorbed nitrous 
 acid. 
 
 Nitrates are sometimes detected in water by converting 
 them into ammonia by some reducing agent, e. g., by addi- 
 tion of aluminum wire and pure sodium hydroxide, after 
 which the ammonia is identified and measured by the Ness- 
 ler test. Among other good methods is one illustrated in 
 principle by the following experiment. Prepare first a so- 
 lution of phenolsulphonic acid by adding to 50 Cc. of pure, 
 strong sulphuric acid, 4 Cc. of water and 8 Gm. of pure 
 phenol. 
 
 Ex. 126. Evaporate 50 or 100 Cc. of water to dryness 
 in a beaker, add 1 Cc. of the reagent and mix it with the 
 water residue. Then add 1 Cc. of pure distilled water and 
 a few drops (3 or 4) of pure strong sulphuric acid. Warm 
 the beaker by placing it for a few minutes in hot water, then 
 dilute the contents to 25 or 30 Cc, add ammonia to give a 
 good alkaline reaction (as shown by the smell), and finally 
 water enough to bring the volume of liquid up to 100 Cc. 
 The presence of nitrate is shown by a more or less deep 
 yellow color. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 141 
 
 In this experiment picric acid is formed at first if a ni- 
 trate is present, and the ammonia makes ammonium picrate 
 which has a strong yellow color. 
 
 When used as a quantitative test the color is duplicated 
 by treating water containing known amounts of nitrate in 
 the same manner until the same shade is reached. 
 
 The simple exercises outlined above, while not intended 
 as directions for the carrying out of exact scientific proc- 
 esses, are probably sufficient to give the student a general 
 idea of the principal chemical tests employed at the present 
 time in sanitary water examinations. 
 
 While physicians are seldom called upon to make chem- 
 ical examinations their opinions are frequently asked in 
 explanation of chemical reports and the work of the last 
 few pages will impart some degree of familiarity with the 
 usual terms employed. 
 
 Tests of Expired Air. 
 
 Experiment has shown that expired air differs essen- 
 tially from that taken into the lungs by containing much 
 less oxygen and a greatly increased amount of carbon 
 dioxide. The average volume of the latter gas expired is 
 over one hundred times as great as the volume taken in with 
 the atmosphere. 
 
 Expired air contains, also, traces of ammonia and or- 
 ganic gases and vapors, which sometimes have a very 
 disagreeable odor. 
 
 Several interesting tests may be made by blowing air 
 from the lungs through distilled water and testing the lat- 
 ter. In order to prevent the passage of saliva into the 
 water, which would vitiate some of the tests, the air is 
 made to pass through a small empty bottle or test-tube 
 first, with tubes leading in and out as in an ordinary wash 
 bottle, the tube from the mouth being the longer, however. 
 
142 ELEMENTARY CHEMICAL PHYSIOLOGY 
 
 Ex. 127. Pour some pure water into two small flasks 
 or beakers. To one add some clear lime water and to the 
 other five drops of tenth normal sodium hydroxide solution 
 and a few drops of phenol phthalein indicator which pro- 
 duces a red color. Blow air into both flasks. In the one 
 containing the lime water a white precipitate appears from 
 the formation of calcium carbonate. In the other the red 
 color, characteristic of an alkaline reaction, soon disappears 
 as the alkali added becomes saturated by the carbonic acid. 
 On the second reaction is based a very simple and accurate 
 method of measuring quantitatively the carbon dioxide in 
 crowded rooms. 
 
 Ex. 128. Force the expired air into a small flask con- 
 taining fifty Cc: of carefully distilled water, to which 
 has been added 1 Cc. of potassium permanganate solution, 
 such as was used in the water test (oxygen consumed), 
 and a small amount of pure sulphuric acid (about 1 Cc. of 
 acid, 1 to 3.). Heat the flask nearly to boiling, and ob- 
 serve that as the air is forced through, the pink perman- 
 ganate color fades, and gradually disappears, owing to 
 the reducing power of the organic vapors of the breath. 
 
 Ex. 129. Into 50 Cc. of fresh distilled water in a 
 small, clean flask blow air five or ten minutes. Then add 
 about 2 Cc. of Nessler solution to the water and notice the 
 yellow color formed, indicative of the presence of ammo- 
 nia. The amount of nitrogen given off from the lungs 
 in the form of ammonia is minute, but can usually be 
 recognized readily by the above test. 
 
 Before beginning any of the above experiments the 
 mouth should be thoroughly rinsed out with water in order 
 to remove any organic matter left from the food or from 
 other source. 
 
Chapter IX. 
 
 SPECIAL PROBLEMS. 
 
 IN this chapter a number of special exercises will be given 
 * of a more advanced character than are those of the 
 preceding pages. They are intended for the use of stu- 
 dents who wish to prepare themselves for independent in- 
 vestigation in chemical physiology, but still are of such a 
 nature that they properly belong in a course of undergrad- 
 uate study, as given in the best of our American schools. 
 
 Problem i. The Preparation of Urea. 
 
 The preparation of urea is from several standpoints an 
 exercise of interest and practical importance. Represent- 
 ing, as it does, the final stage in nitrogenous metabolism in 
 the human body, a study of the methods by which urea can 
 be produced and of the compounds into which it can be 
 transformed must naturally lead to some knowledge of the 
 reactions taking place in the body itself. Chemically 
 speaking, a long distance separates the nitrogen of the food 
 from the nitrogen of the urine, and the course from one to 
 the other is being explored from both directions. In the 
 laboratory urea may be prepared from urine itself, or by 
 several synthetic reactions. 
 
 a. From Urine. Evaporate two to three liters of 
 urine, on the water-bath, to a volume of about 100 to 150 
 Cc. Cool this residue to 0° by surrounding it with ice and 
 salt. Then add 300 Cc. of pure nitric acid diluted to a 
 strength of 50 per cent, and cooled likewise to 0°. The 
 
144 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 acid should be added slowly with stirring, after which the 
 mixture is allowed to stand several hours, or over night, 
 at a low temperature. 
 
 Urea nitrate being very much less soluble at a low tem- 
 perature than at the ordinary one is given an opportunity 
 o crystallize out. 
 
 The mass of crystals is then thrown on an asbestos 
 filter and drained by means of a vacuum pump and then 
 washed several times with strong, well cooled nitric acid, 
 to remove chlorides, phosphates and traces of other sub- 
 stances without dissolving much of the urea. 
 
 The crystals are transferred from the funnel to a 
 beaker and dissolved in a very small quantity of boiling 
 water. The solution is reprecipitated by adding pure cold 
 concentrated nitric acid, and the crystalline and nearly 
 pure nitrate now obtained is thrown on the same asbestos 
 filter and drained thoroughly with the pump. The crys- 
 talline mass is dissolved in a small quantity of warm water 
 and to the solution is added barium carbonate in slight 
 excess, which forms barium nitrate with liberation 
 of urea. The carbonate is added slowly as long as gas is 
 given off on shaking and then a little excess. The mixture 
 is evaporated to dryness on a water-bath and then treated 
 with pure, strong alcohol, which dissolves out the urea but 
 leaves the barium nitrate and excess of barium carbonate. 
 
 The alcoholic filtrate so obtained is usually colored. It 
 may be purified by heating it gently with washed animal 
 charcoal and filtering again. The final filtrate is evap- 
 orated at a low temperature and then allowed to crystal- 
 lize spontaneously in the form of short needles. 
 
 By careful work pure urea can be obtained by the above 
 process, but more readily by the synthetic method next to 
 be given. This method is interesting as being essentially 
 the one by which it was first demonstrated that organic 
 compounds can be built up synthetically in the laboratory. 
 
 b. From Ammonium Cyanate. Fuse loo Gm. of 
 high grade potassium cyanide at a low red heat and oxidize 
 it to cyanate by adding red lead, a little at a time, with 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 145 
 
 constant stirring, while the mass is kept liquid. About 500 
 Gm. in all should be added. When the reaction is com- 
 plete allow the mass to cool, powder it and extract with 
 cold water. Treat the filtrate with solution of barium ni- 
 trate as long as a precipitate (barium carbonate) forms and 
 filter again. The solution now contains potassium cya- 
 nate and several impurities. Add to it a slight excess of 
 solution of lead nitrate which throws down a fine 'white 
 precipitate of lead cyanate. Allow it to settle, wash by 
 decantation and then on a filter with cold water, and finally 
 dry at a low temperature. 
 
 By heating equivalent molecular weights of the pure 
 lead cyanate and ammonium sulphate with a small quanti- 
 ty of water lead sulphate is precipitated and ammonium 
 cyanate formed, which, however, on evaporation is trans- 
 formed completely into urea. Extract the evaporated mass 
 with absolute alcohol and crystallize as before. 
 
 The experiment is also made by starting with potassium 
 ferrocyanide, which is dehydrated by heat and then fused 
 with manganese dioxide to oxidize it to cyanate. The crude 
 potassium cyanate is extracted by water and then the solu- 
 tion is treated directly with ammonium sulphate. On 
 evaporating to dryness urea may be separated from the 
 potassium sulphate by strong alcohol. 
 
 Pure urea melts at a temperature of 133° C. When 
 heated to 150° a large part of it is converted into biuret, 
 which can be recognized by the test already described. Its 
 solution treated with sodium hypochlorite yields free nitro- 
 gen, carbon dioxide and water, while with nitrous acid a 
 very similar decomposition is produced. The nitrogen of 
 the acid escapes here, too. 
 
 These reactions form the basis of a method for the 
 determination of urea, which will be explained in the 
 section on urine analysis. Urea heated with glycocoll 
 (amido-acetic acid) to about 200° C. yields uric acid but 
 the reaction is probably devoid of physiological importance 
 unless it can be shown that it can be brought about at a 
 low temperature. 
 
146 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Problem 2. The Synthesis of Uric Acid. 
 
 The production of uric acid by the method just referred 
 to may be illustrated by the following experiments. 
 
 We take up first the production of the glycocbll which 
 can be accomplished by several methods, but most con- 
 veniently by decomposition of hippuric acid by means of 
 sulphuric or hydrochloric acid by aid of heat. 
 
 Hippuric acid, which is the chief nitrogenous substance 
 excreted in the urine of the herbivora, is in effect a com- 
 bination of benzoic acid and glycocoll, or benzoyl glyco- 
 coll. The action of the aqueous acid in splitting it is one 
 of dehydration as illustrated by this equation. 
 
 C0 8 H.CH 2 NH.(C 6 H 6 CO)+HOH = 
 
 C0 8 H.CH 2 NH 2 +C 6 H 6 C0 2 H. 
 
 That is, benzoic acid and glycocoll are obtained as 
 products. On the large scale the reaction is carried out 
 in Germany in the commercial manufacture of benzoic 
 acid. 
 
 a. To Prepare Glycocoll. Boil 10 Gm. of hippuric 
 acid with 40 Cc. of dilute sulphuric acid (1 part of acid 
 with 5 parts of water), ten to fifteen hours in a flask fur- 
 nished with a return condenser for condensation of vapor. 
 The benzoic acid separates as an oily layer, which solidi- 
 fies on cooling. At the end of the boiling, while the liquid 
 is still warm it is poured out into an evaporating dish and 
 allowed to stand over night in order to let the benzoic acid 
 settle out as completely as possible. The liquid is poured 
 through a filter and the residue washed in water which is 
 poured through the same filter. 
 
 The mixed filtrate is concentrated to a small volume 
 which drives off some of the remaining benzoic acid. The 
 rest is removed by shaking with ether in which the glyco- 
 coll sulphate is not soluble. The liquid is diluted with 
 200 Cc. of water, and neutralized by addition of barium 
 carbonate, which is added in slight excess. A precipitate 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 147 
 
 of barium sulphate separates. The liquid is decanted and 
 the precipitate washed several times with boiling water, 
 the washings are poured through a filter, and finally, with 
 that poured off first, evaporated to a small volume. If the 
 liquid is not clear it must be filtered before the concentra- 
 tion is carried far. 
 
 When reduced, finally, to a small volume the solution 
 of glycocoll, now nearly pure is allowed to stand over 
 night for crystallization. From the mother liquors several 
 further crops of crystals can be obtained by concentration. 
 The several products are mixed and purified by recrystal- 
 lizafion from pure water. 
 
 b. The Formation of Uric Acid. Mix l part of 
 glycocoll with 10 parts of pure urea and heat the mixture 
 to a temperature between 200° and 230° C. The mixture 
 darkens and becomes finally pasty and then hard. It is 
 removed from the heat and allowed to cool, then broken 
 up and dissolved in a boiling hot weak ammonia solu- 
 tion. An insoluble part is separated by filtration. From 
 the filtrate the uric acid is precipitated by addition 
 of a solution containing magnesia mixture and ammonia- 
 cal silver nitrate. The precipitate is washed several times 
 with weak ammonia water, then stirred up with hot dis- 
 tilled water and decomposed by addition of solution of 
 sodium sulphide. A precipitate of silver sulphide is fil- 
 tered off. Hydrochloric acid is added in slight excess to 
 the filtrate which is concentrated and allowed to cool. 
 Uric acid crystallizes out in forms which can be recog- 
 nized under the microscope. This crude product can be 
 dissolved in weak ammonia, and precipitated again with 
 the ammoniacal silver-magnesia mixture. By completing 
 the process as before a purer acid is obtained which yields 
 the tests described in the sections on urine analysis. 
 
 Uric acid can be obtained in quantity best from the 
 excrement of birds or reptiles. It can also be precipi- 
 tated from human urine. When pure it is perfectly white, 
 but when thrown down from urine is always yellow or 
 reddish. 
 
148 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Problem 3. Separation of Glycocholic Acid. 
 
 Some simple tests for the acids existing in bile have 
 already been given. The separation of glycocholic acid in 
 nearly pure condition is shown by the following experiment. 
 
 Glycocholic Acid. Evaporate 1000 to 1500 Cc. of ox 
 bile to one-fourth its volume, then add some washed ani- 
 mal charcoal and evaporate with frequent stirring until the 
 mass becomes practically dry. The whole evaporation 
 must be carried out on the water-bath. The dry residue is 
 rubbed up in a mortar then put in a flask on a water-bath 
 "and covered with twice its volume of absolute alcohol. 
 
 The flask is connected with an upright Liebig's conden- 
 ser. By keeping the alcohol at the boiling temperature 
 fifteen to twenty minutes the acids go into solution. The 
 liquid is filtered and the mass left washed with warm 
 alcohol. 
 
 If the mixed filtrate with washings is highly colored it 
 must be heated again with fresh animal charcoal. The 
 alcoholic solution is evaporated to a thick syrup. This 
 is dissolved in a small quantity of absolute alcohol, the so- 
 lution filtered and treated with absolute ether until a per- 
 manent turbidity appears. The mixture is allowed to 
 stand in a closed vessel in a cool place until the sodium 
 salts of the acids crystallize out in fine needle shaped 
 crystals. These crystals are dissolved in a small amount 
 of water and to the solution is added dilute sulphuric acid 
 until it becomes turbid from precipitation of the free acid. 
 On adding ether to the liquid the glycocholic acid separ- 
 ates in small needles on standing. These needles are 
 pressed out and dissolved in hot water and the solution 
 filtered. On cooling, the acid appears again. It may be 
 made quite colorless by crystallizing from hot water sev- 
 eral times. (Drechsel.') 
 
 It was shown by Huefner that a crystallization of nearly 
 pure glycocholic acid may often be secured by adding hydro- 
 chloric acid and ether to bile directly. The method, as 
 ■worked out by Dr. J. Marshall with American ox bile, is 
 given here. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 149 
 
 Measure out 100 Cc. of bile, add a few drops of hydro- 
 chloric acid, shake and filter without delay. Then add 5 
 Cc. of strong ether (or petroleum spirit) and then 30 Cc. of 
 strong hydrochloric acid. Shake the mixture and allow it 
 to stand a day, corked, in a cool place. 
 
 Crystals soon appear which are pressed out and re- 
 crystallized from hot water. 
 
 This reaction is not given by all biles, and to secure a 
 good result it is necessary to use perfectly fresh bile. The 
 acid and ether must be added within half an hour of the 
 time the gall bladder was removed. The crystals are al- 
 most colorless when formed. Taurocholic acid is much 
 more soluble and is not so easily obtained. 
 
 Problem 4. Preparation of Leucin and Ty rosin. 
 
 It has already been shown that in the prolonged action 
 of the pancreatic secretion on albuminoids these two sub- 
 stances appear among the important end products. Their 
 purification, however, when prepared in this way is a mat- 
 ter of some difficulty and other processes are resorted to 
 when material for experimental purposes is desired. By 
 the prolonged action of hot dilute acids on albuminous or 
 kindred substances the two compounds are made in con- 
 siderable quantity. Horn shavings are now frequently em- 
 ployed as the starting material. 
 
 Preparation. In a large flask boil 500 Gm. of horn 
 shavings with 1,200 Gm. of thirty per cent sulphuric acid 
 during ten to twelve hours, replacing evaporated water 
 from time to time. Then neutralize the excess of acid with 
 barium carbonate and finally with barium hydroxide in 
 very slight excess. Filter through muslin, press out the pre- 
 cipitate, boil it with water and filter again hot. The mixed 
 liquids contain a little baryta which is removed by careful 
 addition of dilute sulphuric acid and filtration. Now 
 evaporate the filtrates until a film of crystals appears, allow 
 to cool, remove the crystals by filtration and evaporate 
 
150 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 again until crystals appear. Separate these and repeat the 
 operations as long as crystals can be obtained. The first 
 crystallizations consist largely of tyrosin, the last of leu- 
 cin. The crude products are mixed and purified as fol- 
 lows: Dissolve in weak ammonia by aid of heat and add 
 solution of basic acetate of lead as long as a precipitate 
 forms and in slight excess, the solution being kept hot. 
 Filter from the precipitate and through the filtrate pass 
 hydrogen sulphide to remove lead in solution. Filter 
 again and allow the liquid to cool. Usually, without 
 further concentration, the tyrosin crystallizes out on 
 cooling. 
 
 If crystals do not appear, concentrate slightly and allow 
 to stand. 
 
 After the tyrosin has separated the mother liquor is 
 concentrated by evaporation to a small volume. To this 
 is added an excess of freshly precipitated copper hydroxide 
 which gives a blue solution with leucin on boiling. Filter 
 hot and concentrate the filtrate which precipitates a com- 
 pound of copper and leucin. This is collected, mixed 
 with water and treated with hydrogen sulphide, which pre- 
 cipitates the copper and leaves the leucin now in solution 
 relatively pure. On concentration crystallization follows. 
 The crystals can be purified further by dissolving in hot 
 alcohol and setting aside in a cool place for a second crys* 
 tallization. 
 
 As leucin is readily soluble in water its purification is 
 by no means a simple matter. Under favorable conditions 
 from 100 parts of horn shavings, 3.6 parts of tyrosin and 
 10 parts of leucin may be obtained. Other nitrogenous 
 substances have been shown to yield even greater quanti- 
 ties of leucin. In general tyrosin is formed in much 
 smaller amount than leucin. 
 
 Problem 5. The Use of the Spectrophotometer. 
 
 Under the head of blood tests the value of the spectro- 
 scope in the qualitative examination of blood was pointed 
 out. In a modified form the instrument has become 
 equally valuable in quantitative investigations and a short 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 151 
 
 explanation of such applications will be given here. Used 
 as a quantitative instrument the spectroscope is converted 
 into a spectrophotometer, and forms have been devised by 
 Vierordt, Huefner, Glan, Wild and others which render 
 good service. Only one of these will be described here, 
 the Vierordt form, as made by Kruess, of Hamburg. 
 
 The essential features of this instrument are: 
 First, a double slit instead of the single slit of the ordi- 
 nary spectroscope. The common slit is divided into two 
 portions, an upper and a lower half, each controlled by its 
 own micrometer screw. In the later instruments these 
 halves open symmetrically, that is from both sides of a 
 central line, instead of from one side as in the common 
 Bunsen spectroscope. The width of each slit can be 
 accurately measured by a micrometer screw. The con- 
 struction and operation of the slit are shown in the figure 
 above. 
 
 Second, the arrangement of the ocular tube by means 
 of which a definite portion of the spectrum can be brought 
 
152 
 
 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 into the field of vision. The ocular tube can be given a 
 lateral motion by means of a fine micrometer screw so that 
 light of perfectly definite wave length can be brought be- 
 fore the center of the eyepiece at will. 
 
 In the eyepiece itself there is a movable framework 
 which, when shoved to the left, brings two fine cross-hairs 
 exactly in the center of the field and when shoved to the 
 extreme right brings an adjustable slit in the same position. 
 
 FIG. 36. 
 
 This slit can be opened or closed by a micrometer screw 
 symmetrically, but its center has the position of the cross- 
 hairs in the previous adjustment. Opening or closing this 
 slit has the effect of taking in more or less of the spectrum. 
 In the above figure is shown the instrument as a whole 
 and in the following one the measuring arrangements of 
 the observation tube just referred to. C is the observation 
 tube which is moved by the micrometer screw, r t , shown in 
 detail in Fig. 37. The ocular slit is opened by the microm- 
 eter screw r 2 , which moves also the cross-hairs, when 
 necessary, and which is shown in detail in Fig. 3V. Be- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 153 
 
 neath and firmly attached to the observation tube is a scale 
 /,, which moves past the fixed pointer with mark at /,. 
 One revolution of the screw r 1 moves l t one division. The 
 head of the screw is divided into 100 parts so that the posi- 
 tion of the tube can be noted in scale divisions and hun- 
 dredths, in four figures in all, as 2,852. 
 
 FIG. 37. 
 
 Suppose now with the ocular slide shoved to the left, 
 that is, with the cross-hairs in the center of the field in 
 perfectly definite position, the instrument is directed to- 
 ward the sunlight and focused with the Fraunhofer lines 
 sharply defined, it is plain that by moving r l any one of 
 these lines can be brought to the center of the cross-hairs. 
 When this is done the exact position of the tube can be 
 read off on l x and r t . If, with the D line, for instance, on 
 the cross-hairs the position of the observation tube in scale 
 division is 1,946, we know that always when we bring the 
 tube in this position we have light of wave length 588.9 in 
 the center of the field of view. 
 
154 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Before using the instrument for practical measurements 
 it must be graduated by a method indicated by what has 
 just been said. Sunlight is thrown directly into the colli- 
 mator slit, S, and one by one the principal and character- 
 istic Fraunhofer lines are brought to the center of the 
 cross-hairs. The position of the observation tube for each 
 line in the center of the field is then read off, giving finally 
 a table connecting the arbitrary scale divisions with light 
 of definite wave length. Expressing wave lengths in mil- 
 lionths of a millimeter a table like the following can be 
 easily made : 
 
 Fraunhofer Scale Wave 
 
 Line. divisions. lengths. 
 
 C 1750 656.2 
 
 D 1946 588 9 
 
 (Ca, green) 2079 558.7 
 
 (Tl) 2168 534.9 
 
 E 2203 526.8 
 
 b 2256 517.1 
 
 F 2446 486.0 
 
 G 2916 430.6 
 
 Between b and F, and F and G, there are many sharp 
 lines which can be easily distinguished. These are brought 
 also to the cross-hairs, and the corresponding position of 
 the observation tube noted. Our table becomes extended 
 so as to embrace many lines from the middle part of the 
 spectrum. With this done we are able to bring any part 
 of the middle spectrum under observation in the center 
 of the field by simply moving r t to the corresponding po- 
 sition. If it is wished to examine light of wave length 
 534.9, that is light similar to the Thallium green, the ob- 
 server moves the micrometer screw until l x and r t show the 
 position 2,168. If light of wavelength 546.7 is wanted, 
 this being midway between the neighboring Ca and Tl. 
 greens, the observation tube is brought to the position 
 2,123. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 155 
 
 For wave lengths near together this interpolation is 
 satisfactory, but for greater differences it will not answer 
 as the differences between the scale readings are not pro- 
 portional, strictly, to the differences between wave lengths 
 corresponding. Interpolations in longer stretches are best 
 made from an interpolation curve, obtained by plotting the 
 wave lengths as ordinates, and the scale divisions as ab- 
 scissas. (The scale divisions given in the above table, as 
 illustrations, are for a particular instrument only. The 
 divisions corresponding to certain wave lengths would dif- 
 fer in different instruments.) 
 
 In order, now, to bring into the center of the field of 
 view a certain color only, the ocular slide is shoved to the 
 right, bringing the slit k in the center instead of the cross- 
 hairs. This slit k may be made narrow or wide by motion 
 of the micrometer screw r 2 , but its center keeps the posi- 
 tion which the cross-hairs formerly had and the light com- 
 ing through it has the mean wave length of that corres- 
 ponding to the scale divisions / 1; r t . 
 
 As the ocular slide, and consequently the cross-hairs, can 
 be moved by the screw r % it is possible to express the 
 width k in wave lengths by this procedure. With the 
 cross-hairs in position in the eyepiece bring a clearly de- 
 fined Fraunhofer line to the center. Meanwhile, r s and / a 
 must be at zero position. Then, by means of the screw r t 
 the line is moved away from the center of the hairs, say 
 fifty divisions on r x . Next, by means of r^ bring the line 
 and center of hairs to coincide again, and note how many 
 divisions r 2 must be moved through to do this. If a cer- 
 tain motion of r % moves the cross-hairs here through a dis- 
 tance corresponding to x scale divisions, the slit k would 
 be opened to correspond to twice this number of divisions, 
 because its two sides move symmetrically and only one is 
 moved parallel with k. These adjustments are very care- 
 
156 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 fully made on the instrument and enable the observer to 
 limit with great exactness the extremes of wave length 
 brought into view. 
 
 With the explanations here given other details can be 
 learned from a study of the instrument itself. We turn 
 next to a discussion of the principles involved in its use. 
 
 It was shown in the simple spectroscopic tests of blood 
 in different degrees of dilution that the amount of light ab- 
 sorbed in passing through the liquid is closely dependent on 
 the concentration, or in other words on the amount of 
 haemoglobin present. Strong solutions are nearly opaque. 
 With increasing dilution more and more light comes 
 through. Finally, with oxyhemoglobin, much of the light 
 between the red and blue is allowed to pass except in two 
 narrow regions. Here two dark bands still show a strong 
 absorption. If the dilution is carried still further these bands 
 grow fainter but their positions remain unchanged. The 
 points of maximum absorption are constant and character- 
 istic for each substance. 
 
 Something similar is shown with solutions of potassium 
 permanganate. If we observe these in cells with parallel 
 walls, we find, for instance, with a certain dilution a heavy 
 absorption band between the lines E and F, growing fainter 
 from F toward a line with wave length I 470.2. On di- 
 lution of this solution the band observed begins to break 
 up, when finally we can easily distinguish 5 bands between 
 D and a point a little beyond F [X 588.9 — k 486, where 
 X stands for wave length). With increased dilution the 
 bands grow fainter and the spaces between them wider 
 and brighter. There must be, therefore, some simple rela- 
 tion between the amount of light absorbed and the con- 
 centration of the absorbing solution, or in other words, the 
 number of molecules of coloring substance brought be- 
 tween the source of light and the slit. If we know this 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 157 
 
 relation and can measure the extent of the light absorption it 
 is evident that we have a means of arriving at the amount 
 of the absorbing substance in solution. 
 
 Light passing through any medium, air, glass, water, or 
 colored liquids, suffers a certain diminution in intensity. A 
 certain amount of it is absorbed, this varying with the con- 
 stitution of the medium, but following a very simple law for 
 different concentrations or thicknesses of the same medium. 
 
 Increasing the thickness of the layer or column of ab- 
 sorbing substance has the same effect as increasing the 
 number of absorbing molecules. To double the thickness 
 of the cell of blood between the lamp and spectroscope slit 
 amounts to multiplying by two the number of oxyhemoglo- 
 bin molecules which exert an absorbing action on the light. 
 
 But it does not follow from this that increasing the 
 thickness of the blood layer or the number of molecules 
 will diminish the intensity of light passing through in the 
 same proportion. The absorption of light in various media 
 follows a different general law which was first worked out 
 by Lambert, on the assumption of a variation in the thick- 
 ness of the medium rather than of its concentration. The 
 relations found by Lambert for glasses of different kinds 
 and which have been shown by later physicists to hold 
 good for liquids, may be indicated by the following. 
 
 Suppose we have a source of light of intensity, I, and 
 allow it to pass into a substance, which we will assume is 
 divided into layers. In passing through the first layer, 
 that is passing a certain number of molecules, its intensity 
 
 is reduced to — or amounts to I 
 
 n n . 
 
 In passing through the next layer of same thickness it 
 is again reduced by loss of the same fraction and is now 
 
 t 1 1 I 
 I. _ . —or _ 
 
 n n n - 
 
158 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 In passing the next layer it becomes 
 
 I J-.J-.J-^J- 
 
 n n n n 3 > 
 
 and in passing p such layers it becomes 
 
 I 
 
 The remaining intensity of the light after passing p lay- 
 ers of absorbing substance may therefore be expressed by 
 the formula 
 
 v = L (1 > 
 
 n p - 
 
 This relation holds good only for homogeneous light or 
 for light from a small region in the continuous spectrum. 
 
 For purpose of further calculation it may be assumed, 
 arbitrarily, that the orignal light had the intensity 1 = 1. 
 Our formula then becomes 
 
 from which 
 or 
 
 I'=— (2) 
 
 n" v ' 
 
 log I' = — p log n (3) 
 
 logn = _!?£!' (4) 
 
 The light absorbing power of two substances can be 
 compared by noting the thickness of layers of these sub- 
 stances which must be taken to reduce the incident light to 
 a certain fraction of the original intensity after passage. 
 If a certain solution of blood coloring matter through 
 which light passes has a thickness of 20 Mm., and if of 
 another blood solution a thickness of 40 Mm. must be 
 taken to reduce the transmitted light to the same fraction 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 159 
 
 it is evident that the first solution must have a much 
 greater light absorbing power than the second. 
 
 In comparing solutions or transparent solid substances 
 practically it may be agreed to consider a reduction of the 
 intensity of the light to ^ its original value as the basis of 
 comparison, and the shallower the layer of substance 
 required to bring about this reduction to -^ the greater 
 must be its light absorbing power. The light extinguish- 
 ing power of a substance or its coefficient of extinction has 
 been defined by Bunsen and Roscoe as the reciprocal value 
 of the thickness of a layer of the substance necessary to reduce 
 the intensity of the transmitted light to ^ its original value. 
 
 Representing the extinction coefficient by E and the 
 reduced intensity by I' we have from the above formulas 
 
 E = — and I' = TTr 
 
 Therefore 
 
 and logn = — lo l_n; _ E (5) 
 
 E = -*Li' (6) 
 
 If p is given, once for all, the thickness of unity, that is 
 if p = 1 (as 1 Cm.), our formula becomes 
 
 E = - log V. 0) 
 
 It was said above that increasing the thickness of a 
 layer of absorbing substance has the same effect as increas- 
 ing its concentration in the same degree. From this it 
 follows that the extinction coefficient must be directly pro- 
 portional to the concentration. If E and E' represent the 
 extinction coefficients and C and C the concentrations of 
 two solutions of the same substance 
 E : C :: E': O 
 represents their relation. 
 
160 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 The further relations 
 
 E. E- _E» etc 
 C' C C"' 
 
 must be equal and constant, and must serve as a character- 
 istic connecting the light absorbing power of a solution with its 
 strength. 
 
 The term absorption rate has been applied to the ratio 
 C : E by Vierordt and designated by him by A. Therefore 
 
 C 
 
 TT= A 
 
 In illustration of this suppose we prepare a perman- 
 ganate solution containing 0.25 Gm. of the pure crystals in 
 a liter. Its concentration is, therefore, 0.00025. Suppose 
 we further find by properTexamination that the intensity of 
 the light after absorption in a cell through which it is 
 directed is 0.0436, a little more than J T ol its original value. 
 
 From the formula 
 
 E = — log l'= — log 0.0436 = 1.36051 
 we have 
 
 A= T :WH = 0.000184. 
 
 Now, experiment shows that for light of constant wave 
 length, (A494.7 — A486.5 in the above illustration,) A is a con- 
 stant for all strengths of solutions of the same substance. 
 Finding A, once for all, in a definite region of the spectrum, 
 we can use the formula 
 
 C=E.A 
 
 to find the strength of a solution of which, experimentally, 
 we are able to determine the extinction coefficient. Quan- 
 titative spectrum analysis by absorption is based on these 
 principles. 
 
 It remains, now, to explain the methods- for the deter- 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 161 
 
 initiation of the extinction coefficient by means of the ap- 
 paratus described above. This is most commonly done by 
 aid of the divided slit of the spectrophotometer. The liquid 
 under examination is placed in a cell with parallel, clear 
 glass walls, 1 Cm. apart. The cell is half filled and so 
 placed before the slit that the surface of the liquid is even 
 with the dividing line between the upper and lower halves. 
 
 it ! w 
 
 I 1 
 
 
 FIG. 38. 
 
 The light from an oil lamp is allowed to shine through the 
 cell into the instrument, the edge of the flame being 
 directed toward the slit and its center being just level with 
 the center of the latter and in a line with the center of the 
 collimator tube. This position of the lamp is essential to 
 uniform illumination. It may be placed from 1 to 2 deci- 
 meters from the cell, which in turn must be as close to the 
 slit as possible. The general arrangement of the appa- 
 ratus is shown above. 
 
 With this arrangement light from the lamp enters the 
 upper slit through the air and the lower one through the 
 
162 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 solution, which absorbs a part of it. If, before the solution 
 is placed in the cell, the upper and lower halves of the 
 divided slit are opened to the sarne width, which is shown 
 by the graduation on the micrometer screw head, the pro- 
 jection of these in the eye piece will have exactly the same 
 illumination, which can be best seen by cutting out all but 
 a small part of the spectrum as explained above. With 
 the cell, half filled, in position, the light coming through 
 the lower slit is much weakened. The intensity in the two 
 fields can be restored by opening the lower slit or closing 
 the upper one to some extent. A better plan is to give the 
 lower slit a definite standard width to begin with, say that 
 corresponding to one whole turn or 100 divisions on the 
 micrometer screw. Then the upper slit is narrowed until 
 the intensity of the light allowed to pass is equal to that 
 through the lower slit and solution. The reduced intensity 
 of the light, or I' of the formula above, is given by the read- 
 ing on the upper micrometer. If while the lower slit is 
 opened by 100 micrometer divisions, the upper one must be 
 narrowed down to l&idivisions to bring the fields to the 
 same intensity, this shows that only 0.18 of the light 
 entering passes through the solution. 
 
 I' = 0.18, and E = —log. I'. 
 
 This, therefore, gives us E. 
 
 The method as outlined is, however, not very conve- 
 nient because of the difficulty in adjusting the two fields in 
 the instrument. The meniscus unavoidably formed in the 
 partly filled cell projects a broad, dark band across the 
 spectrum, which effectually prevents exact comparison of 
 shades. 
 
 But this difficulty has been, in a great measure, overcome 
 by the use of a device suggested by Schuh. He gives the 
 cell a width of 11 millimeters instead of 10, and drops in 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 163 
 
 its lower half a block of clear glass with two faces parallel 
 and just 10 millimeters apart. 
 
 This rectangular block or prism of glass has such 
 dimensions and is given such a position in the cell that all 
 light entering the lower slit of the spectrophotometer must 
 pass through it and through one millimeter of liquid be- 
 sides. The cell is filled, not half filled as before, and all 
 light entering the upper slit must pass through the 11 
 millimeters of liquid. This is accomplished by having the 
 upper surface of the glass prism perfectly horizontal and 
 
 
 
 
 a- 
 
 v. 
 
 
 ) 
 
 FIG. 39. 
 
 on an exact level with the dividing line between the two 
 shutters of the upper and lower slits. 
 
 The above figure represents a cell with its rectangular 
 glass prism, or Schulz's prism, in position. 
 
 In practice the cell is placed on a platform on the top 
 of a standard, which by aid of a screw can be raised or low- 
 ered at will. The standard is attached to a solid iron base 
 having a leveling screw, which is of value in bringing the 
 Schulz's prism into proper position. 
 
 When the adjustments are properly made the projec- 
 tion of the upper glass surface appears as a line coinciding 
 
164 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 with the dividing line between the upper and lower spec- 
 tra. No broad dark band appears to separate these two 
 spectra to such a degree that their comparison is not 
 readily made. The lamp is placed as before, but it is now 
 the upper slit which is opened to the normal width, while 
 the lower one is closed until the two fields become the same 
 in intensity. The method of reading or making the calcu- 
 lation is not altered by the fact that the cell has now a 
 width of 1.1 centimeters instead of 1, because in its lower 
 part the light must pass through 1 millimeter of liquid as 
 well as through the clear glass. The light absorbed by 
 one millimeter below is equivalent to that absorbed by one 
 millimeter of solution above, and the absorption of the re- 
 maining 10 millimeters above, alone comes into comparison. 
 
 In practice substances are dissolved in water, alcohol, 
 ether, or other liquid for observation, and preliminary to 
 actual tests the absorption relations of the solvent and the 
 glass prism must be determined. 
 
 To do this, fill the cell with the clear menstruum, place 
 it in position and examine with the lower slit opened to 
 twenty or twenty- five divisions. Then adjust the width of 
 the upper slit until equal intensities are secured. With 
 water in the cell, and the Schulz prism of the usual glass, it 
 has been found that with the lower slit at twenty-five di- 
 visions, the upper one must be brought to about 22.6 di- 
 visions to give the same intensity. The experimenter 
 should make these tests for himself, however, with each 
 new solvent used and with each Schulz glass body, as they 
 are not absolutely uniform in their absorption power. 
 
 The absorption ratios of a number of physiologically 
 important substances, for certain regions in the spectrum 
 have been determined by Vierordt, Huefner and others and 
 placed on record. The following table contains some of 
 these results. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 165 
 
 
 Name of Substance. 
 
 Spectral region. 
 
 6 
 
 Ed 
 
 re "S 
 
 
 
 a 
 
 3 
 _o 
 
 "5o 
 o 
 
 a 
 « 
 
 id 
 
 S3 
 
 ■S o 
 
 ID'S) 
 
 6 
 
 B c 
 
 5 ° 
 
 8^ 
 
 a B 
 5 go 
 
 o 
 
 i'4 
 
 A569.3— A555.5... 
 
 0.00133 
 0.00100 
 
 0.00122 
 0.00150 
 
 0.00260 
 0.00199 
 
 0.00131 
 0.00115 
 
 
 
 549.9— 540.0... 
 
 
 
 558.1— 534.3... 
 
 0.00113 
 0.0000598 
 0.0000356 
 0.0000209 
 
 0.000215 
 
 501.2— 494.3... 
 
 
 
 
 
 0.000142 
 
 494.3— 486.1... 
 
 
 
 
 
 0.000116 
 
 486.1— 480.6... 
 
 
 
 
 
 0.000102 
 
 480.6— 474.4... 
 
 
 
 
 
 0.0000148 
 0.0000126 
 0.0000118 
 
 0.0000842 
 
 474.4— 468.4... 
 
 
 
 
 
 0. 0000700 
 
 468.4— 461.7... 
 
 
 
 
 
 0. 0000667 
 
 
 
 
 
 
 
 In this table the absorption ratios are mean values of 
 several determinations made with solutions of different 
 concentrations. 
 
 Problem 6. The Determination of Nitrogen. 
 
 The determination of the amount of nitrogen in an 
 organic compound is a problem of the highest importance, 
 and fortunately one of no great difficulty. Many processes 
 have been in use for the purpose, some of the earlier ones 
 requiring elaborate apparatus. These will not be described 
 here. At the present time chemists apply the method of 
 Kjeldahl in the great majority of analyses, and this alone 
 will be explained. 
 
 The Kjeldahl Process. This. is founded on the follow- 
 ing facts. Nearly all organic compounds containing nitro- 
 gen, and probably all with which the physiologist has to deal, 
 are decomposed when heated with strong sulphuric acid. 
 If with the acid certain oxidizing agents are used the nitro- 
 gen becomes wholly converted into ammonia. As origin- 
 
166 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 ally carried out by Kjeldahl and others, the substance is 
 heated for an hour or more in a thin glass flask with sul- 
 phuric acid to a temperature near the boiling point of the 
 latter.' In this heating the organic substances decompose, 
 and the solution becomes nearly colorless. Then, while 
 still hot, powdered potassium permanganate is added in 
 small amount, until a permanent green color is obtained in 
 the oxidized liquid. It is then allowed to cool, diluted 
 with water, neutralized with pure sodium hydroxide in ex- 
 cess and then distilled. The ammonia formed is caught in 
 standard acid and measured as explained below. 
 
 The original process has been modified in several 
 directions, and mainly so as to shorten the time required 
 in oxidation. Kjeldahl himself suggested the use of strong 
 fuming acid and the addition of phosphoric anhydride. 
 Others have used certain metallic salts, and also mercury 
 as an addition, with the result of reducing the time of 
 digestion to less than an hour. More recently the process 
 has been modified by Gunning in a manner which consti- 
 tutes an improvement for most purposes. He found that 
 the oxidation is much more perfectly carried out if to the 
 sulphuric acid about half its weight of pure potassium sul- 
 phate is added, and that the subsequent addition of per- 
 manganate is then rendered unnecessary. Some few 
 nitrogenous bodies are not decomposed by this mixture, 
 but all that we have to consider here are completely oxi- 
 dized. 
 
 The process is conducted in the following manner : 
 Weigh out a gram or more of the substance to be analyzed. 
 If the substance is in moist condition or in solution, con- 
 centrate nearly to dryness, after addition of a little pure 
 sulphuric acid, in a platinum dish, or best in the flask in 
 which the digestion is afterward made. If the solution is 
 very weak it may be evaporated in platinum first, and then 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 167 
 
 transferred to the digestion flask for the final concentra- 
 tion. To the known amount of the nearly dry substance 
 in the digestion flask add 15 Cc. of pure strong sulphuric 
 acid and 10 Gm. of pure potassium sulphate. Place the 
 flask on a sand-bath or gauze, and heat with the Bunsen 
 burner, gently at first, and afterward to a high tempera- 
 ture, until the liquid ceases to froth and becomes colorless 
 or pale yellow. Round bottom, Bohemian flasks with 
 long necks are best for this purpose, and they should be 
 supported on the gauze in an inclined position to prevent 
 loss by spirting. 
 
 The heating may last from half an hour to two hours, 
 the time depending on the nature of the substance and its 
 amount. The liquid in the flask is allowed to cool, and 
 is then diluted with about 200 Cc. of water. The mixture 
 is poured into a distillation flask, and to it a few drops of 
 phenol phthalein solution are added. Then enough 50^ 
 per cent sodium hydroxide solution is added to more than 
 neutralize the sulphuric acid present and liberate the am- 
 monia. This alkali solution must itself be perfectly free 
 from ammonia. Add some small bits of ignited pumice 
 stone (about half a gram of fragments which will pass 
 through a sieve with sixteen meshes to the linear inch) to 
 the flask, connect with the condenser and distill, while 
 the further end of the condenser dips beneath the sur- 
 face of a measured volume of dilute standard sulphuric 
 acid to catch the ammonia. 
 
 The distillation apparatus for that purpose should have 
 the following construction : The flask should contain from 
 500 to 700 Cc, and should be closed with a rubber stopper 
 through which passes a glass tube with an internal diame- 
 ter of at least 12 Mm. This glass tube extends 5 Cm. or 
 more into the neck of the flask and is cut off diagonally. 
 Above the flask it should have a vertical length of about 30 
 
168 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 Cm., in the middle portion of which it is widened to a bulb 
 of about 3 Cm. diameter. Following the vertical part the 
 tube is bent to give a horizontal length of 15 to 20 Cm., 
 and then is bent down. This downward limb passes 
 ■through a rubber stopper into the wide part of the con- 
 densing tube of a short, upright Liebig, or a spiral con- 
 denser. The lower end of the condensing tube dips 
 beneath the surface of the weak acid which catches the 
 distilled ammonia. The glass tube which leads from the 
 distillation flask is continuous until the ^condenser is 
 reached. With this arrangement there is no danger of 
 losing ammonia or of carrying the fixed alkali over mechan- 
 ically. 
 
 As standard acid it is convenient to use one'tenth nor- 
 mal sulphuric acid, which is colored with a single drop of 
 dilute methyl orange solution, as indicator. The distilla- 
 tion is continued until about 150 to 200 Cc. has passed over 
 into the standard acid, which must still show a pink color. 
 The excess of acid is then titrated with weak standard am- 
 monia, and the amount of acid found subtracted from that 
 originally taken shows how much was required to combine 
 with the ammonia in the distillation. 17 parts of ammo- 
 nia, NH S , correspond to 49 parts of sulphuric acid, 
 H 3 S0 4 . For each 17 parts of ammonia found we calculate 
 14 parts of nitrogen, and thus learn the amount of this ele- 
 ment in the weight of substance originally taken for analy- 
 sis. As dry albuminous substances contain in the mean 
 about 16 per cent of nitrogen, and if we know that we are 
 dealing with a body of this class, we obtain the amount of. 
 albumin by multiplying the nitrogen found by the factor, 
 6.25. In illustration, if 50 Cc. of standard sulphuric acid, 
 with 4.9 Gm. to the liter, had been measured out in a cer- 
 tain experiment to catch the ammonia, and if at the end of 
 the distillation 18 Cc. of tenth normal ammonia solution is 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 169 
 
 found necessary to neutralize the excess of acid it shows 
 that the amount of ammonia distilled over is 32x1.7, or 
 54.4 Mg. This is equivalent to 44.8 Mg. of nitrogen in the 
 original substance, which multiplied by the factor, 6.25, 
 gives .280 Gm. as the corresponding amount of albu- 
 minoid. 
 
 It is not always possible to obtain sulphuric acid or 
 potassium sulphate absolutely free from ammonia. In this 
 case it is necessary to make a blank experiment, using the 
 acid mixture alone, and determine the amount of ammonia 
 which can be distilled from it in the usual way as de- 
 scribed. This amount can then be subtracted from that 
 found in the actual test. 
 
 The addition of pumice stone, above recommended, 
 prevents bumping in the distillation of the heavy liquid. 
 Various other substances have been recommended, but 
 this is probably the most satisfactory as with it the libera- 
 tion of vapor is uniform. The 150 or 200 Cc. of distillate 
 may be collected in an hour. 
 
 Problem 7. The Separation of the Proteids, as Illus- 
 trated by the Analysis of Meat Extract. 
 
 Some of the characteristic reactions by which the im- 
 portant proteid compounds may be separated or recognized 
 have been given in a former chapter. These, with others, 
 may now be employed in systematic order for the quanti- 
 tative analysis of a complex mixture such as we find in 
 products of digestion or in meat extract. A general 
 method to be followed in such cases has been given by 
 Stutzer, and this will be applied here, with slight modifica- 
 tions only. 
 
 The Analysis. If the substance for examination is in 
 dry, or nearly dry form weigh out 5 Gm.; if in paste form 
 
170 ELEMENTARY CHEMICAL PHYSIOLOGY. 
 
 take 10 Gm., and if a liquid take 25 Gm. Dissolve it in 
 about 150 Cc. of lukewarm water, and if a part remains in- 
 soluble, filter through a weighed Gooch crucible, wash the 
 residue with distilled water and make the filtrate up to 
 500 Cc. The residue may be dried slowly at a low tem- 
 perature and then at 105° C., and weighed, or while still 
 moist, it may be washed into a flask with a little water, 
 concentrated again and then analyzed for nitrogen accord- 
 ing to the Kjeldahl method. Calculate the nitrogen to 
 albumin, as explained in the last problem. 
 
 If the product contains fat this will appear as an oily 
 layer on the water in which it is dissolved, and after the 
 filtration will be found in the contents of the Gooch cruci- 
 ble. By drying the crucible, extracting with anhydrous 
 ether, evaporating and weighing the residue the fat is 
 determined. 
 
 We have now to examine the aqueous filtrate of 500 
 Cc, which may contain soluble albumin, albumose, peptone 
 and other compounds of nitrogen of less complexity. 
 Measure out 100 Cc. of this filtrate, add to it two or three 
 drops of acetic acid and heat to boiling on wire gauze. If 
 soluble albumin is present it appears as a coagulum, which 
 may be collected on the Gooch crucible, dried and weighed, 
 or it may be transferred to a digestion flask and be treated 
 by the Kjeldahl method for albumin. 
 
 The filtrate and washings from the soluble albumin are 
 concentrated to a small volume, about 10 Cc, and when 
 cold treated with 100 Cc. of a cold saturated solution of 
 pure ammonium sulphate. As has been explained this 
 reagent precipitates albumose, but not peptone. The pre- 
 cipitate is allowed to settle and is then collected on a 10 or 
 12 Cm. filter and thoroughly washed with saturated am- 
 monium sulphate solution. The filter paper used for this 
 purpose must be free from soluble nitrogen compounds. 
 Munktell's washed Swedish paper answers very well. 
 
ELEMENTARY CHEMICAL PHYSrOLOGY. 171 
 
 The albumose precipitate is next treated on the filter 
 with lukewarm water, the solution running through being 
 collected in a clean flask. Make the filtrate up to 250 Cc. 
 It contains the albumose and also some ammonium sul- 
 phate. In order to determine the albumose concentrate 
 150 Cc. of this filtrate to a small volume and find its nitro- 
 gen by the Kjeldahl method. A part of this nitrogen, 
 however, comes from the ammonium sulphate, and its 
 amount must be found and deducted. This may be done, 
 most conveniently, in the following manner. Take the 
 remaining 100 Cc. of the albumose filtrate, add to it a few 
 drops of hydrochloric acid and heat to boiling on gauze. 
 Add now a slight excess of solution of barium chloride and 
 boil some minutes longer. We obtain here a precipitate 
 of barium sulphate which corresponds, of course, to the 
 ammonium sulphate and consequently to the nitrogen of 
 its solution. This precipitate settles well and can be 
 readily collected on a Gooch crucible in the usual manner. 
 Weigh it and reduce the barium sulphate found to corre- 
 sponding nitrogen. Subtract this nitrogen, calculated for 
 150 Cc, from that found in the Kjeldahl test. The re- 
 mainder is that due to the albumose. To obtain the latter 
 multiply by the factor, 6.25, and calculate the amount for 
 the 250 Cc. of filtrate. This gives us now the albumose in 
 100 Cc. of our first filtrate, or in one-fifth of the substance 
 originally taken. 
 
 Of the important nutritious bodies found in meat 
 extract, peptone remains to be determined. No reagent is 
 known which precipitates this leaving the other proteids, 
 hence the following method has been suggested by Stutzer. 
 He finds that while peptone, albumose and albumin are 
 completely precipitated by phospho-tungstic acid and are 
 insoluble in excess, kreatin and kreatinin, precipitated at 
 first, dissolve when more of the reagent is added. Leucin 
 
172 ELEMENTARy CHEMICAL PHYSIOLOGY. 
 
 tyrosin, urea, and taurin, are not precipitated at all. Xan- 
 thin and hypoxanthin behave as do the proteids, that is, 
 they form with the reagent insoluble precipitates. They 
 are only slightly water soluble, however, and can be pres- 
 ent in meat extracts or similar products in but small pro- 
 portion. For our purpose their presence may be neg- 
 lected. Therefore, as appears from what has just been 
 said, if we precipitate peptone, albumose and albumin with 
 phospho-tungstic acid, determine the total nitrogen in the 
 precipitate, and from this subtract the nitrogen of albu- 
 mose and albumin found by other methods we have left the 
 peptone nitrogen, which, multiplied by 6.25 gives approxi- 
 mately the peptone. 
 
 To find the peptone in the case before us, we use the 
 aqueous filtrate obtained after filtering out the insoluble 
 albumin and other matters. Take 50 Cc. of this filtrate, 
 acidify it strongly with sulphuric acid and add to it an ex- 
 cess of a reagent made by dissolving 100 Gm. of crystallized 
 sodium tungstate and 25 Gm. of glacial phosphoric acid in 
 500 Cc. of water, to which afterward enough sulphuric acid 
 is added to give a strong acid reaction. By this treatment 
 the proteids, with traces of xanthin and hypoxanthin possi- 
 bly, precipitate, while other nitrogenous bodies are left in 
 solution. Allow the mixture to stand some hours and then 
 filter it through a nitrogen-free filter paper. Wash the pre- 
 cipitate with the reagent and finally with a little dilute 
 sulphuric acid. Allow it to drain and then transfer, with 
 the filter paper, to a digestion flask and treat with the 
 Kjeldahl oxidizing mixture in the usual manner. The ni- 
 trogen of albumin and albumose have been already de- 
 termined. We subtract this from the nitrogen of the last 
 determination and calculate the remainder to peptone by 
 multiplying by 6.25, as the percentage composition of pep- 
 tone is practically the same as that of the other proteids. 
 
ELEMENTARY CHEMICAL PHYSIOLOGY. 173 
 
 The water in the original sample may be determined by 
 weighing about 2 to 5 Gm. into a small platinum dish and 
 heating on a water-bath until constant weight is obtained. 
 The loss in weight represents the water. 
 
 The mineral matter or ash may be found by incinerat- 
 ing the above dry residue and heating it until it becomes 
 colorless. There is usually a small loss by volatilization 
 here. 
 
 By adding together the water, mineral matters and 
 proteids, and subtracting from 100 per cent, we have a 
 remainder which represents the percentage of nonproteid 
 nitrogen bodies. If fat is present it must be subtracted 
 too. 
 
 A further idea of the amount of these nonproteid 
 nitrogenous matters may be obtained by making a deter- 
 mination of total nitrogen in the original substance by the 
 Kjeldahl method. By subtracting from this total nitrogen 
 that of the proteids, found as just explained, we have the 
 nitrogen of the kreatin, xanthin and other bodies. This 
 multiplied by 3.12 gives, approximately, the amount of these 
 substances because they contain in the mean about 32 per 
 cent of nitrogen. 
 
Part II. 
 Urine Analysis. 
 
Chapter X. 
 
 OUTLINE OF TESTS. PRELI11INARY TESTS. 
 
 THE importance of an accurate knowledge of the bodies 
 excreted by the urine has long been recognized and 
 elaborate investigations have been carried out to determine 
 the nature and quantities of these substances, some of 
 which appear normally in health, while others are found 
 only during the progress of disease. 
 
 Experiment shows that normally certain products occur 
 in the urine in relatively large amounts, and give to it its 
 prominent characteristics, while of other products the 
 amounts present are so minute that their detection is a 
 matter of no little difficulty. 
 
 Certain grave disorders are accompanied by the appear- 
 ance of certain substances in the urine, and where the 
 chemical or microscopic tests for the latter are simple and 
 unquestionably correct we have at hand a convenient aid 
 to diagnosis. In many cases, however, it is true that we 
 are unable to trace the relation between small amounts of 
 substances occasionally appearing in urine and any specific 
 disorder or condition of the body. The detectioji of such 
 substances is naturally without value in diagnosis, at the 
 present time. 
 
 Yet it would be unwise to neglect the study of such 
 traces because, as medical science progresses, new relations 
 are from time to time brought to light which give value 
 to data which at one time may have been considered 
 wholly unimportant. Complete handbooks on the urine 
 give prominence to many topics which will not be touched 
 
178 
 
 THE ANALYSIS OF URINE. 
 
 upon in what follows because we are here concerned with 
 phenomena everywhere recognized as important and the 
 bearings of which, in the main at least, are understood. 
 
 In the practical analysis of urine such as is customary 
 for clinical purposes comparatively few tests are required 
 and little apparatus is necessary beyond that already used 
 for other examinations. Frequently a single test . is suffi- 
 cient to determine all the physician needs to know, for 
 instance, regarding the presence or absence of sugar or 
 albumin. 
 
 In the following pages those tests and processes will be 
 described which have been shown by experience, to be 
 amply sufficient for all practical requirements. Some of 
 these are qualitative, others quantitative and may be tabu- 
 lated as follows: 
 
 1. Observation of color and odor. 
 
 2. The reaction, whether acid or alkaline. 
 
 3. The tests for albumin. 
 
 4. The tests for sugar. 
 
 5. The tests for the characteristic biliary 
 
 acids and coloring matters. 
 
 6. The various tests for blood. 
 
 7. Tests for other coloring matters. 
 
 8. The examination of the sediment. 
 
 Qualita- 
 tive tests- 
 
 Quantita- 
 tive tests. 
 
 9. Determination of specific gravity. 
 
 10. 
 11. 
 12. 
 13. 
 14. 
 15. 
 
 " the amount of albumin. 
 " " " sugar. 
 
 " " " uric acid. 
 
 " " " urea. 
 
 " " of phosphates. 
 " " chlorides. 
 
 The above includes the usual and important tests. A few 
 
THE ANALYSIS OF URINE. 179 
 
 others will be given in the proper place, for instance, tests 
 for acetone and diacetic acid, which under circumstances 
 may have importance. 
 
 Normally, urine contains as its most important con- 
 stituents urea, sodium chloride, certain phosphates and 
 urates, and smaller amounts of other substances as hip- 
 puric acid, xanthin, krea.tinin, traces of phenols, etc. 
 
 Pathologically there may appear albumin, sugar, blood, 
 pus, bile pigments and acids, and a number of other 
 bodies insoluble, or of slight solubility, which usually ap- 
 pear as a sediment. 
 
 We turn now to an explanation of the various prelim- 
 inary tests employed. 
 
 Specific Gravity. 
 
 The density or specific gravity of the urine secreted in 
 twenty-four hours, varies in health between rather wide 
 limits, probably between 1.005 and 1.030. 1.020 may be 
 taken as about the mean value at 15° C. 
 
 The specific gravity depends primarily on the amounts 
 of liquid arid solid food taken, and on the loss of water 
 •from the skin by perspiration. When this loss is great 
 the specific gravity of the urine is correspondingly in- 
 creased, other things being equal. 
 
 In disease the density may be lowered below or in- 
 creased above the normal value. 
 
 For an absolutely exact determination of the density 
 the use of the pycnometer, Mohr-Westphal balance, or 
 other apparatus is necessary. But for our purpose the 
 urinometer, or density bulb, is sufficiently accurate. This 
 little instrument is shown in the following figure. The 
 urine to be tested is poured into a narrow jar, about one 
 Cm. wider than the bulb, and after the air bubbles have 
 .escaped, the urinometer is immersed in it. When it comes 
 
180 
 
 THE ANALYSIS OF URINE. 
 
 to rest the degree at which it stands is read off below the 
 surface. Usually the last two figures only of the den- 
 sity are marked on the stem, as 25 y instead of 1.025, and 
 these are often given as the density. 
 
 As the density of" urine de- 
 creases about one degree for an 
 increase in temperature of 3° 
 to 5° C. it is important that 
 the test be made at a definite 
 known temperature, as 15° or 
 25° C. Urinometers have usu- 
 ally been graduated to give the 
 correct reading at a tempera- 
 ture of 15.5° C. (60°F.). But 
 at the present time we have 
 them for the temperature of 25° 
 C. (11° F.), because this is with ' 
 us a more common house tem- 
 perature than the lower one. 
 It is convenient to have the 
 instrument indicate the correct 
 specific gravity without the ne- 
 cessity of cooling. The specific 
 gravity of urine varies approxi- 
 mately as does that of water, 
 with changes of temperature. 
 A table in the appendix shows 
 the rate for water, and by the 
 use of this a correction can be 
 made. 
 
 By noting the amount of 
 urine passed in twenty- four 
 
 hours, and the density of the mixed liquid, a rough deter- 
 mination of the solid matters contained in it can be made. 
 
 
 
 
 i 
 
 
 s 
 
 tl 
 
 
 
 MM 
 .ill 
 
 
 i 
 
 f Mgjji 
 
 
 1 
 
 III 
 
 
 
 
 
 J 
 
 wm 
 
 
 i 
 
 i 
 
 il 
 
 iff* 
 
 
 
 III 
 
 i 
 
 H. 
 
 li* 5 ! 
 
 I 
 
 
 
 Hi 
 
 
 FIG. 40. 
 
THE ANALYSIS OF URINE. 181 
 
 For this purpose it is simply necessary to multiply the last 
 two figures of the density by 2.33 (known as the coefficient 
 of Haeser), which gives the approximate number of grams 
 in a liter. By proportion the amount for the day can be 
 calculated from this. 
 
 For example, 1,400 Cc. of urine was passed, and its 
 density was found to be 1.024, 
 
 Then, 24x2.33=55.92, 
 and, 1,000 : 1,400 :: 55.92: x — 78.288. 
 
 This calculation is frequently of service. 
 
 As indicated above a variation in the specific gravity of 
 normal urine may be due to several causes, the most im- 
 portant of which are changes in the volume of water drunk 
 or the weight of nitrogenous food and salt digested. The 
 amount of urine excreted daily may be taken as 1,500 Cc. 
 in the mean. Assuming that fifteen grams daily is the 
 salt consumption and that it is all excreted with the urine 
 we have through this factor alone a specific gravity of 
 about LOO?. Assuming further that 150 grams of nitrog- 
 enous food (considered as pure albumin) is consumed 
 daily and that four-fifths of this amount is daily excreted 
 as urea, the weight of the latter in the urine would be forty- 
 three grams. This alone would produce a density of near- 
 ly 1.008 and give a percentage composition of 2.84. Com- 
 bined with the other substances in urine the relative effect 
 of the addition of urea would be greater. All of the solids 
 of the urine have a specific gravity greater than that of 
 water and their presence therefore adds to the specific 
 gravity of the excretion, but changes in the density, due to 
 changes in the amounts of uric acid, phosphates, sulphates, 
 etc., passed are of less importance because of the relatively 
 small quantities of these substances normally present. 
 
 If, with the food consumed normal, the water taken is 
 
182 THE ANALYSIS OF URINE. 
 
 small in amount, or if a large amount is lost from the skin 
 as perspiration then the density of the excreted urine must 
 be correspondingly higher. A large volume of water con- 
 sumed or little evaporation from the skin will give a urine 
 of lower density. It is plain, therefore, that great varia- 
 tions in the specific gravity of the urine may occur and 
 from perfectly normal causes. 
 
 In disease even greater variations may occur, one of the 
 most characteristic and important being that due to the 
 presence of sugar in diabetes mellitus. Here the density 
 may reach 1.040 or higher, while the volume of urine is 
 above 1,500 or 2,000 Cc. in the twenty-four hours. A high 
 specific gravity with large volume is always suspicious and 
 suggests presence of dextrose although occasionally it may 
 be due to presence of large doses of soluble salts taken into 
 the system as remedial agents. A low specific gravity with 
 small volume of urine must also call for investigation, as 
 this points to the absence of or marked decrease in the 
 normal constituents from some cause. A lower density is 
 observed in diseases where the elimination of urea is slow- 
 er because of hindered tissue changes, in conditions of 
 malnutrition in general, and in any disease involving the 
 structure of the liver itself. In acute yellow 'atrophy of the 
 liver, for instance, urea is much diminished, and the speci- 
 fic gravity low. 
 
 The diminution in excreted chlorides, with normal con- 
 sumption, in certain diseases is also a factor in causing low 
 specific gravity. This may follow when the salt consumed 
 is eliminated temporarily in various exudations or effusions 
 rather than by the normal channel. 
 
 Some of these indications will receive attention later 
 during the discussion of the tests for the common normal 
 and abnormal urine constituents. 
 
THE ANALYSIS OF UKINE. 183 
 
 Reaction. 
 
 In health the reaction of the mixed urine passed through 
 twenty-four hours is always acid. 
 
 This normal acid reaction is supposed to be due to the 
 presence of acid phosphates and to small amounts of uric 
 acid and to other free organic acids. The reaction can be 
 observed by the aid of sensitive litmus paper, but the abso- 
 lute amount of free acid is very small. 
 
 Occasionally urine is passed which gives the so-called 
 amphoteric reaction with litmus; that is it turns blue paper 
 red and red paper blue. It has not been found possible to 
 connect this phenomenon with certainty with any definite 
 pathological condition; it has, therefore, no special clinical 
 significance at the present time. Some hours after a hearty 
 meal an alkaline reaction is frequently obse"rved, giving 
 place soon to the usual acid condition. This alkaline reac- 
 tion may be due to the presence of small amounts of tri- 
 sodium phosphate formed during active digestion. The 
 administration of alkali carbonates, or of certain organic 
 salts, as malates, acetates, tartrates or citrates, which yield 
 carbonates by final decomposition, may also occasion an 
 alkaline condition. Sometime after it is voided urine 
 always becomes strongly alkaline in reaction, but this 
 change may be delayed for days or weeks even. It js 
 brought about by the decomposition of urea, which is 
 usually a result of bacterial action. In this decomposition 
 ammonium carbonate fs formed, the odor of which often 
 becomes very strong. Anything which prevents or im- 
 pedes the bacterial ^activity tends to maintain the ordinary 
 acid or neutral reaction. Salicylic acid, thymol, chloro- 
 form, volatile oils and other antiferments behave in this 
 manner, and are frequently added to specimens of urine to 
 preserve them for investigation. For the preservation of 100 
 Cc. of urine one-fourth of a Gm. of salicylic acid is enough. 
 
184 THE ANALYSIS UF URINE. 
 
 Sometimes the decomposition of the urea takes place 
 in the bladder, the voided urine having then a very marked 
 alkaline reaction and strong odor, usually. Such a change 
 may be brought about by the progress of disease, or may 
 be induced by the introduction of a dirty catheter into the 
 bladder. This carries the organism capable of splitting up 
 the urea, and the condition once established may be main- 
 tained for a long time. 
 
 Ammonium carbonate results from the reaction. This 
 may be distinguished from the fixed alkalies (hydroxide 
 or carbonate of sodium or potassium) by a very simple 
 rest. A piece of .sensitive red litmus paper immersed in 
 alkaline urine becomes blue. On drying the paper the 
 color due to the nonvolatile alkalies persists, while that of 
 ammonium carbonate disappears. The test has some 
 practical value as it is necessary to distinguish between 
 the alkalinity of urea fermentation and that of an excess of 
 fixed alkali occasionally present. For these tests only 
 fresh sensitive paper can be safely used. The conversion 
 of urea into ammonium carbonate is represented by this 
 equation: 
 
 CON 2 H 4 + 2 H s O = (NH 4 ) 2 C0 3 
 
 In highly colored urines it is not always easy to observe 
 the reaction with litmus paper. In this case the method 
 described under the blood tests in an earlier chapter may 
 be applied. This consists in immersing small discs of 
 plaster of Paris in neutral litmus solution and then drying 
 them. A few drops of urine are placed on a disc and 
 allowed to remain some minutes. The urine is then washed 
 off leaving a bluish or reddish spot indicating the reaction. 
 
 Odor. 
 
 The odor of urine is not easily described, as in health it 
 is sui generis and characteristic. Normal urine contains 
 
THE ANALYSIS OF URINE. 185 
 
 traces of complex aromatic bodies the exact nature of 
 which cannot in all cases be given. These substances are 
 more abundant after a vegetable than after an animal diet, 
 and are especially noteworthy in the urine of persons 
 whose food contains such vegetables as cabbage, radishes, 
 parsnips, asparagus or the spices. It is well known that 
 certain substances given as remedies give rise to distinct 
 odors in the urine. The administration of turpentine im- 
 parts to the urine an odor of violets. 
 
 As the odor so largely depends on the nature of the 
 food it may be much modified even in health, and in dis- 
 ease may be characteristically changed. The ammoniacal 
 odor of urea decomposition in the bladder has been referred 
 to, and the peculiar sweetish odor of diabetic urine has long 
 been noticed. 
 
 But it must be remembered that many strong odors 
 may be developed in the urine soon after passage by the 
 action of ferments other than the micrococcus urea which 
 yields ammonium carbonate. In some cases these give rise 
 to what may be called a putrefactive odor. 
 
 Color. 
 
 The color of urine is described as straw yellow. Many 
 causes, however, may produce a change in this shade, 
 leaving the urine still normal. * As can be readily seen the 
 color is closely dependent on concentration and must, 
 therefore, vary with the amount of liquid taken into the 
 stomach. 
 
 Certain foods from the vegetable kingdom possess 
 characteristic coloring matters which pass, more or less 
 changed, into the urine. As long as the latter is acid the 
 presence of these may not be noticed, but with a change of 
 reaction a change of color may follow, usually to reddish. 
 
 Santonin imparts a yellowish color to urine, reddened 
 
186 THE ANALYSIS OF URINE. 
 
 by alkalies. In pathological conditions the color of urine 
 is often characteristic and of great importance in diagnosis. 
 The presence of blood, for instance, is indicated by a more 
 or less sharp shade of red, bile by a peculiar greenish 
 brown, especially noticeable in froth produced on shaking. 
 The urine of diabetes mellitus is generally very pale, while 
 the urine of fevers is usually highly colored not only from 
 the diminution of water but also from the presence of ab- 
 normal coloring matters. Different shades are produced 
 by the presence of altered blood and bile constituents, 
 which will be referred • to later. The real color of the 
 urine is often obscured by loss of transparency due to pre- 
 cipitation. Normal urine is generally perfectly transparent 
 when, passed, but sometimes cloudy from presence of a 
 suspended precipitate of mucus or phosphates. On be- 
 coming alkaline a precipitation of earthy phosphates usu- 
 ally follows. A precipitate of urates, without change of 
 reaction, often takes place by simply lowering the temper- 
 ature of the urine. This precipitate, however, disappears 
 on the application of a slight heat to the urine, and leaves 
 the latter clear for examination of color. 
 
 Clinically, the color indications of greatest importance 
 are those due to the presence of derivatives from the blood 
 or bile. These may be unaltered elements of the blood or 
 bile or decomposition products of their essential coloring 
 matters. 
 
 In a following section on the tests for abnormal coloring 
 matters in urine this question will be again taken up. 
 
Chapter XI. 
 
 THE TESTS FOR ALBUHINS. 
 
 A LBUMINOUS bodies do not occur in normal urine ex- 
 **■ cept, perhaps, in mere traces. Numerous investiga- 
 tions have been published on this subject, and while some 
 of the recent ones would seem to show the probability of a 
 physiological albuminuria, others, seemingly as thorough, 
 lead to quite the opposite conclusion. 
 
 Temporarily, it is true, albumin may be found in the 
 urine of healthy individuals, as after the consumption of 
 large quantities of egg albumin, or after the action of some 
 cause producing a sudden alteration of the blood pressure, 
 but the amounts found in such cases are too small, and 
 their occurrence too rare to permit them to be classed as 
 anything but accidental. It is certain that the presence of 
 any appreciable amount of albumin in the urine and the 
 persistence of the same must be looked upon as a patho- 
 logical phenomenon and one of the greatest importance to 
 the physician. 
 
 Albumins may appear in urine from several sources, 
 most frequently, probably, because of some structural 
 change in the tubules of the kidneys which permits a filtra- 
 tion from the blood. But this is not always the case as 
 they may appear from sources in no wa'y dependent on 
 renal disorder, from lesions of the ureters, bladder or 
 urethra, for instance, in which case blood or pus may be 
 present. Ordinary serum albumin is the usual, but not the 
 only proteid body which may appear in urine. Half a 
 dozen or more modifications have been described as Occur- 
 
188 THE ANALYSIS OF URINE. 
 
 ring under different circumstances, but the evidence for 
 some of these appears to be of doubtful character. Cer- 
 tain forms are not readily detected or identified. In what 
 follows tests will be given for those proteid bodies which 
 can be detected with certainty and whose presence has 
 some definite clinical importance. 
 
 i. Serum Albumin. 
 
 The presence of serum albumin in the urine is a char- 
 acteristic of what is ordinarily termed albuminuria. As 
 intimated above albuminous bodies may appear in the 
 urine from different sources. The presence of serum albu- 
 min suggests {a), a functional or structural disorder of some 
 part of the essential tissue of the kidney, in which case we 
 have renal albuminuria or true albuminuria, or (b~), a lesion 
 of some part of' the urinary tract below the kidney, in which 
 case we have what is called false or accidental albuminuria. 
 Renal albuminuria is the condition appearing in Bright's 
 disease or acute parenchymatous nephritis and in other patho- 
 logical conditions in which a change of the diffusion mem- 
 brane is involved. It is also frequently induced by derange- 
 ments in the circulation due to heart diseases, high fevers, 
 etc., which in turn may react and give rise to a derange- 
 ment of the kidney itself. That is to say, the causes pro- 
 ducing certain febrile conditions may extend to the struc- 
 ture of the renal filtering apparatus and so alter its condi- 
 tion that the passage of albumin is no longer hindered but 
 becomes continuous. 
 
 Under all such circumstances the albumin passing 
 through the kidney is generally accompanied by some- 
 thing which suggests its origin. There may be here an 
 excessive amount of the epithelial lining of the tubules, or 
 plugs of coagulated albumin, mucus, or of the wax-like, 
 partially degenerated albumin known as lardacein, all in 
 
THE ANALYSIS OF URINE. 189 
 
 the form of "casts" of the uriniferous tubules. These 
 may' be readily seen and recognized by the microscope. 
 
 As intimated above, false or accidental albuminuria can 
 originate from several causes and in general is a condition 
 of far less clinical importance than the other. It is usually 
 possible to determine by a few examinations the real 
 nature of the disorder by aid of the facts just mentioned. 
 
 Because of the very great importance of the subject to 
 the physician, much attention has been given to the ques- 
 tion of albumin tests, and the number of reactions pro- 
 posed for its detection reach, possibly, the hundreds. 
 Many of these are of such extreme delicacy and so easy of 
 execution that to make a choice of a few is by no means a 
 simple matter. 
 
 The best of them depend on the fact that the soluble 
 serum albumin, which finds its way into the urine, can be 
 coagulated and made visible as white flocculi, or as a white 
 cloud when present in small quantity. Of the various 
 methods of producing this coagulation, only those will be 
 mentioned which are most characteristic, and practically 
 the most useful. 
 
 Qualitative Coagulation Tests. 
 
 Coagulation by Heat. When a sample of urine 
 is boiled a precipitate usually forms. This in 
 most cases consists of earthy phosphates, and is often 
 sufficient to conceal a precipitation of albumin possibly 
 present. If now to the boiled sample about one-tenth 
 its volume of strong nitric acid be added, the precipi- 
 tated phosphates will disappear, while the albumin will 
 remain coagulated. It is necessary to add as much nitric 
 acid as is here indicated, because a small amount may some- 
 times dissolve coagulated albumin, forming soluble acid-albu- 
 min. This acid albumin is broken up on the addition of 
 more acid. 
 
190 THE ANALYSIS OF URINE. 
 
 Even when boiling does not throw down a precipitate, 
 the addition of nitric acid cannot be omitted, as under cer- 
 tain circumstances the heating may produce a soluble com- 
 bination between alkalies present and albumin, which is 
 stable. Nitric acid in sufficient quantity will break up this 
 combination, and bring about coagulation. 
 
 Under most circumstances this heat test, as outlined, is 
 sufficient, and the possibility of making a mistake is very 
 small. It has been shown in an earlier section of the book 
 that small amounts of albumin combine readily with weak 
 acids and alkalies, forming soluble and stable combinations 
 known as acid-albumin and alkali- albumin. 
 
 If the urine has a neutral or alkaline reaction to begin 
 with a small amount of alkali-albumin would escape detec- 
 tion by heating alone. On addition of just the proper 
 amount of acetic acid to neutralize the alkali, the applica- 
 tion of heat will cause a coagulation, but a slight excess of 
 this acid might convert the alkali- albumin into acid-albumin, 
 equally hard to precipitate. Traces of nitric acid, and in a 
 marked degree hydrochloric acid, behave in the same 
 manner, but the addition of larger amounts of nitric acid is 
 free from this objection because in proper amount this acid 
 is able to decompose both acid and alkali-albumin. 
 
 When taken for examination, urine is frequently cloudy 
 from the presence of precipitated urates or earthy phos- 
 phates. Heat is sufficient to dissipate the cloud if due to 
 the urates, but the phosphate cloud is rendered heavier. It 
 is always a good plan to carefully filter the urine, if in the 
 least degree turbid, before undertaking the test. 
 
 With old samples of urine which have undergone the 
 urea fermentation and have become alkaline, the test by 
 heat and subsequent addition of acid is not always satisfac- 
 tory or convenient. In such cases it is best to proceed at 
 once to a method which disposes of the excess of alkali at 
 the start and in such a manner as to cause no confusion. 
 
THE ANALYSIS OF URINE. 191 
 
 Coagulation by Nitric Acid. As indicated above, 
 nitric acid can coagulate albumin, and this test is frequently 
 employed without previous boiling. When applied to fresh 
 urine the test may be made in this manner. 
 
 Several Cc. of the strong acid are warmed in a test- 
 tube, and over this is carefully poured an equal volume of 
 uiine, so as to overlie without mixing. If albumin is pres- 
 ent a white ring appears at the surface between the two 
 liquids. When the urine contains an excess of coloring 
 matter the ring is variously tinted. 
 
 If urine is poured over cold acid, a precipitate may 
 appear which is not albumin. This can happen when the 
 urine is highly charged with urea, in which case crystal- 
 line nitrate of urea will separate out, or where urates are 
 abundantly present, in which case the ring will consist of 
 very fine crystals of uric acid, or acid urates. Both of 
 these precipitates are dissipated by heat, and if the nitric 
 acid is previously warmed, they cannot appear. It is bet- 
 ter to make the test as just suggested than to use cold acid, 
 and then try to warm a ring formed, as this would cause 
 an admixture of the liquids sufficient to obscure a slight 
 amount of albumin. 
 
 It is sometimes recommended to pour the urine in a 
 test-tube, and by means of a pipette, or dropping-tube, 
 allow the acid to flow under it. This is an excellent 
 method of performing the test, but the acid should be 
 slightly warm as before. If only a trace of albumin is 
 present the ring will not appear immediately, but only 
 after standing. It is well, therefore, in doubtful cases, to 
 set the tube aside for twelve hours and then observe it. If 
 a ring is now found it should be very gently and carefully 
 warmed to determine its behavior toward heat, because on 
 standing in the cold a ring of urates might appear. 
 
 When this test is applied to old, cloudy or alkaline 
 
192 THE ANALYSIS OF URINE. 
 
 urine it should be preceded by this preliminary prepara- 
 tion : 
 
 Boil the urine with half its volume of 10 per cent potas- 
 sium hydroxide. solution and filter. This will usually give 
 a bright, clear liquid, but if not add two drops of the 
 "magnesia mixture" employed in qualitative analysis and 
 described in the appendix, boil and filter again. The fil- 
 trate is now suitable for testing. 
 
 The action of the reagents is this : The strong alkali 
 forms a bulky precipitate of the earthy phosphates present 
 which usually settle and leave the supernatant liquid clear. 
 The amount of alkali taken is sufficient to prevent the 
 coagulation and precipitation of the albumin on boiling, 
 while it serves also to expel ammonia which may be pres- 
 ent. If the first filtrate is not perfectly clear the addition 
 of the magnesia mixture accomplishes this by making a 
 new precipitate of phosphates in traces which now leaves 
 it bright. 
 
 With the clear filtrate the tests by addition of nitric 
 acid may now be carried out. It must be remembered, 
 however, that as the urine is now strongly alkaline, a rela- 
 tively large volume of the strong nitric acid must be em- 
 ployed. 
 
 The text-books abound in minute descriptions concern- 
 ing the best methods of conducting this comparatively 
 simple test. The few sources of error which may mislead 
 will now be pointed out. It is, of course, understood that 
 these appear only in the search for small amounts of albu- 
 min, that is for amounts less than one-tenth of one per cent 
 by weight. For greater quantities the reactions, even 
 when not conducted with extreme care, are usually sharp. 
 
 When urine is poured over nitric acid or when the acid 
 is introduced under the urine a layer of some kind always 
 appears at the junction of the two liquids. The problem is 
 
THE ANALYSIS OF URINE. 193 
 
 to decide what this is. The peculiar appearance of a rela- 
 tively large amount of coagulated albumin is so characteris- 
 tic that any one who has ever seen it will recognize it again. 
 But a faint cloud or haziness is, at the start, somewhat con- 
 fusing. A colored layer or ring, which is very common, 
 must not be mistaken for a precipitate or cloud. The nor- 
 mal urine coloring matters may produce a highly colored 
 ring, and the bands with biliary colors are even deeper. 
 But these color bands or zones are transparent which can 
 be determined by holding the test-tube in the proper light. 
 Urine very highly charged with urea may give a crystal- 
 line precipitate of urea nitrate. This is a very unusual 
 reaction, and the precipitate may be quite easily recognized 
 through the form and size of the crystals, which are large 
 flat plates readily seen by the naked eye or by a common 
 magnifying lens. If a urine suspected, to contain such an 
 excess of urea be diluted with an equal volume of water be- 
 fore testing the crystals will not appear. Besides, they do 
 not appear when the liquids are warm. The finely granular 
 precipitate of acid urates or hydrated uric acid appears 
 only in a cold liquid, therefore cannot be present to mis- 
 lead if the test is conducted as directed. 
 
 If the special tests indicate the presence of unusually 
 large quantities of urates the urine may be diluted with an 
 equal volume of water before adding the nitric acid. It oc- 
 casionally happens that a yellowish white cloud or band 
 appears in this test which is not due to albumin or uric 
 acid. Such a cloud may be caused by the presence in the 
 urine of bodies taken into the system as remedies and 
 which are excreted in but slightly changed form. Deriva- 
 tives of turpentine and certain resinous bodies are spec- 
 ially liable to behave in this manner. After the use of 
 copaiba balsam nitric acid throws out from the urine insol- 
 uble resin acids, which are not dissipated by heat. The 
 
194 THE ANALYSIS OF URINE. 
 
 precipitate formed by such acids dissolves readily in alco- 
 hol and can thus be readily distinguished from albumin 
 which is not soluble. It must be remembered that while 
 pure albumin precipitated by nitric acid is white, that 
 thrown down from urine may be more or less colored from 
 the presence of normal or abnormal coloring matters. 
 
 Attention must be called to a method of conducting the 
 nitric acid test which is frequently employed, but which for 
 small quantities of albumin is very untrustworthy. This 
 method consists in mixing about equal volumes of strong 
 acid and urine and boiling. This is open to the grave ob ; 
 jection that by it the albumin sought may be decomposed 
 and so lost from view. Nitric acid is a very strong oxidiz- 
 ing agent, and albumin a substance easily decomposed. 
 Traces may therefore be lost even by a very short boiling, 
 as may be readily -determined b]' the student by a few 
 experiments with weak albumin solutions. 
 
 Tanret's Mercuric- Potassium Iodide Test. A so- 
 lution of this compound precipitates albumin from acidi- 
 fied urine and is on the whole an extremely delicate re- 
 agent. Among the general albumin tests of Chapter IV. 
 it was shown that many of these bodies are thrown out 
 from their solutions in the form of complex basic com- 
 pounds by addition of salts of certain heavy metals. Solu- 
 ble salts of mercury, lead and copper give characteristic 
 reactions. 
 
 Tanret prepared a well-known and popular test solu- 
 tion in the following manner:: 
 
 Dissolve 33.12 Gm. of pure potassium iodide in about 
 200 Cc. of distilled water. Add 13.54 Gm. of powdered 
 mercuric chloride and warm until, with sufficient stirring, 
 the red precipitate of mercuric iodide disappears, leaving a 
 rclear, slightly yellowish solution. Dilute this with distilled 
 
THE ANALYSIS OF URINE. 195 
 
 water to about 800 Cc, and add 100 Cc. of pure, strong 
 acetic acid. Allow to stand over night if not absolutely 
 clear, and decant from any small precipitate which may 
 have settled out. Dilute then to one liter with distilled 
 water. This solution contains the two salts in the propor- 
 tion of 4 KI to Hg Cl 2 . 
 
 The test with the reagent so prepared is carried out as 
 follows : 
 
 Filter the urine to make it perfectly clear, and add 
 enough acetic acid to give it a good acid reaction. To 
 about ten or fifteen Cc. in a test-tube add a very little of 
 the reagent, a drop at a time, from a pipette or dropping- 
 tube. In all not more than five drops should be added, as 
 this is sufficient to give a strong precipitate if albumin is 
 present. The precipitate is flocculent, and appears as a 
 white cloud or streak, as the first drop of the heavy mercu- 
 ric solution settles and mixes with the urine. As each fol- 
 lowing drop mingles with the urine the hazy cloud grows 
 to a precipitate in case the urine contains more than a 
 mere trace of albumin. 
 
 The delicacy of the reaction is remarkable. It is said 
 that by it one part of albumin in one hundred thousand 
 parts of urine may be detected. This, however, is proba- 
 bly excessive. One part in twenty-five thousand in a series 
 of tests is nearer the average result. It has been claimed 
 that where the solution is to be kept a long time it is 
 best prepared without the addition of the acetic acid, as 
 this is liable to produce slight decomposition in time. 
 It is likely that the danger of this has been overestimated. 
 
 In any event, unless the urine is fresh and slightly acid 
 the addition to it of acetic acid should not be neglected. 
 The use of the acid is said by some writers to be unneces- 
 sary, but it has the advantage of disclosing the presence of 
 any quantity of mucin which might interfere with the test. 
 
196 THE ANALYSIS OF URINE. 
 
 If the acid throws out a cloud of mucin it should be filtered 
 off and then the reagent added. 
 
 While this is an exceedingly valuable test certain pre- 
 cautions must be observed in its use. The mercuric solu- 
 tion is similar to one used as a test for alkaloids, and in 
 fact precipitates many of these bodies. Quinine and other 
 alkaloids given as remedies and excreted by the urine 
 would therefore be shown by the test. 
 
 Alcohol dissolves these precipitates, however, but is 
 without solvent action on that formed by albumin. Uric 
 acid and urates give precipitates with the reagent if pres- 
 ent in large amount, but such precipitates can be avoided 
 by diluting the urine before testing, or if formed can be 
 dissipated by slight heat. 
 
 Mistaking mucin for albumin can be avoided as shown 
 above. Small amounts of peptones are precipitated by the 
 reagent, but the coagulum disappears by application of 
 heat. 
 
 The Ferrocyanide Test. A reagent of great del- 
 icacy is potassium ferrocyanide in presence of acetic acid. 
 It shows not only serum albumin, but globulin and per- 
 haps other proteids. It does not give a reaction with 
 peptones. 
 
 The test is applied in this manner. The urine must be 
 made as clear and bright as possible by filtration and then 
 strongly acidulated with acetic acid. If a precipitate or 
 cloudiness from mucin appears now filter again and to the 
 filtrate add four or five drops of Afresh clear solution of the 
 ferrocyanide. With even traces of albumin this gives a 
 flocculent yellowish white precipitate. 
 
 One of the advantages claimed for this test is that it 
 gives no reaction with the vegetable alkaloids and there- 
 fore can be used as a check upon, some of the others, the 
 
THE ANALYSIS OF URINE. 197 
 
 mercuric-potassium iodide test for instance. The precipi- 
 tate formed, although flocculent, is very fine and can be 
 observed therefore only in clear solution. 
 
 A modified form of the test is the following: Mix five 
 drops of the ferrocyanide solution with five Cc. of 30 per 
 cent acetic acid. Pour this carefully over an equal volume 
 of clear urine in a test-tube and allow to stand a short time. 
 A white zone at the junction of the liquids shows the albu- 
 
 The Picric Acid Test. Picric acid solutions, pure, 
 or combined with citric, acetic or other acids, have long 
 been used as reagents for the detection of traces of albumin 
 in urine. In its simplest form the test liquid employed is 
 a saturated aqueous solution of pure picric acid. It gives 
 a very characteristic yellowish flocculent precipitate with 
 even traces of albumin. Another solution frequently em- 
 ployed contains in one liter 10 Gm. of picric acid and 20 
 Gm. of citric acid. It must be made clear by filtration, if 
 necessary, and is applied to clear urine in small quantity 
 by means of a pipette, so as to show a cloudiness as the 
 liquids mingle. The reagent is added gradually to the 
 urine and in all not more than one-half the volume of the 
 latter for a qualitative test. 
 
 The real practical value of this test is, in some quarters, 
 still in dispute. It is certainly very delicate, but as it gives 
 precipitates with peptones, alkaloids, urates, mucin, kreati- 
 nin, and perhaps other bodies, the first result observed is 
 subject to revision. The precipitates formed with these 
 substances and picric acid are dissipated by heat, but there 
 is risk of getting the temperature too high, in which case 
 other precipitates are liable to be formed, especially with 
 the plain solution without the citric acid. A urine which 
 yields a precipitate of earthy phosphates by warming will 
 
198 THE ANALYSIS OF URINE. 
 
 give it at the same temperature in the presence of picric 
 acid. It seems to be true, however, that with citric acid 
 added the interference from phosphates is eliminated, and 
 there remains only mucin as a disturbing element. The 
 danger here is not great, and it is likely that in all cases it 
 can be avoided by adding the citric acid first, filtering if 
 necessary, and then adding the picric acid. 
 
 Other Tests. A number of other tests are in use which 
 show very minute traces of albumin. But they seem to possess 
 no advantages over those enumerated above. One of these 
 depends on the precipitation of albumin by phenol and 
 acetic acid, in another picric and hydrochloric acids are 
 used, in a third a strong solution of common salt and 
 hydrochloric acid, and so on. But, practically, no one will 
 find it necessary to go beyond the five tests given. Indeed, 
 two are by most authorities generally thought sufficient, 
 viz., the heat test and the nitric acid test. What cannot 
 be shown by these reactions is so minute that for practical 
 purposes it can be neglected usually. 
 
 The Amount of Albumin. 
 
 It is not alone sufficient that we are able to detect the 
 presence of albumin in urine; we often need to know its 
 amount to determine the practical value of a line of treat- 
 ment pursued from day to day. To be of the greatest pos- 
 sible service, a method must be so easy of execution that 
 approximately correct results may be obtained by it by the 
 use of simple apparatus and in a short time. Several 
 methods are known by which the amount of albumin in 
 urine can be found. One of these, and the best, may be 
 called the gravimetric method, as by it the albumin is pre- 
 cipitated, collected and weighed. In another, the albumin 
 is precipitated and its volume measured, while in a third 
 
THE ANALYSIS OF URINE. 199 
 
 process the amount of albumin is estimated from the degree 
 of turbidity caused by its precipitation in the urine. 
 
 Only the first and second methods will be described. 
 The first is employed in exact investigations, and the sec- 
 ond in clinical estimations. 
 
 The Gravimetric Method. If the qualitative test has 
 shown only a small amount of albumin 100 Cc. of the urine 
 should be measured out into a beaker for precipitation. If 
 the qualitative test has given a strong indication 50 or 25 Cc. 
 should be taken and diluted to 100. Enough dilute acetic 
 acid is added to the urine to give it a faint acid reaction, 
 after which it is brought up to a temperature of 80° or 90° 
 C. on the water-bath, being stirred, meanwhile, frequently. 
 From time to time the beaker is held up against the light* 
 so that the operator may determine whether the coagula- 
 tion is complete or not. A satisfactory coagulation is 
 shown by the precipitation of the albumin in large flakes, 
 leaving the surrounding liquid nearly clear. If this is not 
 the case a little more acid should be added, but very care- 
 fully, and in all but three or four drops, unless the urine 
 was strongly alkaline to begin with. 
 
 When the reaction seems to be complete on the water- 
 bath the beaker is placed on gauze and the contents 
 brought to boiling. The precipitate is then allowed to 
 settle. Meanwhile, a small filter of well-washed filter paper 
 is dried and weighed in a weighing tube. It is plaited and 
 put in a funnel and then the albumin precipitate is col- 
 lected on it. The precipitate is washed with hot distilled 
 water until it gives no chlorine reaction to the wash water, 
 then with absolute alcohol, and finally with ether. The 
 funnel with contents is placed in an air oven and dried at 
 120° C. The filter is transferred to the weighing tube, and 
 when cold is weighed. The increase in weight gives the 
 
200 
 
 THE ANALYSIS OF URINE. 
 
 albumin. Instead of collecting on paper, a much better 
 plan is to collect on a Gooch funnel of asbestos, when it 
 can be had, which simplifies the test, besides 
 adding to its accuracy. This method con- 
 sumes a good deal of time, but gives results 
 which are near the truth when it is properly 
 conducted. The best results are obtained 
 when the weight of the precipitate does not 
 exceed .3 Gm. 
 
 Volume Method. One of the simplest of 
 these is the one proposed by Esbach. In this 
 a special tube is used, called the Esbach albu- 
 minometer, and a special solution or reagent 
 made by dissolving 10 Gm. of pure picric acid 
 and 20 Gm. of pure citric acid in a liter of 
 distilled water. The solution must be filtered 
 if it is not perfectly clear, and is the same as 
 the one used for the qualitative test. The 
 principle involved in the employment of the 
 method is this. The precipitate of albumin 
 and picric acid settles in coherent manner 
 and in a compact volume proportional to its 
 weight provided certain definite amounts of 
 the reagent and urine are taken. The albu- 
 minometer, or measuring tube used, resembles 
 a test-tube of heavy glass about six inches 
 long, and is graduated, empirically, to show 
 how much urine and reagent to take and the 
 amount of albumin obtained expressed in 
 grams per liter, or tenths of one per cent. The 
 annexed cut shows the tube and its markings. 
 
 The test is carried out in this manner. Urine is poured 
 in, to the mark U, and then the reagent, described above, 
 
 in 
 
 FIG. 41. 
 
THE ANALYSIS OF URINE. 201 
 
 to the mark R. The tube is closed with the thumb and 
 tipped backward and forward eight or ten times until the 
 liquids are thoroughly mixed. It is then closed with a rub- 
 ber stopper and allowed to stand in a perpendicular posi- 
 tion twenty-four hours. This will give the precipitate time 
 to settle thoroughly, after which the amount can be read off 
 on the scale. The results are accurate enough for clinical 
 purposes and by practice can be made to agree moderately 
 well with those found by the gravimetric method. 
 
 But to obtain this close agreement a number of precau- 
 tions musit be observed. The volume of the precipitate is 
 in a marked degree variable with the temperature, and with 
 the time given it for subsidence. The empirical gradua- 
 tion is based on the supposition that the test will be made 
 at a temperature of 15° to 20° C, and that the reading will 
 be made at the end of twenty-four hours. If the reading 
 is delayed to two or three days the volume of the precipi- 
 tate will be found much smaller. At the present time 
 small centrifugal machines are rapidly coming into use to 
 settle urine sediments. Some of these are operated by the 
 Edison electric light current, and give the rotating tubes a 
 high velocity. Where these machines are employed to 
 settle the picric acid-albumin precipitate, the volume of 
 the latter may be rendered abnormally small and the read- 
 ing, therefore, prove erroneous. 
 
 The volume of the precipitate will depend here, not 
 only on time and temperature, but also on velocity of rota- 
 tion and the effect of this factor must be determined for 
 each instrument before it can be accurately used. 
 
 In any case in applying this test the urine should not 
 be highly concentrated. The best results are obtained with 
 urine of low specific gravity and with the albumin not over 
 .3 of one per cent. If a test shows an amount greatly in 
 excess of this the urine should be diluted with a known 
 
202 THE ANALYSIS OJ> URINE. 
 
 proportion of water and tested again. On long standing 
 in the cold a yellowish red precipitate of uric acid some- 
 times settles out. This need not mislead the student, as 
 its color and general appearance are quite distinct from 
 those of albumin. 
 
 The precipitates of albumin from urine by whatever 
 means obtained are bulky and lead to the impression that 
 the amount present is much larger than is actually the 
 case. It was at one time customary to speak of 25/30 and 
 50 per cent of albumin, these numbers representing the 
 apparent volume of the precipitate in the test-tube. When 
 these light precipitates are collected, properly dried and 
 weighed a very different volume is obtained. A urine with 
 one per cent of albumin contains an unusually large 
 amount, and in any case seldom more than 10 or 15 grams 
 of albumin occur in the day's urine. Between 1 gram and 
 5 grams are more common amounts, even in cases of acute 
 albuminuria. 
 
 2. Serum Globulin. 
 
 This is an albuminous body resembling the serum al- 
 bumin in many respects and often, perhaps generally, asso- 
 ciated with it. 
 
 At the present time globulin in the urine has the same 
 clinical significance as serum albumin. Although similar 
 to each other in most points there are several characteristic 
 differences which are taken advantage of as qualitative 
 tests. The general relations of albumin and globulin were 
 pointed out in a former chapter of this work and a number 
 of reactions given for each. Not all of the tests for globu- 
 lin which we find given in the books are suitable for use in 
 the examination of urine, however, as we are here limited 
 by the presence of other substances. Most of the reac- 
 tions given above for albumin apply equally well to globu- 
 
THE ANALYSIS OF URINE. 303 
 
 lin and it is only within recent years that attempts have 
 been made to detect one in the presence of the other. 
 Among the methods applicable in the examination of urine 
 the following may be given as most characteristic: 
 
 Qualitative Tests. 
 
 Dilution Test. Globulin is insoluble in water, but 
 soluble in dilute salt solutions. Hence its solubility in 
 urine. If the latter is diluted until the specific gravity is 
 1.002 or 1.003 the globulin may separate out. At any rate 
 the addition of a few drops of dilute acetic acid will pro- 
 duce the desired result. A current of carbon dioxide 
 passed into the diluted liquid for several hours accom- 
 plishes the same end. 
 
 The test may be modified in this manner. Filter the 
 urine if it is not perfectly clear, and then pour it, drop by 
 drop, into a tall, narrow beaker of distilled water. If glob- 
 ulin is present it is thrown out as a white cloud, which 
 shows as the drops pass down through and mix with the 
 lighter, clear water. The globulin ma}" afterward be con- 
 firmed by adding a small amount of salt solution which 
 will cause the precipitate to disappear. 
 
 Sulphate Test. Globulin may also be detected by 
 reason of its insolubility in strong salt solutions. To this 
 end treat the urine with enough ammonia water to give an 
 alkaline reaction. This precipitates phosphates and some- 
 times other salts, which after a time are filtered off, leaving 
 a clear liquid. To this add an equal volume of saturated 
 solution of ammonium sulphate which, in presence of 
 globulin, produces a white flocculent precipitate. 
 
 Magnesium sulphate is frequently used for the same pur- 
 pose, and under some circumstances may possibly be pref- 
 erable. 
 
304 THE ANALYSIS OF URINE. 
 
 In this test there is some danger of confounding albu- 
 mose with globulin, as the former is also precipitated by 
 ammonium sulphate. But the danger of confusion is small 
 if the conditions given are adhered to, i. e., mix equal vol- 
 umes of the clear filtered urine and saturated ammonium 
 sulphate solution. For the precipitation of albumose 
 higher concentration is necessary, as will be further ex- 
 plained below. 
 
 3. Albumose or Memialbumose. 
 
 We have here a representative of an important class of 
 proteid compounds which are derived from the albumins 
 proper. It has already been explained that in the diges- 
 tion of native albumins the albumoses appear as one of the 
 stages, and are found therefore, among the products of pepr 
 tic and pancreatic action in the body. It is likely that 
 normally albumoses are always converted into peptones 
 before leaving the alimentary tract. 
 
 The special significance of the bodies called albumose 
 in the urine is by no means clear. While certainly a patho- 
 logical appearance it has not yet been found possible to 
 definitely connect it with any one disease. Its presence 
 has been reported in the urine in several cases of osteo- 
 malacia but it appears by no means to be a constant ac- 
 companiment. Observers have called attention to its 
 occurrence during the progress of several other diseases, 
 but without being able to point out any definite relation. 
 
 Qualitative Tests. 
 The recognition of albumose is not a matter of diffi- 
 culty, as it is distinguished from the other proteid com- 
 pounds sometimes found in the urine by several well 
 marked characteristics. It is not coagulated by heat or by 
 the addition of acetic or warm nitric acid, and is very solu- 
 
THE ANALYSIS OF URINE. 305 
 
 ble in hot water. It is much less soluble in cold water, 
 but the presence of small amounts of salts seems to in- 
 crease its solubility here in marked degree. 
 
 In presence of albumin or globulin it can be found by 
 the following process unless it is in very small amount. 
 The urine is saturated with pure sodium chloride, and then 
 enough acetic acid is added to give a strong acid reaction. 
 The mixture is boiled and filtered hot. This treatment 
 throws out both albumin and globulin, but does not pre- 
 cipitate any albumose present. The latter would therefore 
 be found in the clear filtrate and sometimes in amount 
 sufficient to precipitate as this cools. The filtrate should 
 therefore be allowed to remain at rest until quite cool. If 
 much albumose is present it will appear as a white cloud. 
 Sometimes, however, it will be necessary to concentrate 
 the filtrate before looking for this reaction, and this is done 
 by evaporating slowly on the water-bath to half the vol- 
 ume. On now cooling salt will quickly settle out, while 
 the albumose precipitates later in flocculent form. 
 
 Another test is this : Separate the albumin and globu- 
 lin by boiling with a small amount of acetic acid without 
 the salt. Filter while warm and concentrate the filtrate to 
 a volume of one-third. Allow to cool thoroughly and add 
 a large excess of saturated solution of ammonium sulphate. 
 This gives a white flocculent precipitate of albumose, if 
 present. The precipitate can be collected on a filter and 
 washed with the saturated ammonium sulphate solution 
 and then dissolved in a little distilled water, poured on the 
 filter. This filtrate gives tests with picric acid, potassium 
 ferrocyanide and acetic acid and other albumin reagents. 
 
 If the original urine shows no reactions for albumin or 
 globulin the albumose tests can be applied directly after 
 concentration. The method by precipitation by means of 
 picric acid gives good results. The biuret test is also 
 delicate and clear. 
 
306 THE ANALYSIS OF URINE. 
 
 The Amount of Albumose. 
 
 This can be best found by collecting the ammonium 
 sulphate precipitate on a filter and washing it thoroughly 
 with more of the same saturated solution to remove urea 
 and other nitrogen compounds. The neck of the funnel, 
 with the moist precipitate, is then placed in the neck of a 
 quarter-liter flask and distilled water poured on to dissolve 
 the precipitate. From 50 to 100 Cc. will be amply sufficient 
 to dissolve and wash the whole of the albumose into the 
 flask below. The funnel is removed and distilled water 
 added to bring the volume up to exactly 250 Cc. Of this 
 volume 150 Cc. is transferred to a beaker, acidified with 
 pure sulphuric acid and evaporated slowly down to a vol- 
 ume of 5 to 10 Cc. This is poured into a Kjeldahl diges- 
 tion flask, the beaker being rinsed out with 10 Cc. of pure, 
 strong sulphuric acid. Finally 10 Cc. more of the acid is 
 added to the flask and then 10 grams of pure potassium 
 sulphate. The flask is heated on the hot plate and the 
 operation of determining nitrogen completed as described 
 in a former chapter where the Kjeldahl method is ex- 
 plained. 
 
 The remaining 100 Cc. in the measuring flask is used 
 for the determination of the amount of ammonium sul- 
 phate in the solution. This must be done in order to sub- 
 tract the nitrogen corresponding from the result of the 
 Kjeldahl distillation. The determination is best made by 
 precipitating the sulphate with barium chloride in the 
 usual manner. 
 
 4. Peptones. 
 
 As has been explained, peptones are proteid compounds 
 formed from native albumins, globulins, fibrin, etc. in the 
 digestive process. In this respect they are related closely 
 to the albumoses, both differing from the other proteids in 
 
THE ANALYSIS 01- URINE. 207 
 
 important respects. But peptones have further character- 
 istic properties by which they are differentiated in turn 
 from the albumoses. 
 
 Peptones may occur in the urine as a result of various 
 abnormal conditions of the body ; but their appearance 
 does not depend, like that of albumin, on changes in the 
 circulation or on pathological conditions of the kidney. 
 Their clinical significance, therefore, is very different from 
 that of albumin or globulin. 
 
 In cases of phosphorus poisoning the urine has fre- 
 quently been found to contain peptones, but their presence 
 can usually be connected with the disintegration of pus 
 somewhere in the body. The peptone substances are, 
 therefore, frequently, or perhaps generally, found in the 
 urine in cases of purulent meningitis, purulent pleurisy, 
 in the termination of pneumonia by resolution, and in gen- 
 eral under circumstances in which products of suppura- 
 tion can find their way into the circulation to be eliminated 
 afterward by the kidneys. 
 
 Peptones have been reported in erysipelas, pulmonary 
 tuberculosis, acute articular rheumatism, carcinoma of the 
 gastro-intestinal canal, catarrhal jaundice, and in numer- 
 ous other disorders. The condition in which the urine 
 contains peptones as a result of the breaking up of puru- 
 lent products is spoken of as pyogenic peptonuria, in contra- 
 distinction to that in which there is no indication of the 
 existence of a suppurative process. It is claimed that in 
 Certain cancerous conditions of the stomach and intestine 
 the peptones of digestion may find their way into the 
 circulation. 
 
 Normally, peptones do not exist in the blood in more 
 than traces, and during absorption from the healthy sur- 
 faces of the alimentary tract a change into albumin, seems 
 to take place. It is held by some writers that if taken up 
 
208 THE ANALYSIS OF URINE. 
 
 from an unhealthy surface (ulcerated) this conversion 
 may not take place and peptone unchanged enters the cir- 
 culation to disappear finally by way of the kidneys and 
 through other channels. 
 
 It has been shown also that peptones are a normal con- 
 stituent of the urine of women in the puerperal state and 
 their occurrence has been pointed out under still other con-, 
 ditions not connected with a suppurative process. How- 
 ever, in the great majority of cases in which their existence 
 is shown the connection with the latter is clear, and the 
 detection of these substances becomes important as an aid 
 to diagnosis. 
 
 Some of the reactions already referred to under "Chem- 
 ical Physiology" can be applied to the urine, and addi- 
 tional ones are also available. The following tests may 
 be applied: 
 
 Qualitative Tests. 
 
 Biuret Test. Peptones give this test as do other forms 
 of albumin, but the final color is more nearly red than with 
 the latter. 
 
 The direct treatment of the urine with strong alkali and 
 the copper sulphate solution is seldom sufficient, it being 
 usually necessary to subject it to a preliminary treatment 
 to remove substances which interfere with the reaction. A 
 large amount of peptone substance unmixed with albumin 
 or globulin may give a very characteristic color, but in a 
 highly colored urine this may be unsatisfactory and it is 
 therefore safest to apply first a purifying process. The 
 nature of the preliminary treatment will depend on the 
 presence or absence of albumin or globulin. First, sup- 
 posing these bodies absent we may proceed as in the fol- 
 lowing: 
 
 Hofmeister's Tests. In order to prove the presence 
 of peptone in urine at least half a liter must be taken and 
 
THE ANALYSIS OF URINE. 209 
 
 treated with neutral acetate of lead in amount sufficient to 
 produce a heavy flocculent precipitate, which is separated 
 by filtration. An excess of the lead must not be used, but 
 the solution is added carefully, a few drops at a time, giving 
 the liquid meanwhile opportunity to settle so that a fresh 
 precipitate can be seen after the addition of more of the 
 reagent. By using proper care the right point can be 
 found when addition of the acetate must cease. 
 
 Supposing now that we have a clear filtrate and that no 
 albumin is present we next add to the filtrate a little hydro- 
 chloric acid and then a solution of phospho-tungsticacid in 
 hydrochloric acid as long as a precipitate forms. If peptone 
 is present it is contained in this precipitate which must be 
 separated immediately by filtration, and washed on the 
 filter with dilute sulphuric acid (.5 per cent) until this 
 passes through without color. The moist precipitate is then 
 transferred to a small dish and mixed with a slight excess 
 of barium carbonate or with enough crystalline barium hy- 
 droxide to give a slight alkaline reaction after thorough 
 stirring. A little water is added and the whole is heated on 
 the water- bath about ten minutes and filtered. The filtrate 
 is then tested for peptone by the biuret test, or by the picric 
 acid solution. 
 
 If albumin is present in the original urine it cannot be 
 completely removed by lead acetate as just explained. The 
 reaction with acetic acid and potassium ferrocyanide will 
 usually show it in the filtrate from the lead and it must be 
 removed as follows, this being accomplished by precipita- 
 ting it with ferric oxide. Add to the 500 Cc. of filtrate a. 
 small amount of sodium acetate solution and then some 
 ferric chloride, .after which the urine is made neutral with 
 sodium hydroxide and boiled. The iron should be com- 
 pletely precipitated, carrying with it the albumin. The 
 solution is filtered, allowed to cool and tested for albumin. 
 
210 THE ANALYSIS OF URINE. 
 
 If free from this it is ready for the peptone test, beginning 
 with the addition of hydrochloric acid. If albumin is still 
 present, add a little more iron or alkali, as experiment will 
 decide, and boil again. 
 
 The phospho-tungstic acid solution is made up by vari- 
 ous formulas, but as a reagent for urine the following method 
 is recommended. A solution of pure sodium tungstate is 
 made of about twenty per cent strength. To this is added 
 either glacial phosphoric acid or syrupy phosphoric acid 
 and the mixture boiled, the acid being added in sufficient 
 amount to give a strong acid reaction. With glacial phos- 
 phoric acid the proportion should be about one part to four 
 of the tungstate. The boiled mixture is allowed to cool 
 thoroughly and to it is added about one-fifth its volume of 
 strong hydrochloric acid. Allow the mixture to stand and 
 pour off the clear liquid from any precipitate which may 
 settle out. The complex phospho-tungstic acid is one of 
 the few reagents which completely precipitate peptones 
 and other albuminous bodies. In this case it is applied 
 after the other albumins are thrown out by other means and 
 serves to take the peptone away from coloring and equally 
 objectionable substances. 
 
 Negative Tests. Peptones are not precipitated by 
 heat or by the addition of hydrochloric, sulphuric, nitric or 
 acetic acid. The reaction with potassium ferrocyanide 
 and acetic acid, characteristic for the other albumins, is not 
 given by the peptones. 
 
 They differ from the albumoses in not being thrown 
 down by excess of ammonium sulphate, from which it fol- 
 lows that the test below can sometimes be" applied for the 
 detection of peptones in presence of albumin or albumose. 
 
 Precipitate 50 to 100 Cc. of urine, acidulated with a few 
 drops of acetic acid, by boiling. Filter, concentrate the 
 
THE ANALYSIS OF URINE. 211 
 
 filtrate to a volume of 5 Cc. allow to cool and add 50 Cc. of 
 cold saturated solution of ammonium sulphate and then 
 some of the pure crystals so that the whole liquid is com- 
 pletely saturated. Then filter and to the filtrate apply the 
 biuret test with a large excess of alkali and trace only of 
 copper sulphate, or the phospho-tungstic acid test. 
 
 If the latter test is used the filtrate from the albumose 
 should be diluted with two volumes of distilled water. 
 The solution of phospho-tungstic acid gives a precipitate 
 with normal urine, and also with the undiluted ammonium 
 sulphate filtrate. 
 
 But reduced with water as directed no precipitate of 
 the ordinary urinary constituents appears. A slight opal- 
 escence only may result, while in presence of even traces 
 of peptones there is a marked precipitate. The reaction 
 between phospho-tungstic acid and peptone solutions is 
 one of extraordinary delicacy, so that traces show even 
 after the above treatment. 
 
 5. Mucin. 
 
 In small amount mucin is probably present in all nor- 
 mal urines, in the case of women, coming especially from 
 the vagina. In moderate amount it has, therefore, no 
 pathological significance. If coming from the urinary tract 
 in more than these traces it usually indicates an irritated 
 condition or catarrh of the passages and then has clinical 
 interest. 
 
 Urine containing mucin in large amount is turbid when 
 passed; with a smaller amount it may be clear at first but on 
 standing deposits a cloud which settles nearly to the bot- 
 tom of the vessel and there floats in loose form, instead of 
 compact as with other sediments. This cloud does not 
 clear up by the addition of acetic acid or dilute nitric acid. 
 
 In clear urine containing mucin a flocculent, hazy pre- 
 
213 7W£ ANALYSIS OF URINE. 
 
 cipitate is formed by the addition of acid. The test is best 
 made by pouring some acetic acid into a test-tube and then 
 carefully an equal volume of urine so as to mix the two 
 liquids as little as possible. A mucin cloud appears in 
 the urine layer above the zone of contact of the liquids. 
 An albumin cloud makes its appearance lower, or at the 
 zone. In testing for mucin in presence of albumin the 
 main portion of the latter should be precipitated first by 
 boiling and filtering. The mucin test can be applied to 
 the cold filtrate by addition of acetic acid. 
 
 Mucin as well as albumin is precipitated from urine by 
 the addition of an excess of strong alcohol, (three volumes 
 to one). After some hours the precipitate may be collected 
 on a filter and washed with alcohol. It is then washed 
 with warm water which dissolves the mucin. This may be 
 recognized in the aqueous solution by the addition of acetic 
 acid. 
 
 Small amounts of mucin are so frequently mistaken for 
 traces of albumin that attention must be paid to the 
 methods of distinguishing between them. From what has 
 been said it will be understood that the cloud which ap- 
 pears as a diffused haze in testing for albumin by an ex- 
 cess of acetic acid or a very small trace of nitric acid may 
 be due to mucin and not to albumin as frequently assumed 
 by mistake. A proper excess of nitric acid redissolves 
 mucin, but not albumin in the cold. 
 
Chapter XII. 
 
 THE TESTS FOR SUGAR. 
 
 ^~\N the question of the occurrence of sugar in the urine 
 ^^ avast amount has been written. At one time, indeed 
 until within quite recent years, it was generally assumed 
 that normal urine contains no sugar or carbohydrate of any 
 kind. But present methods of research seem to throw 
 doubt on the truth of this view. It is not possible to sep- 
 arate small traces of sugar from a complex liquid like the 
 urine so that the body separated may be recognized by its 
 sensible properties. On the contrary we must depend on 
 the results of certain reactions given by sugar solutions 
 and in many instances by other organic bodies, and it is on 
 the proper interpretation of these reactions that the author- 
 ities differ. Some of these reactions for traces will be 
 explained below. In this place it suffices to say that the 
 leading physiological chemists of the present time are near- 
 ly unanimous in holding that traces of the sugar known as 
 dextrose exist normally in urine, in other words, that there 
 may be such a condition as physiological glycosuria as dis- 
 tinguished from the well-known pathological condition 
 characterized by the presence of relatively large amounts 
 of sugar in the urine and named diabetes mellitus. 
 
 The amount of sugar believed to be normally present is 
 very small and cannot be recognized by the first three or 
 four tests given below. An amount of sugar in the urine 
 sufficient to have clinical importance is readily recognized 
 by many tests. 
 
 The characteristics of urine in true diabetes are these. 
 
214 THE ANALYSIS OF URINE. 
 
 It has a specific gravity higher than normal, usually be- 
 tween 1.030 and 1.040, and this with a greatly increased 
 quantity. A high specific gravity with small volume, it has 
 been shown, need have no special clinical importance as 
 such a condition can result from many causes outside of 
 disease. Diabetic urine is usually light in color and prone 
 to speedy decomposition by fermentation. The amount of 
 sugar which can be present in advanced stages of diabetes 
 mellitus may be very large. It is saidthatas much as 1,000 
 grams of dextrose has been passed with the urine in one 
 day in extreme cases. But the amount usually coming 
 under the observation of the practitioner is far below this, 
 10 to 100 grams being much more common amounts. In 
 typical diabetes the percentage amount of the normal urine 
 constituents is usually greatly diminished because of the 
 great dilution with water, but the actual amount excreted in 
 twenty-four hours may be increased. 
 
 It is well known that sugar may temporarily occur in 
 the urine from a variety of causes. It has been found after 
 the absorption of several poisons, and in cases of carbon 
 monoxide poisoning; also in the course of certain diseases. 
 
 The amounts present in these circumstances are usual- 
 ly small, and disappear with other" symptoms of the dis- 
 order. The continued presence of considerable quantities 
 of sugar is characteristic of only one disease, i. e., the dia- 
 betes mellitus. This fact should be borne in mind in the 
 practical examination of urine and tests should be repeated 
 from time to time, unless the other clinical evidence is suf- 
 ficient to immediately confirm the indication of the chemi- 
 cal test. 
 
 Qualitative Tests. 
 
 The tests for sugar in urine depend on several distinct 
 general reactions. The most' common of these reactions is 
 that due to the oxygen absorbing power of alkaline dex- 
 
THE ANALYSIS OF URINE. 215 
 
 trose solutions. The absorption of oxygen may give rise 
 to a solution of characteristic color and odor as in the first 
 one of the tests given below, or to certain precipitates 
 formed by the abstraction of oxygen from metallic com- 
 binations in the test solutions employed. Some of these 
 points have been already referred to in the first part of the 
 book. The sugar tests which are most commonly em- 
 ployed in urine examination are the following : 
 
 Moore's Test. This depends on the reaction between 
 grape sugar and strong alkali solutions. When a solution 
 of .sugar or diabetic urine is mixed, without heating, with a 
 solution of sodium or potassium hydroxide, no change is at 
 first apparent unless the amount of sugar present is large 
 or the alkali very strong. But on application of heat, even 
 with weak sugar solutions, a yellow color soon appears 
 which grows darker, becoming yellowish brown, brown, and 
 finally almost black, while an odor of caramel is quite 
 apparent. The strong alkali-sugar solution absorbs at- 
 mospheric oxygen, giving rise to a number of products 
 among which lactic acid, formic acid, pyrocatechin and 
 others have been recognized. The brown color is due to 
 other unknown decomposition products. 
 
 This is a good reaction for all but traces of sugar, as 
 the intense dark brown color and strong odor are not given 
 by other substances liable to be present in urine. 
 
 But traces of sugar cannot be recognized by this test 
 with certainty, as the color of normal urine even is dark- 
 ened to some extent by the action of alkalies. 
 
 Urine containing much mucin becomes perceptibly 
 darker when heated with sodium, potassium or calcium 
 hydroxide solutions. 
 
 The Trommer Test. This is one of the oldest and 
 best known of the tests for the recognition of sugar in 
 
216 THE ANALYSIS OF URINE. 
 
 urine, and has been referred to before. It is performed by 
 adding to the urine an equal volume of ten per cent solu- 
 tion of sodium or potassium hydroxide and then a very few 
 drops (three or four to begin with) of dilute solution of 
 copper sulphate. 
 
 Solutions of alkali and copper sulphate alone give a 
 blue precipitate of copper hydroxide, but in presence of 
 sugars and certain other bodies a deep blue solution, and not 
 a precipitate is formed. Therefore, if the urine tested 
 contains sugar the first indication is a more or less blue 
 solution, stable for some time in the cold. On standing, 
 however, the liquid turns greenish, and finally deposits a 
 a yellow precipitate. This change takes place immediately 
 on application of heat, the greenish colored precipitate 
 turning yellow, and finally red by boiling. Copper sub- 
 oxide precipitates, and this is the second and character- 
 istic stage of the Trommer reaction. 
 
 Several substances can give the first stage, but dex- 
 trose is the only body liable to be present in the urine 
 which can give a good indication in the second. . 
 
 The test must, however, be used with certain precau- 
 tions. Albumin, if present, must be coagulated and 
 filtered off. The amount of copper sulphate used must be 
 small, because if only a trace of sugar is present and much 
 copper is used the latter will give a blue precipitate which 
 does not redissolve, and which turns black on boiling, thus 
 obscuring a sugar reaction which maybe given at the same 
 time. 
 
 In adding the copper sulphate it is best to pour into 
 the test-tube containing the urine and alkali, first about 
 three drops of a five per cent solution. If this appears to 
 give a yellow color on boiling, which does not turn black 
 more should be added, and this continued until a yellow 
 or red precipitate is formed. A black precipitate on boil- 
 
THE ANALYSIS OF URINE. 217 
 
 ing shows that too much copper has been added, and that 
 probably sugar is absent. 
 
 The active body in producing the reaction is copper 
 hydroxide, but this must be in solution to act as a good 
 oxidizing agent with sugar ; and the test, therefore, be- 
 comes uncertain or unsatisfactory if so much copper is 
 added that the hydroxide formed cannot be dissolved by 
 the sugar which may be present. In doubtful cases it 
 becomes necessary to make several trials before the right 
 proportion between urine, alkali and copper solution is 
 found. In the solution, on completion of the reaction, sev- 
 eral oxidation products of sugar are found, among which 
 are formic acid, oxalic acid, tartronic acid, etc. But the 
 complete reaction is obscure. In order to avoid the indi- 
 cated uncertainty of the Trommer test when used for 
 small amounts of sugar the next one was proposed. 
 
 The Fehling Solution Test. Fehling suggested the 
 use of a solution containing along with the copper sul- 
 phate and alkali a tartrate to dissolve the copper hydroxide 
 formed by the first two. Many substances besides sugars, 
 referred to in the last paragraph, have the power of dis- 
 solving copper hydroxide with a deep blue color. Among 
 these may be mentioned tartaric acid and the tartrates, 
 glycerol, mannitol and others of less value. 
 
 A solution prepared by mixing certain quantities of al- 
 kali, copper sulphate and either one of these bodies with 
 water in definite proportions remains perfectly clear when 
 boiled. But if a trace of dextrose (or several other sugars) 
 is present the usual yellow precipitate forms. The prep- 
 aration of Fehling solution proper is given in the appendix 
 and its general use is explained in Chapter II. Here it 
 suffices to indicate its special applications as a urine test. 
 The great advantage which this solution has over the 
 
218 THE ANALYSIS OF URINE. 
 
 Trommer test is found in the fact that it may always be 
 used safely in excess. With only a trace of sugar there is 
 no danger that the copper will precipitate as black hy- 
 drated oxide. In performing the test a few cubic centi- 
 meters of the Fehling solution (4 or 5) are poured into a 
 test-tube, diluted with an equal volume of water and boiled. 
 The solution must remain clear. Then the urine is poured 
 in, at first about half a cubic centimeter, and the mixture 
 boiled. If sugar is present in amount above one-tenth of 
 one per cent it should show with the volume of urine taken. 
 For smaller amounts of sugar more urine must be added, 
 and the mixture boiled again. 
 
 When normal urine is heated with Fehling solution, a 
 greenish flocculent precipitate usually makes its appearance. 
 This has no significance as it is due to the phosphates nor- 
 mally present which come down when the reaction is made 
 alkaline. Many urines produce a clear dark green solution 
 when heated with the Fehling solution. This is a partial 
 reduction reaction and like the other has no special im- 
 portance as urines free from sugar give it. At other times 
 urines free from sugar yield an almost colorless mixture 
 when boiled with the Fehling solution. These peculiar 
 reduction effects are due to the presence of uric acid, 
 kreatin, kreatinin, pyrocatechin and several other sub- 
 stances and are generally characterized by discharge of the 
 deep blue color of the solution without precipitation of the 
 copper suboxide. Certain substances taken as remedies 
 give rise to products in the urine which exert a similar ac- 
 tion. Occasionally, however, the amount of uric acid is so 
 large that the reduction is accompanied by actual precipi- 
 tation of the copper as red oxide. This fact is of interest 
 as it makes the test, at times, somewhat uncertain, 
 but it is a very simple matter to determine whether or 
 not a great excess of uric acid is present, as will be pointed 
 
THE ANALYSIS OF URINE. 219 
 
 out later. The liability to error in the Trommer test from 
 these causes is less than in the Fehling test, but notwith- 
 standing this the latter must still be regarded as the better 
 test practically, because of its great convenience and 
 the sharpness of the reaction with even traces of sugar. 
 The ingredients of the Fehling test are best kept in separ- 
 ate bottles closed with rubber stoppers. A very conveni- 
 ent arrangement is explained in the following paragraph. 
 
 Two bottles, each holding about 200 Cc, are fitted with 
 perforated rubber stoppers. Through the opening in each 
 stopper the stem of a 2 Cc. pipette with very short tip is 
 passed, and left in such a position that when the bottles 
 are half filled the bulbs and stems to the mark will be 
 covered with the liquid. One bottle contains the standard 
 copper sulphate solution, the other the mixture of alkali 
 and tartrate solution. The rubber stoppers should be 
 covered with vaseline so that they will permit the pipette 
 stems to slide easily in the perforations, and also close the 
 bottles perfectly. When the stoppers are inserted the 
 pipettes should stand full to the mark, ready for use. 
 
 On withdrawing the stoppers with forefinger closing the 
 pipettes, exactly two Cc. of each liquid can be taken out 
 without delay, and on mixing in a test-tube yield the 
 Fehling solution, fresh and ready for use, directly, or after 
 dilution with distilled water, as thought necessary. As the 
 solutions are used the pipette stems are pushed farther 
 through the stoppers so as to leave the marks always at the 
 surface of the liquids. The solutions may be kept in this 
 manner for years, and their use is not attended with any 
 inconvenience. The open ends of the pipette stems should 
 be kept closed with small rubber caps, or a bit of soft paraf- 
 fine wax. The mixed Fehling liquid does not keep well 
 unless prepared with certain unusual precautions, and 
 therefore several other single solutions have been sug- 
 gested, as described in the next paragraph. 
 
220 THE ANALYSIS OF URINE. 
 
 Other Copper Solutions. The original Fehling solu- 
 tion has been modified in various ways. Most of these modi- 
 fications consist in mere changes in the proportions of the 
 ingredients dissolved. Two, however, may be considered 
 as fundamentally different- 
 
 Loewe (1870) recommended a solution made by dissolv- 
 ing copper sulphate in water, adding solution of sodium 
 hydroxide and then glycerol. For certain purposes copper 
 hydroxide was found to possess advantages over the sul- 
 phate. The preparation of the Loewe solutions is described 
 in the appendix. The claim was made by Loewe that the 
 addition of glycerol prevents the spontaneous decomposi- 
 tion of the blue solution, which may, therefore, be kept 
 mixed. While this is not absolutely correct it is true that 
 the glycerol solutions keep much/better than the mixed tar- 
 trate-alkali-copper solutions as usually made, and have 
 therefore, found favor with some physicians. 
 
 Schmiedeberg (1886) described a solution containing in 
 one liter 34.63 Gm. of crystallized copper- sulphate, 16 
 Gm. of mannitol and 480 Cc. of sodium hydroxide solution 
 of 1.145 Sp. Gr. This solution is easily prepared and has 
 also the advantage of permanence. 
 
 Both the Loewe and the Schmiedeberg solutions have 
 suffered slight alterations, without, however, being im- 
 proved. 
 
 The second suggestion of Loewe, i. e., to use copper 
 hydroxide instead of sulphate, has not been generally fol- 
 lowed, but it certainly has in some cases decided advan- 
 tages. The final reaction in all these tests is the same as 
 with the Trommer or Fehling test. 
 
 The Bismuth Test. Boettger found (1856) that in 
 presence of alkali bismuth subnitrate is reduced to the 
 metallic condition by the action of dextrose in hot solution. 
 
THE ANALYSIS OF URINE. , 221 
 
 As a urine test he recommended to make it strongly alka- 
 line with sodium carbonate, and then add a very small 
 amount, what can be held on the point of a penknife, pf 
 the pure bismuth subnitrate. On boiling the mixture the 
 insoluble bismuth compound, which settles to the bottom, 
 turns dark if sugar is present. 
 
 The test is at present carried out by adding to the 
 urine in a test-tube an equal volume of 10 per cent solution 
 of sodium or potassium hydroxide, and then the .subnitrate. 
 Boiling gives the reaction as before. In absence of sugar 
 (or albumin) the bismuth compound remains white. 
 
 In performing this test only a very small amount of the 
 subnitrate should be taken. This is absolutely necessary 
 in the detection of traces of sugar. In this case the reduc- 
 tion is but slight, and not much black powder of bismuth 
 or its oxide can be formed. If a great excess of the white 
 subnitrate is taken it may be sufficient to completely ob- 
 scure the reduction product. It is frequently well to use 
 not more than four or five milligrams of the subnitrate. 
 
 The black precipitate formed was at one time supposed 
 to be finely divided metallic bismuth. Later investigations 
 seem to show that it consists essentially of lower oxides of 
 bismuth. This test has certain advantages over the cop- 
 per tests. It is easily made, and with materials every- 
 where obtainable in condition of sufficient purity. Further- 
 more, the reaction is not given with uric acid, which it will 
 be remembered may act on the Fehling solution if exces- 
 sive. 
 
 Albumin, however, interferes with the test, as it gives, 
 also, a black precipitate when boiled with alkali and the 
 bismuth subnitrate. In this case the albumin gives up 
 sulphur and forms bismuth sulphide. 
 
 If albumin is present in a urine it should be coagulated 
 and filtered out before trying the bismuth test. Bruecke 
 
222 • THE ANALYSIS OF URINE. 
 
 recommends to coagulate by means of a solution of potas- 
 sium bismuth iodide, the excess of bismuth serving to com- 
 plete the sugar test. The reagent for this purpose is made 
 by dissolving freshly precipitated bismuth subnitrate in a 
 hot solution of potassium iodide by the aid of some hydro- 
 chloric acid. This is the solution previously recommended 
 by Fron for the precipitation of alkaloids, and is made by 
 dissolving 1 Gm. of potassium iodide in 20 Cc of water to 
 which after heating 1.5 Gm. of the bismuth subnitrate and 
 1 Cc. of pure strong hydrochloric acid are added. The 
 mixture must be kept hot until all is dissolved, resulting in 
 an orange red solution. 
 
 This reagent precipitates albumin, but as it is rendered 
 turbid by water the amount of acid necessary to prevent this 
 for a given volume must be ascertained -before it can be 
 used with urine. This can be determined by adding a lit- 
 tle of it (a few drops) to some water in a test-tube, and 
 then dilute hydrochloric acid until the precipitate just dis- 
 appears. The test proper is then made by taking the same 
 quantity of urine and adding the same amount of acid and 
 the reagent. 
 
 Albumin and other disturbing substances precipitate, 
 and can be filtered off. The clear filtrate should not be 
 made turbid by acid or the reagent. It is then made 
 strongly alkaline with potassium or sodium hydroxide and 
 then boiled. In presence of sugar a black precipitate is 
 formed as before. 
 
 After adding the reagent it is necessary to wait several 
 minutes for a possible precipitate to form and settle. The 
 addition of alkali to the nitrate produces a bulky white pre- 
 cipitate of bismuth hydroxide which is readily reduced at 
 the boiling temperature by sugar present. If only traces of 
 sugar are present the boiling must be long continued to ob- 
 tain the black precipitate. What was said above about the 
 
THE ANALYSIS OF URINE. 223 
 
 danger of obscuring this precipitate by the white bismuth 
 compounds obtains also here. 
 
 When only a small amount of sugar is suspected it is 
 best to allow the bismuth hydroxide precipitate to partially 
 settle, and then to pour off the supernatant alkaline urine 
 with a little of it. In this manner the amount of the bis- 
 muth compound which finally enters into the test is so 
 small that it should all be reduced by even a trace of sugar, 
 on subsequent boiling. When carefully performed this 
 modification of the original BSttger test is a practically 
 good one. It is not as sensitive as the Fehling test but 
 shows traces of sugar of clinical importance. 
 
 The Phenylhydrazine Test. In this test a reaction 
 discovered a few 'years ago has been applied by v. Jaksch 
 to the examination of urine. Add to about 10 Cc. of urine 
 .2 Gm. of phenylhydrazine chloride and a slightly greater 
 amount of sodium acetate. Warm the mixture gently, and 
 if solution does not take place add half the volume of water 
 and heat half an hour on the water-bath. Then cool the 
 test-tube by placing it in cold water and allow it to stand. 
 If sugar is present a yellow precipitate settles out, which 
 consists of minute needles generally arranged in rosettes, 
 visible under the microscope. Albumin does not obscure 
 this test, but if much is present it is best to coagulate it as 
 well as possible by boiling and filter. The yellow precipi- 
 tate is called phenylglucosazon. 
 
 For the detection of traces of sugar by this method it is 
 necessary to use more urine and more of the reagents. 
 50 Cc. of urine with 2 Gm. of phenylhydrazine and 3 Gm. 
 of sodium acetate may be taken. 
 
 Phenylglucosazon melts at 205° C. and a determina- 
 tion of the melting point may be made as a confirmatory 
 test. For this purpose the supernatant liquid is poured 
 
224 THE ANALYSIS OF URINE. 
 
 off and the fine yellow crystals are washed with water by 
 decantation. They are transferred to a small watch glass 
 allowed to dry over sulphuric acid in a desiccator and are 
 then ready for the test. Melting points are usually found 
 by placing a small amount of the substance in question in 
 a thin narrow tube, which is fastened to a thermometer by 
 means of rubber bands. The substance in the bottom of 
 the tube must be near the bulb of the thermometer. The 
 bulb and bottom of tube are then immersed in a beaker of 
 oil or sulphuric acid which is gradually heated until the 
 substance begins to fuse. The temperature indicated by 
 the thermometer is taken as the melting point. For best 
 methods of working this test of finding the fusing point 
 some standard manual of organic chemistry should be con- 
 sulted. 
 
 On the whole, it must be said that this reaction is of 
 very limited applicability in urine analysis. It has value 
 only when the copper or bismuth methods are insufficient 
 to decide concerning the presence or absence of sugar. In 
 cases having real clinical importance such uncertainty is 
 rare. 
 
 The A-naphthol Test. This depends on the reac- 
 tion between a-naphthol and sugar in presence of sulphuric 
 acid, and was discovered by Molisch. Take about a cubic 
 centimeter of urine, previously diluted with five to ten vol- 
 umes of water, and add to it two drops of a twenty per cent 
 solution of a-naphthol in alcohol. Then add about half 
 a cubic centimeter of strong sulphuric acid and agitate. 
 A blue color indicates sugar. If the acid is carefully 
 added so as to flow under the lighter liquid a blue zone is 
 formed between them. By diluting largely with water and 
 shaking, a violet precipitate is produced. 
 
 This method is exceedingly delicate, but unfortunately 
 
THE ANALYSIS OF URINE. 225 
 
 is not characteristic, as many substances show the same 
 result. The trace of sugar, or similar body, normally 
 present, gives a marked reaction, hence the direction to 
 largely dilute the urine before adding the reagent. 
 
 The a-naphthol may be replaced in this test by a twenty 
 per cent alcoholic solution of thymol. The mixture be- 
 comes dark red and carmine red on dilution with water. 
 It has been shown that these color changes depend on the 
 formation of small amounts of furfurol by action of sul- 
 phuric acid on traces of carbohydrates and the subsequent 
 combination of the furfurol with the a-naphthol or thymol. 
 However, not only carbohydrates, but also albumins and 
 many other substances yield furfurol in this manner and in 
 normal urine some of these substances may be always 
 present. 
 
 Molisch claims that the reaction found with highly 
 diluted urine is a sugar reaction, that in condition of high 
 dilution other bodies which may possibly be present can- 
 not give this test. A color still shows when normal urine 
 diluted 100 times is used, and on this behavior, partly, the 
 claim that sugar is normally always present in urine is 
 made. It will be seen from this that the test is too sensi- 
 tive for ordinary clinical needs. But as a laboratory test it 
 is valuable. By attentive study of the behavior of diluted 
 normal and diabetic urines the chemist soon learns to 
 recognize the deeper colors obtained with the latter and is 
 therefore able to employ the test in the way of confirmation. 
 
 The Fermentation Test. When yeast is added to 
 urine containing sugar and the mixture left in a moderately 
 warm place the usual fermentation soon begins, which is 
 shown by two principal changes. Carbon dioxide is given 
 off, which may be collected and identified, and the mixture 
 becomes lighter in specific gravity. When only traces of 
 
226 THE ANALYSIS OF URINE. 
 
 sugar are present the test by collection and identification 
 of the carbon dioxide frequently fails because of the solu- 
 bility of the gas in the liquid. 
 
 The variation in the specific gravity is an indication of 
 . greater value, as it can be readily observed with proper ap- 
 pliances. The test has practical value, however, only as a 
 confirmation of some other one. If by the copper solutions, 
 for instance, a strong indication is obtained which it is sus- 
 pected may be due to an excess of uric acid, the reaction 
 by fermentation may be resorted to because only sugar will 
 respond to it. The test may be made by pouring 100 Cc. 
 of the urine into each of two bottles or flasks. To one, 
 half a cake of compressed yeast, crumbled, is added; the 
 other is left pure. The bottle with the yeast is closed by 
 means of a perforated stopper (to allow escape of gas), 
 while the other is tightly corked. The two are left, side 
 by side, in a warm place about twenty-four hours. At the 
 end of this time a test of the specific gravity of the con- 
 tents of both bottles is made. If sugar is present to the 
 amount of one-half per cent the specific gravity of the 
 yeast bottle should be perceptibly lower. 
 
 The test is frequently recommended as a quantitative 
 one, as there is a fairly definite relation between amount 
 of sugar and loss in density. 
 
 Other Sugar Reactions. Many other tests have 
 been proposed for the detection of sugar in urine. A few 
 of these will be referred to briefly in this place. 
 
 One of these, the picric acid test, is based on the fact 
 that a urine containing sugar when mixed with solutions 61 
 potassium hydroxide and picric acid and boiled, turns a 
 dark mahogany red from formation of picramic acid. 
 
 When urine is made strongly alkaline with potassium 
 hydroxide and treated with a weak solution of diazoben- 
 
THE ANALYSIS OF URINE. 227 
 
 zene sulphonic acid in water it turns reddish yellow, if 
 sugar is present, and becomes afterward claret-red and 
 finally dark red if much is in solution. The reaction is 
 delicate, but is given by other bodies than sugar. 
 
 Another test depends on the reaction between sugar 
 solutions and indigo-carmine in presence of alkali. The 
 urine is made alkaline with sodium carbonate and treated 
 with indigo-carmine until a deep blue is obtained on heat- 
 ing. If sugar is present on longer heating the color fades 
 to yellow by reduction. The color returns by cooling and 
 shaking with air. 
 
 These tests have given good results in the hands of 
 those who have recommended them, but seem to possess 
 no advantages over the copper and bismuth reactions. 
 
 The Amount of Sugar. 
 
 It is not always sufficient to be able to detect the pres- 
 ence of sugar in urine. A knowledge of the amount is fre- 
 quently of the greatest importance. A number of methods 
 have been proposed by which a quantitative determination 
 can be made, some of them crude and of little practical 
 value, while others give, when properly carried out, results 
 which are accurate. The methods in general may be di- 
 vided into four groups, depending on the 
 
 (1.) Reduction of solutions of heavy metals, and 
 
 measurement of the amount of reduction. 
 (2.) Change of color produced in organic solutions, 
 by action of sugar, the depth of final color being 
 proportional to the amount of sugar. 
 (3.) Results of fermentation with measurement of 
 change in specific gravity of the urine or measure- 
 ment of evolved carbon dioxide. 
 (4.) Observation of rotary polarization of light. 
 
228 THE ANALYSIS OF URINE. 
 
 Methods by Reduction of Metallic Solutions. 
 
 The reduction methods are illustrated in the use of the 
 Fehling solution as a qualitative test and in the bismuth 
 tests. The general principles involved in making a quan- 
 titative determination of sugar by aid of the Fehling solu- 
 tion have already been explained in the chapters on chemi- 
 cal physiology. When applied to the urine, however, the 
 process requires certain modifications because of the fact 
 that this secretion contains always a number of sub- 
 stances which interfere to some extent with the normal re- 
 duction and precipitation of the copper suboxide. The 
 determination of dextrose in aqueous solution by the Fehl- 
 ing liquid is a problem of extreme simplicity, but in urine 
 the case is somewhat different. 
 
 If we measure out 50 cubic centimeters of the mixed 
 Fehling solution, heat it to boiling and then run in the 
 saccharine urine from a burette it frequently happens that 
 a greenish yellow muddy precipitate forms which does not 
 turn bright red and which, instead of quickly settling to 
 the bottom of the flask, remains suspended and makes it 
 impossible to observe the disappearance of the blue color 
 indicating the end of the reduction. This difficulty may be 
 largely obviated by working with solutions of greater di- 
 lution, as explained in the following paragraph. 
 
 Determination by Fehling Solution. Prepare a 
 Fehling solution as shown in the appendix and then ac- 
 curately mix it with four volumes of distilled water. That 
 is, to 100 cubic centimeters add 400 cubic centimeters of 
 water, to 50 add 200, or to 25 add 100. In any event the 
 dilution must be accurately made. One cubic centimeter 
 of this liquid will oxidize almost exactly one milligram of 
 dextrose as shown by a table in Chapter II., provided the 
 sugar is in approximately one per cent solution. For all 
 
THE ANALYSIS OF URINE. 229 
 
 practical purposes of urine analysis the oxidizing power 
 may be considered the same in a solution of one-half per 
 cent strength, and only very slightly increased in still 
 weaker solutions. Therefore, before beginning the test 
 dilute the urine accurately with four or nine volumes of 
 water. This can be done by making 50 Cc. up to 250 or to 
 500 Cc. and mixing well by shaking. 
 
 Now proceed with the analysis exactly as described in 
 Chapter II. Measure out 50 Cc. of the dilute Fehling so- 
 lution, pour it in a flask and heat to boiling on gauze. 
 Fill a burette with the diluted urine and when the solution 
 in the flask is actively boiling run in about 3 Cc. Boil two 
 minutes, remove the lamp and wait half a minute to 
 observe the color. If blue is still visible heat to boiling 
 again and run in 3 Cc. more. After boiling two minutes as 
 before wait a short time and observe the color near the 
 surface of the liquid in the flask. If still blue repeat these 
 operations until on waiting it is found that the blue has 
 given place to a yellow. The urine should be so dilute 
 that at least 10 Cc. must be run in to reduce all the copper 
 hydroxide. 
 
 When the volume required is found to within 2 or 
 3 Cc. a second experiment must be made, the urine being 
 added very gradually now, without interrupting the boiling 
 longer than necessary, until the first of the limits between 
 which the correct result must lie, as shown by the former 
 test, is reached. From this point the addition of the urine 
 is continued, with frequent pauses for observation of color 
 until the reduction is complete. The volume of urine used 
 contains 50 Mg. of sugar. 
 
 If the preliminary experiment shows that the urine is 
 strong in sugar and that the reduction is easy, that is that 
 the cuprous oxide separates and settles readily, the second 
 test may advantageously be made with 50 Cc. of a stronger 
 
230 THE ANALYSIS OF URINE. 
 
 Fehling solution. With many strong diabetic urines it is 
 possible to use the undiluted copper solution with the oxi- 
 dizing power of 4. 15 milligrams of sugar to each Cc. The 
 difficulties in this test have been very much overestimated; 
 with a little practice any one can make a good sugar deter- 
 mination in urine. The important point is to find by a few- 
 simple preliminary tests the best conditions of dilution of 
 Fehling solution and urine to give a precipitate which set- 
 tles readily. With this information, and it can be acquired 
 in a few minutes, the actual quantitative experiment can be 
 easily made. 
 
 The Use of Pavy's Solution. To avoid some of the 
 difficulties in the titration of diabetic urine by the Fehling 
 solution, Pavy suggested a solution containing ammonia. 
 If a solution of dextrose is run into a boiling copper solu- 
 tion containing ammonia in considerable quantity the cop- 
 per is gradually reduced, giving finally a clear, colorless 
 solution instead of a red precipitate. The end of the re- 
 duction is, therefore, indicated by disappearance of color 
 alone. The preparation of the Pavy solution is given in 
 the appendix. Its strength as there described is just one- 
 tenth of that of the common Fehling liquid, that is, 100 
 Cc. oxidizes 50 milligrams of dextrose. The test is per- 
 formed in a flask, as is the Fehling titration ; but as the 
 solution is easily changed by atmospheric oxidation, just 
 as soon as it begins to hold some reduced copper, precau- 
 tions should be taken to exclude the air during titration. 
 This can be done by passing a slow current of illuminating 
 gas or hydrogen through the flask during the test. 
 
 The titration is carried out as follows : Measure 100 
 Cc. of the ammoniacal copper solution into a flask hold- 
 ing about 300 Cc. Throw in some small pieces of pumice 
 stone to prevent "bumping," and then heat to the boiling 
 
THE ANALYSIS OF URINE. 231 
 
 point on wire gauze. The sugar solution must be dilute, 
 and should be contained in a burette with a delivery tip 
 bent to one side and then down, so that the contents of 
 the burette can be added slowly but continuously to the 
 liquid in the flask without interrupting the ebullition. The 
 operation should be carried out where there is a good cir- 
 culation of the air to carry off the evolved ammonia fumes, 
 and meanwhile a slow current of the hydrogen or coal gas 
 should be led down into the flask to keep out the air. As 
 the reduction is very slow the addition of the sugar solu- 
 tion must not be rapid. There is danger of adding too 
 much until the operator becomes familiar with the method. 
 If the precaution of passing in gas is neglected, which is 
 usually the case, the results come out a little too low, be- 
 cause the air reoxidizes some of the ammoniacal cuprous 
 solution, making it necessary to add more of the sugar to 
 complete the reduction, that is, to completely discharge 
 the color. 
 
 The method yields at best only approximate results, 
 and working it subjects the analyst to the annoyance of 
 ammoniacal fumes unless the apparatus is complicated by 
 the addition of a delivery tube to carry the evolved am- 
 monia through a window or into a fume chamber. 
 
 The reducing power of the copper in this solution de- 
 pends to some extent on the amount of ammonia present, 
 and from the fact that this is lost by ebullition during the 
 performance of the test, irregularly and at different rates 
 in different experiments, it follows that the results obtained 
 cannot be perfectly uniform. Besides this the solution 
 does not keep perfectly, its reducing power slowly under- 
 going change. However, the method has value and should 
 be learned, because it can be rapidly worked and the results 
 obtained are sufficiently accurate for clinical purposes. 
 
 The solution has been still further modified by substi- 
 
232 THE ANALYSIS OF VKINE. 
 
 tuting glycerol for the tartrate, giving what may be called 
 the Loewe-Pavy solution. This solution is employed as is 
 the Pavy liquid and has the same advantages and draw- 
 backs. It is claimed for it, however, that it keeps some- 
 what better. Solutions containing ammonia cannot be 
 used for qualitative testing. 
 
 Sugar Test by Solutions of Mercury. In Chapter 
 II. it was explained that certain solutions of compounds 
 of mercury can be used in sugar titration. Two such 
 solutions are frequently used, viz., Knapp's solution, con- 
 taining mercuric-potassium cyanide, and Sachsse's solution 
 containing mercuric-potassium i6dide. See the appendix 
 for the preparation of both of these. 
 
 The solution of Knapp is frequently used in urine titra- 
 tion and is employed in the following manner: 10 Cc. of the 
 solution, corresponding to 25 Mg. of dextrose is diluted 
 with 25 Cc. of water in a flask and heated to boiling. The 
 urine, which has been previously diluted accurately with 
 from four to nine volumes of water, is run from a burette 
 into the hot liquid until the whole of the mercury is pre- 
 cipitated, which can be recognized as follows : Allow the 
 precipitate to settle and then by means of a glass rod place 
 a drop of the yellowish supernatant liquid on a piece of white 
 Swedish filter paper. Hold the paper then over an open hy- 
 drochloric acid bottle containing the fuming acid, and 
 afterward over a beaker containing some strong hydrogen 
 sulphide water. If the drop of transferred liquid contains 
 even a trace of mercury this will be shown by the forma- 
 tion of a brown stain. In this case it will be necessary to 
 add more of the sugar solution, and repeat the operations 
 until the complete reduction and precipitation of the mer- 
 cury compound is accomplished, as shown by negative re- 
 sult with the hydrogen sulphide test. 
 
THE ANALYSIS OF URINE. 233 
 
 This method has been found to give very excellent re- 
 sults, but longer practice is necessary to give proficiency 
 with it than with the other. 
 
 Color and Fermentation Methods. 
 
 The methods of quantitative sugar analysis depending 
 on comparison of colors in sugar solutions acted on by pic- 
 ric acid and alkali or other reagent are neither very con- 
 venient nor accurate. 
 
 The fermentation test is sometimes applied quantitative- 
 ly, but in those cases where it is the most accurate it is least 
 necessary. With very weak sugar solutions it can only be 
 used with the most careful regard to changes in tempera- 
 ture by the method referred to above. With strong dia- 
 betic urines accurate results are more readily reached, but 
 here, by dilution, the copper solutions give the desired in- 
 formation more quickly and accurately. 
 
 When the saccharine urine is fermented as described 
 and a change of specific gravity observed, the percentage 
 of sugar is approximately given by multiplying each .001 
 lost by .23. For instance, if the urine before fermentation 
 had a specific gravity of 1.032, and after fermentation, at 
 the same temperature, a specific gravity of 1.016, we have 
 16x.23 = 3.68 as the per cent of sugar present. 
 
 Sugar Determination by Polarimetry. 
 
 The construction and method of using the polarimeter 
 or polariscope have been explained in Chapter II. It 
 remains now to indicate how the instrument may be used 
 in the examination of urine. 
 
 The direct examination of urine is not always possible 
 because of its color and sometimes because of its 
 slight turbidity. The best results are obtained with color- 
 less and clear solutions. It is therefore sometimes neces- 
 
234 THE ANALYSIS OF URINE. 
 
 sary to prepare the urine by a preliminary treatment before 
 it can be filled into the observation tubes. Diabetic 
 urines light in color may frequently be used after simple 
 filtration to render them perfectly clear, especially with 
 the high class modern instruments of the half-shadow type 
 with which a good illumination can be secured. If the 
 urine is much colored, so that an observation cannot be 
 made with the shortest tube — 100 millimeters in length — 
 which can be determined by a simple trial, resort must be 
 had to precipitation to remove part of the color. Several 
 precipitating agents are used for clarifying sugar solu- 
 tions for the polariscope. The simplest of these is a solu- 
 tion of basic lead acetate which produces a voluminous 
 precipitate that carries down much coloring matter. This 
 is frequently used alone, but perhaps better combined with 
 alum. When the basic acetate is added first and then 
 some aluminum sulphate the mixed precipitate is floccu- 
 lent and very effective in carrying down coloring matters. 
 Use the basic acetate of lead described in the appendix 
 and prepare a solution of aluminum sulphate of about 
 equivalent strength, that is of such strength that one Cc. 
 will precipitate the lead of one Cc. of the other. 
 
 Measure out 100 Cc. of the urine, add 5 Cc. of the lead 
 solution and 2 or 3 Cc. of the alum solution, shake well, 
 add water to bring the volume to 110 Cc. exactly, shake 
 again and allow to stand 10 minutes. Then filter through 
 a dry filter. The filtrate will be found much lighter in 
 color than the original and probably suitable for use. If it 
 is opalescent pour it through the precipitate on the filter 
 when it will be found much brighter. 
 
 There is a slight loss of sugar in this operation as some 
 is carried down by the precipitate. The clarified solution 
 is then filled into the polarization tube and observed in the 
 usual manner. The result obtained must be increased by 
 
THE ANALYSIS OF URINE. 235 
 
 one-tenth because of this dilution of the original urine. 
 As the precipitate formed occupies an appreciable volume 
 when dried, the clarified solution is correspondingly concen- 
 trated and the reading from this cause would be too high. 
 For our purpose, however, we can assume that the gain in 
 concentration is counterbalanced by the loss of sugar in- 
 closed with the precipitate and neglect both sources of error. 
 
 If the urine contains albumin it must be separated by 
 coagulation and filtered out, because it rotates the plane of 
 polarized light to the left, and would therefore make the 
 amount of sugar appear lower. 
 
 A given volume of urine is poured into a beaker and 
 enough dilute acetic acid is added to give a faint reaction ; 
 it is then boiled, and after standing five minutes filtered. 
 As all albuminous bodies, however, are not precipitated by 
 simple coagulation with acetic acid, it has been recom- 
 mended to add to 100 Cc. of the urine, 10 Cc. of the strongly 
 acid solution of phospho-tungstic acid, already described, 
 and filter after ten minutes. The dilution must be allowed 
 for in the final calculation. Some coloring matters are -also 
 removed by this treatment. The urine may contain other 
 'active substances, but in amount so small that their effect 
 may be neglected entirely. 
 
 Calculation of Result. For sodium light the formula 
 r -, 100a 
 
 W = 17 
 
 is used with the factor [a] = 53° 
 
 100a 
 
 Hence we have 
 
 /.53 c 
 
 That is, the number of grams of diabetic sugar in 
 100 cubic centimeters of the solution polarized is equal to 
 the product of the observed angle of rotation multiplied by 
 
236 THE ANALYSIS OF URINE. 
 
 100 and divided by the product of the length of the obser- 
 vation tube in decimeters multiplied by the specific rota- 
 tion, 53° 
 
 If in a given case we find a rotation of 10° 36', with a 
 tube two decimeters long, our formula becomes 
 
 c = 100 X 10.6 _ in 
 2 X 53 
 
 that is, the concentration, c, is 10 grams per 100 Cc. 
 
 With a decimeter tube each degree of rotation corre- 
 sponds to a concentration of 1.8868. With the usual two 
 decimeter tube each degree indicates 0.9434 Gm. in each 
 100 Cc. 
 
 The specific rotation of dextrose as obtained from urine 
 appears to be a little higher than is that of the product 
 made from starch. 
 
 Other Sugars in Urine. 
 
 Pathologically, traces or even larger quantities of 
 several other saccharine bodies are occasionally found in 
 urine. Among these we have first : 
 
 Laevulose, or Fruit Sugar. This is found along with 
 dextrose in some cases of diabetes, but does not appear to 
 occur alone. 
 
 While the recognition of laevulose in the pure state or 
 in simple aqueous solution is a matter presenting no diffi- 
 culty, the certain detection of this body as it occurs in 
 urine is by no means as readily effected. This sugar 
 gives the reduction and fermentation tests as described 
 under dextrose, and therefore, cannot be distinguished by 
 these methods. Laevulose, however, rotates the plane of 
 polarized light to the left, and this property is sometimes 
 of service in aiding the recognition. If the rotation is 
 
THE ANALYSIS OF URINE, 237 
 
 strongly to the left, the presence of laevulose in quantity 
 may be inferred, assuming that albumins are absent. If 
 the quantity of sugar, calculated as dextrose, determined 
 by polarization is much below that found by the copper 
 reduction method the indication is that laevulose is present 
 with the dextrose. An exact measurement of the amounts 
 of the two sugars when mixed in the urine is not possible 
 with present means. 
 
 Lactose, or Milk Sugar, is occasionally found in the 
 urine of nursing women. Its certain detection when in 
 small amount presents even greater difficulties than is the 
 case with laevulose. As its rotation is right-handed the 
 polariscopic test is of little value. 
 
 Milk sugar is more strongly acted on by Fehling solu- 
 tion than is dextrose. While 1 Cc. of the copper solution 
 oxidizes 4.75 Mg. of dextrose, it oxidizes 6.76 Mg. of milk 
 sugar. 
 
 When a solution of milk sugar is boiled with dilute 
 hydrochloric acid it yields dextrose and galactose, the lat- 
 ter resembling dextrose in its behavior with the copper 
 solution. The specific rotation, [a] D , of dextrose is 52.7°, 
 of lactose, 52.5°, while that of galactose is 81.3°. The 
 specific rotation of a mixture of equal parts of dextrose and 
 galactose has been found by experiment to be 67.5°, which 
 agrees closely with the mean of 52.7° and 81.3°. If, there- 
 fore, the rotation of urine is increased after heating with 
 acid and neutralizing, and its copper reducing power dimin- 
 ished, we have data suggesting the presence of milk sugar. 
 Experiments to show these points with certainty must be 
 very carefully conducted, consuming no little time in manip- 
 ulation. They are, therefore, of little value from a clinical 
 standpoint. 
 
 Inosite, or Muscle Sugar, has been found in urine, in 
 
238 THE ANALYSIS OF URINE. 
 
 diabetes, and also with albumin. There is no simple 
 method by which it may be separated in the small quantity 
 in which it occurs in urine. 
 
 Dextrine and a body termed animal gum have been re- 
 ported as occurring in some cases of diabetes, but as their 
 clinical significance is not clear nothing more need be said 
 about them here. 
 
 Acetone. 
 
 This is a substance which frequently is found in urine 
 in small amounts. Indeed, it may be true, as has been as- 
 serted, that it is normally always present in traces. This 
 physiological acetonuria has no clinical significance. Under 
 some circumstances, however, it may be found in larger 
 quantity, sometimes in amount sufficient to be detected by 
 the odor alone, which fact first called attention to it. At 
 one time it was supposed to be related to the sugar found 
 in urine, but it is now established that it more generally 
 accompanies albumin and is frequently observed in many 
 febrile conditions. 
 
 Acetone in urine is believed to be a decomposition prod- 
 uct of albumins. It has been shown that in health, even, 
 it can be much increased by a diet rich in nitrogenous ma- 
 terials. 
 
 But, occurring as it does in fevers and in advanced 
 stages of diabetes mellitus, a certain interest attaches to 
 its detection, and numerous methods have been proposed 
 by which it may be identified in small, amount. Those 
 which depend on its direct recognition in the urine are 
 mostly uncertain. It is always safer to distill the liquid 
 and apply the test to a portion of the distillate. Half a 
 liter, or more, of the urine is poured in a retort attached to 
 a Liebig's condenser, and after addition of a little phos- 
 phoric acid is subjected to distillation. One hundred cubic 
 
THE ANALYSIS OF URINE. 239 
 
 centimeters of distillate will be enough. A portion of this 
 can be taken for each test as follows: 
 
 Legal's Test. Add to 25 Cc. of the fluid a small 
 amount of a fresh solution of sodium nitroprusside, and a 
 few drops of a fifty per cent potassium hydroxide solution. 
 If a ruby red color appears which slowly gives place to 
 yellow, and if the addition of acetic acid changes this to 
 purple, or violet red, the presence of acetone is indicated. 
 
 Lieben's Test. This depends on the production of 
 iodoform, and is carried out in this manner. To about 5 
 Cc. of the distillate add a few drops of a solution of iodine 
 in potassium iodide (the " compound solution of iodine," 
 Lugol's solution), and then a small amount of potassium 
 hydroxide, to marked alkaline reaction. If acetone is pres- 
 ent a yellowish white precipitate soon appears, which, on 
 standing, becomes crystalline and more deeply colored. 
 The test is said to be sharper and more characteristic if 
 ammonia is used instead of the fixed alkali. The liquid is 
 first made strongly alkaline with ammonia, and then the 
 iodine solution is added until the brownish precipitate 
 formed at first dissolves very slowly. In a short time the 
 yellowish iodoform precipitate makes its appearance. A 
 rough quantitative measure of the amount of acetone pres- 
 ent is given by noting the smallest volume of the distillate 
 with which a distinct iodo%>rm reaction can be seen. It is 
 said that .0001 Mg. in one Cc. tsan be detected. .5 Mg. in 
 10 Cc. can be recognized by the nitroprusside reaction. 
 
 Kreatinin gives a ruby red color as does acetone when 
 the nitroprusside reaction is directly applied to urine, but 
 after adding acetic acid a green or blue color results. 
 
240 THE ANALYSIS OF URINE. 
 
 Aceto-acetic or Diacetic Acid. 
 
 This compound is very frequently found associated with 
 acetone in the urine of fevers and in diabetes mellitus. 
 While acetone may occur in very small amount normally it 
 is believed that aceto-acetic acid is always pathological. 
 In the past few years much has been written on the sub- 
 ject of this substance and its clinical significance. It 
 appears from the discussion that its presence in diabetes is 
 of especial importance and that any increase in its amount 
 should be carefully followed by analytical tests. What is 
 known as the coma of diabetes is closely associated, accord- 
 ing to eminent authority, with the presence of aceto- 
 acetic acid in the blood. 
 
 Just how this body is produced in the blood from which 
 it passes into the urine cannot be explained satisfactorily 
 at present. Several theories have been offered to account 
 for the phenomenon, but they are scarcely definite enough 
 to be presented in an elementary work on practical tests 
 like the present. 
 
 Urine containing aceto-acetic acid always contains 
 acetone. The latter is probably derived from the former, 
 and both may be derived from a still more complex sub- 
 stance, /3-oxybutyric acid: It has lately been claimed that 
 many of the reactions supposed to be due to aceto-acetic 
 acid are in reality due to this substance. For our purpose, 
 however, it will be sufficient to follow the important tests 
 by which the aceto-acetic acid may be recognized. 
 
 The simpler acetone test should be made first because 
 the other appears to be present only with this, and because 
 further, the aceto-acetic acid gives these tests as well as 
 does acetone. 
 
 Ferric Chloride Test. Our main test for aceto-acetic 
 acid depends on a reaction with ferric chloride with which 
 
THE ANALYSIS OF URINE. 241 
 
 it strikes a red color. Normally, there is nothing in urine 
 which gives the same reaction, so that if on the addition of 
 a few drops of solution of ferric chloride to fresh urine a 
 wine red color results the presence of aceto-acetic acid may 
 be inferred. At the present time, however, many. coal tar 
 products are given as remedies which oxidize to com- 
 pounds that, on elimination with the urine, give a red or 
 purple color with ferric chloride when added, To detect 
 the aceto-acetic acid with certainty under these conditions 
 it is necessary to proceed with greater care. To this end 
 add to the urine, which should be fresh, a few drops of 
 ferric chloride or enough to precipitate the phosphates 
 present. Filter and add a little more of the chloride. A 
 red color indicates the acid. Divide the liquid into two 
 portions; boil one and allow the other to stand a day or 
 more. In the boiled portion the color due to aceto-acetic 
 acid should disappear within a few minutes, while in the 
 other it should remain about twenty-four hours. 
 
 Acidulate another portion of the urine with dilute sul- 
 phuric acid and extract it with ether which takes up aceto- 
 acetic acid. Remove the ethereal layer and shake it with 
 an aqueous solution of ferric chloride. The red color should 
 appear as before and disappear on boiling, which behavior 
 distinguishes the acid from other substances likely to be 
 present. 
 
Chapter XIII, 
 
 THE COLORING MATTERS IN URINE. 
 BILIARY ACIDS. 
 
 Normal Coloring Matters. 
 
 ALTHOUGH many investigations have been carried out 
 on the subject of the normal urinary pigments we are 
 yet unable to give a very definite account concerning them. 
 This is partly due to the fact that the coloring substances 
 exist in the urine in minute traces only, which makes their 
 separation and recognition exceedingly difficult, and partly 
 to another fact that some of them are easily altered or de- 
 stroyed by the action of the reagents employed in their in- 
 vestigation. By proceeding according to different methods, 
 physiologists have obtained very different results indicating 
 the existence of several colors, or at any rate modifications 
 of colors. It is generally admitted, however, that at least 
 two distinct coloring matters exist in the urine, and the 
 others may be classed as derived from these by oxidation 
 processes. One of these colors is known as urobilin and 
 the other as indican. 
 
 Among the products related to or derived from urobilin 
 the following may be mentioned as described by different 
 writers. Urohsematin, Harley, urophain, Heller, hydrobili- 
 rubin, Maly, urochrome, Thudichum. Among the colors 
 related to indican, uroxanthin, Heller, may be mentioned. 
 
 Urobilin. This has been obtained as a reddish brown 
 amorphous substance, but probably not in absolutely pure 
 condition. It is slightly soluble in water, readily soluble 
 
THE ANALYSIS OF URINE. 243 
 
 in alcohol and chloroform. The neutral alcohol solutions 
 are characterized by a marked greenish fluorescence which 
 is an important means of recognition. The acid alcohol 
 solutions are reddish in color, the shade varying with the 
 concentration. 
 
 If present in more than minute traces in urine it gives 
 characteristic absorption bands in the spectrum which have 
 been referred to before. In acid urine the center of the 
 dark band is near the Fraunhofer line F ; in alkaline urine 
 the center is about midway between b and F. 
 
 Urobilin is generally much increased in fevers and in 
 some diseases of the liver and heart. Any cause tending 
 to break up the red corpuscles, increases urobilin. It is 
 not always present in sufficient quantity in normal urine to 
 be easily recognized. If the quantity is abnormally large 
 the following test will show it : 
 
 Add ammonia water to strong alkaline reaction and filter 
 if necessary. Then add a few drops of solution of zinc 
 chloride, but not enough to give a permanent precipitate. 
 In this way a zinc salt is formed, which shows a peculiar 
 greenish fluorescence. 
 
 Ammonia generally causes a precipitate of phosphates, 
 hence the direction to filter. If the characteristic fluores- 
 cence fails to appear the following modification may be tried, 
 which is sufficient to give the reaction with most urines. 
 
 Precipitate 200 Cc. of urine with basic lead acetate, 
 collect the precipitate on a filter, wash it with water and 
 dry it. Then wash it with alcohol. 
 
 Finally, digest with, alcohol containing a little sulphuric 
 acid, and filter. The filtrate is usually fluorescent. Make 
 it strongly alkaline with ammonia, and add solution of 
 zinc chloride. This will give the fluorescence referred to 
 above if but little is added, while if an excess of the zinc 
 chloride is added, a reddish precipitate falls. 
 
244 THE ANALYSIS OF URINE. 
 
 Urophain. This is the name given by Heller to a 
 substance identical with, or similar to, urobilin. Heller 
 gives this test : Take a few Cc. of strong sulphuric acid 
 in a conical glass and pour on it, drop by drop, about 
 twice as much urine. As the two mix, a deep garnet red 
 is produced. 
 
 This reaction is not, however, characteristic, as several 
 other matters may give it. 
 
 Urohaematin is the name given by Harley to a color- 
 ing matter similar to the above. He applies this test: 
 Dilute or concentrate the urine so that it is equivalent to 
 1,800 Cc. for the twenty- four hours. Take a few Cc. in a 
 test-tube or wine glass, and add one-fourth of its volume 
 of strong nitric acid. No change of color can be observed 
 if the urohaematin is present in normal amount. If more 
 than this is present various shades from pink to red may 
 be produced. The test should be made with cold urine, 
 as with increased temperature darker colors result. 
 
 Indican and its Reactions. Although a normal con- 
 stituent of urine indican is found greatly increased during 
 the progress of certain diseases and becomes therefore a 
 substance of clinical importance. It is formed along with 
 other complex compounds in the oxidation of indol in 
 presence of sulphuric acid. Indol is one of the common 
 products of putrefaction, a change brought about in al- 
 buminous bodies, usually by bacterial agency. Such 
 changes may take place in the alimentary canal, and the 
 indol formed becomes oxidized to indoxylsulphuric acid or 
 indican, and appears as such in the urine. The sulphuric 
 acid necessary for the production of this body is present in 
 combination in the system. 
 
 If much indican is found it suggests that abnormal 
 
THE ANALYSIS OF URINE. 245 
 
 putrefaction is taking place somewhere in the body. In 
 diseases accompanied by the formation of putrid secretions 
 indican usually appears in increased amount, and hence the 
 inference derived from its ready detection. It is found in 
 increased amount in cancer of the stomach or liver, in peri- 
 tonitis, in some stages of pleurisy, in intestinal invagination 
 (whereby the normal passage of albuminous and other food 
 products is hindered, thus making putrefaction possible) 
 and in other diseases. 
 
 Indican is found in normal urines in very small amount 
 only. It may, under favorable circumstances, be detected 
 as here given: Take about four Cc. of pure hydrochloric 
 acid in a test-tube and add about half as much urine, shak- 
 ing well. A blue or violet color shows indican. 
 
 A more generally applicable method is this: To 10 Cc. 
 of urine and the same volume of strong pure hydrochloric 
 acid, add 2 or 3 Cc. of chloroform. Then add, drop by 
 drop, solution of sodium hypochlorite, shaking after each 
 addition. The hypochlorite acts as an oxidizing agent, 
 liberating the coloring matter, which is then taken up by 
 the chloroform. 
 
 The oxidation must not be carried too far; that is, too 
 much hypochlorite must not be added, as it would then 
 destroy the color as fast as formed. 
 
 Albumin must be separated by coagulation before ap- 
 plying either of these tests, as it develops a blue color 
 with hydrochloric acid. The amount of indican normally 
 present in urine is said to vary between 5 and 20 milli- 
 grams daily. The chloroform layer in the bottom of the test- 
 tube in the above test shows roughly by the depth of color 
 developed the amount of indican present. It is neces- 
 sary to use good hypochlorite for this test as with a weak 
 solution the oxidation mav fail to take place. 
 
246 THE ANALYSIS OF URINE. 
 
 Abnormal Coloring Matters. 
 
 In disease several other coloring matters may appear in 
 urine, the most important "of which are those of the bile 
 and blood. 
 
 As abnormal colors must be classed, also, many prod- 
 ucts taken into the stomach with the food or as remedies 
 and which appear directly in the urine or give rise to 
 marked coloration on the addition of reagents. 
 
 Biliary Coloring Matters. 
 
 These are found in the urine in jaundice and may be 
 traced to the stoppage of the bile ducts of the liver as in 
 common jaundice and to other causes having no connection 
 with a disorder of the liver. The appearance of these 
 coloring matters in the urine is therefore a symptom of dif- 
 ferent diseases, although perhaps most commonly associ- 
 ated with an abnormality in the flow of the bile. Jaundice 
 may sometimes be traced to a disintegration of the red cor- 
 puscles in the blood and consequent liberation of derived 
 coloring matters. 
 
 Biliary urine has generally a characteristic greenish 
 yellow color sometimes tinged with brown. The froth from 
 such urineis readily recognized by its yellow color, which 
 is often a sufficient test in itself. Among the chemical tests 
 the following are the best known. 
 
 Gmelin's Test. This has been referred to before and 
 depends on the oxidation of bilirubin, the pigment com- 
 monly present in fresh jaundice urine, by nitrous acid. 
 Pour in a test-tube about 5 Cc. of the urine under exami- 
 nation and by means of a pipette introduce below it an 
 equal volume of strong nitric acid mixed with nitrous. 
 This should be carefully done so as to avoid mixing the 
 liquids much. At the junction of the two liquids, if bile 
 
THE ANALYSIS OF URINE. 247 
 
 is present, several colored rings appear of which the 
 green due to biliverdin is most characteristic. Bands of 
 blue, violet, red and yellow may appear above the green, 
 but this, next to the acid where the oxidation action is 
 strongest, is essential. It must be remembered that nitric 
 acid gives the other colors at times with urine free from 
 bile, but green is characteristic of the latter. 
 
 Fleischl modified this test by mixing the urine with a 
 strong solution of sodium nitrate and then adding strong 
 sulphuric acid carefully. This settles below the urine and 
 decomposes the nitrate at the point of contact liberating 
 the necessary nitric and nitrous acids for the oxidation as 
 before. This method is a very good one. 
 
 In another modification, urine is dropped on a plaster of 
 Paris disc and then a few drops of the oxidizing mixture of 
 nitric and nitrous acids is placed in its center. The same 
 play of colors appears as before. 
 
 Trousseau's Test. Add to some urine in a test-tube 
 a few drops of tincture of iodine, allowing the iodine to 
 float on the urine. If bile pigments are present a green 
 color is produced when the iodine touches the urine, and 
 persists some hours. Care must be taken to avoid using 
 an excess of the iodine if the fluids are allowed to mix. In 
 this case with the proper amount of the tincture the whole 
 urine appears green. 
 
 Heller's Test. Take 5 or 6 Cc. of pure strong hydro- 
 chloric acid in a conical glass and add enough of the urine 
 to give it a faint color on mixing. Now add pure nitric 
 acid by means of a pipette so as to bring the latter under 
 the mixture of hydrochloric acid and urine. The colored 
 rings appear as in the Gmelin test and on shaking can be 
 followed through the liquid. 
 
248 THE ANALYSIS OF URINE. 
 
 The Detection of Traces. To 100 Cc. of the urine 
 add 10 Cc. of pure chloroform and shake gently until the 
 latter is colored. By means of a pipette withdraw a small 
 part of the chloroform and mix it in a test-tube with 10 Cc. 
 of strong pure hydrochloric acid. Add nitric acid as in the 
 other tests and shake. With bile present the oxidation 
 colors appear slowly in the chloroform, the green being the 
 deciding tint. 
 
 Blood Coloring Matters. 
 
 As these appear in the urine they may be derived from 
 different sources. We may have, first, color due to the pres- 
 ence of blood corpuscles themselves sometimes in nearly 
 fresh condition. There may be enough blood present to 
 impart to the urine a marked red color and it may be de- 
 rived from the kidney, bladder, urethra or other part of the 
 urinary tract. In blood from a fresh lesion the corpuscles 
 usually appear in clearer outline than is the case when they 
 have remained long in contact with the urine. 
 
 The presence of blood may be detected by several 
 methods. The corpuscles are often easily recognized by 
 the microscope in the sediment deposited when the urine 
 is allowed to stand, as will be explained in a following 
 chapter. Then we can make use of the spectroscope by 
 which means the characteristic absorption bands of oxy- 
 haemoglobin are detected, as was shown in the earlier chapter 
 on the blood. If urine containing blood is treated with a 
 few drops of ammonium sulphide and very gently warmed 
 the spectrum of reduced haemoglobin is given. 
 
 Sometimes the coloring matters alone without the cor- 
 puscles can be found. This is the case when the latter 
 become disintegrated, the more stable and soluble haemo- 
 globin passing into solution while the stroma disappears 
 by decomposition. The condition in which blood itself is 
 
THE ANALYSIS OF URINE. 24"9 
 
 present, and can be recognized by the microscope, is 
 known as hamaturia, while the condition characterized by 
 the presence of the coloring substance only is called 
 h amoglobinuria. 
 
 In urine, haemoglobin frequently undergoes two decom- 
 positions. It may become converted into methmmoglobin, 
 as was explained in Chapter V., or it may suffer a complete 
 modification, breaking up into haematin and a body re- 
 sembling globulin. Haematin is best recognized by spec- 
 troscopic examination, as it gives a spectrum different from 
 haemoglobin. This modified product is said to occur in 
 urine in cases of poisoning by hydrogen arsenide. 
 
 The following are the best chemical tests for the recog- 
 nition of these bodies : 
 
 Heller's Test. Treat the urine with solution of 
 sodium or potassium hydroxide, and heat to boiling. This 
 produces a precipitate of the earthy phosphates which in 
 subsiding carry down coloring matters. If a precipitate 
 does not separate readily it may be hastened by adding 
 two or three drops of magnesia mixture. Haemoglobin, 
 when present, is decomposed by this treatment with sepa- 
 ration of haematin, which in turn settles down with the 
 phosphates, imparting a red color to the precipitate. 
 
 Struve's Test. Make the urine slightly alkaline with 
 sodium hydroxide solution, and then add enough solution 
 of tannic acid in acetic acid to change the reaction. If 
 haemoglobin is present a dark brown precipitate of haema- 
 tin tannate settles out. The test is a good one, and 
 easily performed. This precipitate can be used for the 
 production of Teichmann's haemin crystals by moistening 
 with salt and hydrochloric acid by the method described 
 in Chapter V. 
 
250 THE ANALYSIS OF URINE. 
 
 Almen's Guaiacum Test. In a "test-tube mix equal 
 volumes of fresh tincture of guaiacum and ozonized 
 turpentine. Two or three Cc. of each will suffice. The 
 mixture, if made of proper materials must not show a 
 green or blue color after thorough shaking. Now add a 
 few Cc. of the urine to be tested, a drop at a time, and 
 agitate after each addition. If haemoglobin is present it 
 causes the oxidizing material of the ozonized turpen- 
 tine (probably hydrogen peroxide) to act on the precipi- 
 tated guaiacum resin, imparting to it first a greenish, and 
 finally a blue color. Old and alkaline urine must be made 
 faintly acid before performing the test. Pus in the urine 
 gives a somewhat similar reaction, and a few other bodies, 
 very seldom present, interfere. The test is very delicate, 
 and if it gives a negative result it is safe to conclude that 
 blood is absent. 
 
 Vegetable and Other Colors. 
 
 It has long been known that many peculiar coloring 
 matters enter the urine from substances taken as remedies 
 and sometimes as food. A few of the more common of 
 these colors will be mentioned here. 
 
 Chrysophanic Acid. This complex organic acid is 
 found in the root of several kinds of rhubarb, in senna 
 leaves, in certain lichens and elsewhere. After the admin- 
 istration of any of these substances the urine becomes 
 more highly colored, being a brighter yellow if acid and 
 yellowish red when made alkaline. When phosphates are 
 precipitated by addition of alkali they appear red in pres- 
 ence of chrysophanic acid, as they do with blood. But 
 the latter can be easily distinguished by the other tests 
 already given. 
 
 Santonin. This crystalline principle is found in the 
 unexpanded flowers of Levant wormseed, and when ad- 
 
THE ANALYSIS OF URINE. 251 
 
 ministered as a remedy produces a characteristic change 
 in the color of the urine. The color becomes a deep yel- 
 low which turns red with alkalies, as in the case of chryso- 
 phanic acid. If the colored alkaline urine is shaken with 
 amyl alcohol the coloring matter from the santonin leaves 
 the urine and passes into the alcohol, but the color from 
 chrysophanic acid is only very slightly soluble in amyl al- 
 cohol and remains with the urine when the same treatment 
 is applied. 
 
 Salicylic Acid- The urine of persons taking this sub- 
 stance has usually a grayish smoky tinge which becomes 
 blue on addition of solution of ferric chloride if more than 
 traces are present: 
 
 Salicylic acid is excreted in the free state or as a sal- 
 icylate of sodium or potassium mainly; a small portion 
 seems to pass into other compounds. But as the iron re- 
 action is very delicate minute amounts of the free or com- 
 bined acid can be found. 
 
 Enough ferric chloride must be added to be in excess of 
 what would combine with the phosphates present, other- 
 wise a sharp reaction may not be secured. 
 
 Phenols. Several phenol bodies as carbolic acid, hy- 
 droquinol, resorcinol, pyrocatechol and others sometimes 
 find their way into the urine, to which they impart a dark 
 color on standing exposed to the air. This change of 
 color is said to be due to the formation of oxidation 
 products of hydroquinol. From urine darkened in this 
 manner phenols have been recovered by making acid with 
 sulphuric acid and then distilling with steam. 
 
 Alkapton. In some cases described in the literature 
 in the last few years the urine has had a brownish tint turn- 
 
252 THE ANALYSIS 01 URINE. 
 
 ing darker on exposure to the air. The substance giving 
 rise to this color was called alkapton, but has been shown 
 to be a mixture, probably, of hompgentisinic acid with one 
 or more other complex aromatic products. The color is 
 very marked in presence of alkali and can become almost 
 black, absorbing oxygen in this condition rapidly. The 
 clinical significance of this substance is not well under- 
 stood as yet. 
 
 Other Colors. Blueberries, carrots and several other 
 common vegetable foods give deep color to the urine. It 
 is not always possible to recognize the coloring substances 
 in these cases. Such urine usually becomes yellow with 
 acids and reddish with alkalies. It is occasionally possi- 
 ble to identify the color by means of the spectroscope, as 
 the absorption spectra of some of these products have been 
 studied. 
 
 The Detection of the Bile Acids. 
 
 It sometimes happens that the physician desires in- 
 formation regarding the presence of the biliary acids as 
 well as the pigments in the urine. This information, how- 
 ever, is not easily secured because there is no simple test 
 which can be applied directly to the urine which will give 
 a certain indication of the presence of these acids. They 
 must first be separated from the large amount of other 
 substances present, which can be done in this way (JVeu- 
 komiti) : 
 
 Evaporate 300 to 500 Cc. of urine nearly to dryness; 
 extract with ordinary alcohol, evaporate this solution, and 
 extract the residue with absolute alcohol. 
 
 Evaporate this and take up the residue with water. 
 Precipitate the solution by lead acetate, avoiding excess; 
 allow to settle, wash with water on a filter, and dry by 
 pressing between bibulous paper. This leaves an impure 
 
THE ANALYSIS OF URINE. 253 
 
 lead salt of the acids. Extract it with hot alcohol, and 
 filter; add sodium carbonate to the filtrate, evaporate to 
 dryness and extract the sodium salt, thus formed, with 
 absolute alcohol. Evaporate again, add some water and 
 apply the Pettenkofer test, as follows: 
 
 To the solution add one or two drops of a 20 per cent 
 cane sugar solution, and then some strong sulphuric acid, 
 slowly to avoid heating. 
 
 It is best to immerse the test-tube in water to keep the 
 temperature below 60° C. As the acid mixes with the liquid 
 a violet or purple color is produced. It has been shown that 
 this, like the naphthol test for dextrose is a furfurol reaction, 
 the furfurol formed from the mixed sugar and acid combin- 
 ing with the acids of the bile. It has even been proposed 
 to use a dilute solution (one-tenth per cent) of furfurol 
 instead of the sugar in the test. 
 
 Kuelz recommends to evaporate the solution on a water- 
 bath to dryness, to moisten the residue with a drop of 
 dilute sugar solution, and then with a drop of the strong 
 acid. The color appears almost immediately, but can be 
 sharpened by heating the evaporating dish a few seconds 
 on the water-bath. 
 
 Applying either of these tests directly to urine is un- 
 safe, as the coloring and other matters present would inter- 
 fere very much with the reaction. 
 
Chapter XIV. 
 
 DETERniNATION OF URIC ACID. 
 
 T TRIC acid occurs normally in urine combined with 
 *— ' sodium, potassium, magnesium, or ammonium. The ab- 
 solute amount excreted daily is small but quite variable, 
 depending on many conditions not well understood. In 
 health the amount passed daily seems to vary between 0.2 
 Gm. and 1 Gm. These limits may not be correct, however, 
 as many of the older determinations were made by inac- 
 curate methods. 
 
 Regarding the clinical significance of variations in the 
 amounts of uric acid passed our knowledge is still very 
 defective. It is generally held that there is a considerable 
 increase in the excreted uric acid in fevers and in diseases 
 characterized by diminished respiration and consequently 
 imperfect oxidation. In leucaemia there is a pronounced 
 and characteristic increase of uric acid. Certain writers 
 have attempted to connect a decreased elimination of uric 
 acid with an accumulation of the same in the blood, giving 
 rise to numerous disorders of which gout may be mentioned 
 as one in which the connection has been, apparently, 
 clearly shown. Great variations in the excreted uric acid 
 seem to be characteristic of a train of disorders, rather 
 than of a single one. 
 
 From recent investigations it appears that the ratio of 
 excreted urea to uric acid is in health not far from 50:1, 
 and that variations in this ratio are of greater moment 
 than are variations in the absolute amount of the acid. 
 Both must be considered as normal end products of nitro- 
 
THE ANALYSIS OF URINE. 255 
 
 genous metabolism, contrary to the older view that uric 
 acid is the antecedent of urea, and that the amount of the 
 former found in the urine represents merely that which 
 failed to be completely oxidized. A marked change in the 
 above ratio, 50:1, by increase of the uric acid is charac- 
 teristic of a condition which is somewhat indefinitely 
 called the uric acid diathesis. 
 
 In the recognition of uric acid the following points may 
 be noted. When present in large amount it frequently 
 precipitates from the urine in the free form, or as acid 
 urates which have a yellowish color. When the amount 
 present is small it may be found by acidifying with hydro- 
 chloric acid and then allowing the urine to stand some 
 hours in a cool place; uric acid crystals separate. In mixed 
 sediments it may be recognized by this test : 
 
 Murexid Test. Throw the sediment on a filter and 
 wash once with water. Place the residue in a porcelain 
 dish, add a drop of strong nitric acid, and evaporate to 
 dryness on the water-bath. A yellow or brown mass is ob- 
 tained, and this touched with a drop of ammonia water 
 turns purple. 
 
 Unless the uric acid or urate is present in the sediment 
 in fine granular form its recognition by the microscope is 
 very simple. Illustrations of the forms of uric acid and 
 certain urates are given in the chapter on the .sediments. 
 
 The Amount of Uric Acid. 
 
 For the determination of the amount of the acid in the 
 urine we have the choice of several methods, not one of 
 which is very convenient or of great accuracy. The first 
 of these depends on the fact referred to above, that hydro- 
 chloric acid liberates uric acid from its combinations, pre- 
 cipitating it in crystalline form. 
 
256 THE ANALYSIS OF URINE. 
 
 Precipitation Test. Measure out 200 Cc. of urine and 
 add to it 20 Cc. of strong hydrochloric acid. Mix thor- 
 oughly and set aside in a cool place for about 48 hours. 
 At the end of this time collect the reddish-yellow deposit 
 on a weighed filter, wash it with a little cold water dry and 
 weigh. Not over 30 or 40 Cc. of water should be used in 
 the washing. The precipitated uric acid is not pure, hold- 
 ing coloring and other substances which increase its weight. 
 On the other hand, it is soluble to some extent even in cold 
 acidulated water so that not the whole of it is obtained on 
 the filter and a correction must be made. It is usually 
 recommended to add to the weight obtained 4.8 Mg. for 
 each 100 Cc. of filtrate and washings. 
 
 If the urine under examination contains albumin, the 
 latter must be coagulated by heating with a drop or two of 
 acetic acid and filtered out, before the test is made. If the 
 urine is very cold to begin with and has a sediment of 
 urates the latter must be brought into solution by warm- 
 ing before beginning the test. To prevent precipi- 
 tation of phosphates during the warming a few 
 drops of hydrochloric acid may be added. This method 
 is at best only a rough approximation, but is the 
 one by which most of our results have been obtained. 
 The following gives better results: 
 
 Salkowski-Ludwig Method. The determination 
 here is based on the fact that uric acid gives a very insolu- 
 ble precipitate with ammoniacal solution of silver nitrate, 
 from which precipitate after filtration and washing the acid 
 may be readily separated, brought into concentrated solu- 
 tion, reprecipitated and weighed. 
 
 In using the method the following solutions are 
 required. 
 
THE ANALYSIS OF URINE. 257 
 
 (a.) Ammoniacal Silver Nitrate. Dissolve 25 Gm. 
 of silver nitrate in 100 Cc. of distilled water, add 
 ammonia water until the precipitate which ap- 
 pears at first is completely redissolved, leaving a 
 clear solution. Make this up to 1,000 Cc. with 
 distilled water and keep in a dark bottle or away 
 from the light. 
 
 (b.) Magnesia Mixture. Made as described in the 
 appendix. It must be strongly alkaline and clear, 
 or nearly so. 
 
 (V.) Solution of Potassium or Sodium Sulphide. 
 
 The pure crystals of sodium sulphide obtained 
 from dealers in chemicals may be used by dissolv- 
 ing 25 to 30 Gm. (Na^S, 9H 2 0) in 1,000 Cc. of 
 distilled water. A solution may be made, also, by 
 dissolving 10 Gm. of pure sodium hydroxide in 
 1,000 Cc. of water, and converting this into sul- 
 phide which is done as follows: Divide the solu- 
 tion into two equal portions. Saturate one 
 thoroughly with hydrogen sulphide and to this 
 then add the other half. Keep in a glass stop- 
 pered bottle, the stopper paraffined. 
 
 To make the test measure out 200 Cc. of the urine and 
 transfer to a beaker. Add 20 Cc. of the silver solution, (a), 
 to an equal volume of the magnesia mixture, (J>), and then 
 ammonia enough is added to clear up any precipitate which 
 forms. This clear mixture is now poured into the urine in 
 the beaker and the whole well stirred. A precipitate of 
 silver urate forms along with silver and earthy phosphates. 
 The excess of ammonia prevents the precipitation of silver 
 chloride. Silver urate is quite insoluble in ammonia; it is 
 gelatinous alone and does not settle very well but the phos- 
 
258 THE ANALYSIS OF URINE. 
 
 phate precipitate corrects this difficulty to some extent. 
 The beaker is allowed to stand at rest about an hour, after 
 which the contents are filtered and the precipitate washed 
 with weak ammonia on the filter. To do this the ammonia 
 is sprayed into the beaker from a wash bottle and rinsed 
 around thoroughly. This is done several times, the liquid 
 being poured on the filter. Where available a Gooch cru- 
 cible serves admirably for the collection of the precipitate 
 as the filtration is slow on paper without aspiration. It is 
 not necessary to remove any of the precipitate which clings 
 to the beaker, as will be seen. When the washing is com- 
 plete transfer the precipitate and filter paper, or asbestos if 
 the Gooch crucible is used, back to the beaker and pour 
 over it a boiling mixture of 20 Cc. of the sulphide solution, 
 (<r), and 20 Cc. of distilled water. Stir up thoroughly, al- 
 low to stand some time and then add 50 Cc. of boiling 
 water. Place the beaker on a sand-bath or gauze and bring 
 the contents to boiling, stirring continually. Keep hot 
 some minutes and then allow to stand until cold, the pre- 
 cipitate being stirred meanwhile occasionally. 
 
 The treatment with the sulphide solution decomposes 
 the silver urate with precipitation of black insoluble silver 
 sulphide, the uric acid remaining in solution as soluble 
 urate. The cooled liquid is filtered into a porcelain dish, 
 and the precipitate washed with warm water, the washings 
 going also into the dish. Enough hydrochloric acid is now 
 added to combine with all the bases present and liberate 
 the uric acid, which is the case when the liquid becomes 
 acid in reaction. It is now slowly evaporated to a volume 
 of about 10 Cc, best on a water-bath, and then allowed to 
 stand an hour for the complete separation of the uric acid. 
 This is then collected on a weighed Gooch crucible, the 
 crystals being transferred gradually by aid of the filtered 
 liquid. When the crystals are on the asbestos they are 
 
THE ANALYSIS OF URINE. 259 
 
 washed with a little acidulated water several times. The 
 crucible is then dried at 100°, put back in the funnel and 
 treated with a small amount of pure carbon bisulphide to 
 remove traces of sulphur separated on decomposing the 
 alkali sulphide. Finally, wash with ether, dry at 100° C. 
 and weigh. The results are always a little low, but fairly 
 regular. 
 
 As the acid is finally precipitated from a very small 
 volume of liquid and but little water is used in washing no 
 correction need be made for solubility, as in the first 
 process described. While simple enough in principle and 
 easily carried out considerable time is required for the per- 
 formance of all the operations involved in the method. 
 
 The following method is free from this objection and is 
 equally accurate: 
 
 Haycraft Method. This depends on the precipitation 
 of the uric acid, as silver urate, and its subsequent titration 
 by standard solution of ammonium sulphocyanate. The 
 following solutions are required: 
 
 («.) Ammoniacal Solution of Silver Nitrate. Dis- 
 solve 5 Gm. of crystals in 100 Cc. of water and then 
 add enough ammonia water to give the solution a 
 strong alkaline reaction. Make up to 200 Cc. 
 with the ammonia. 
 
 {b.) Ammonium Sulphocyanate. This solution is 
 made as described later for the determination of 
 chlorides in urine by the Volhard method. It is 
 given just one-fifth the strength there described 
 and may be made by diluting 100 Cc. of that so- 
 lution to 500 Cc. in a measuring flask. 
 
 (c.) Ammonium Ferric Sulphate, (ferric alum), 
 saturated solution as indicator. Described under 
 the chlorine test. 
 
260 THE ANALYSIS OF URINE. 
 
 It has been shown by Dr. Haycraft that silver combines 
 with uric acid in constant and definite proportion, viz., one 
 atom of silver to one molecule of the acid, or 108 parts of 
 silver to 168 of the acid, giving the formula AgC 6 H 3 N 4 3 . 
 
 This precipitate dissolves in dilute nitric acid and if the 
 solution so obtained is treated with the ammonium sulpho- 
 cyanate the following reaction takes place: 
 
 AgC 6 H 3 N 4 3 +NH 4 SCN= 
 
 AgSCN+NH 4 C 6 H 3 N 4 3 . 
 
 From this it follows that one cubic centimeter of a fif- 
 tieth normal, (tt)> solution of the sulphocyanate liberates 
 and indicates .00336 Gm. of uric acid. 
 
 It is fully explained under the chlorine test that if a so- 
 lution of a sulphocyanate is added to a solution of a sil- 
 ver salt containing nitric acid and ferric sulphate a com- 
 plete reaction takes place between the sulphocyanate and 
 silver before the characteristic reaction between the former 
 salt and the ferric compound appears. In other words, 
 the sulphocyanate and the silver combine first and then 
 any further amount of sulphocyanate added unites with the 
 iron, producing a red color (of ferric sulphocyanate) indi- 
 cating the completion of the first reaction. 
 
 With these general explanations the process will now be 
 understood. 
 
 Measure out 50 Cc. of the urine and warm it gently if 
 it contains a sediment of urates. Add 3 to 4 Gm. of pure 
 sodium bicarbonate and then ammonia enough to give a 
 strong alkaline reaction. This may give a precipitate of 
 phosphates which need not be heeded. Next add 5 Cc. of 
 the silver solution, («), and mix thoroughly. This pro- 
 duces a precipitate of silver urate along with the bulky 
 phosphates thrown down by the ammonia. Allow to stand 
 half an hour and then filter. A paper filter and funnel 
 
THE ANALYSIS OF URINE. 261 
 
 may be used in the usual manner, but much better results 
 are obtained by the use of the Gooch crucible and asbestos 
 with aid of an aspirator. Rinse the sides of the beaker 
 thoroughly with weak ammonia and pour this on the pre- 
 cipitate in the funnel or crucible. Continue the washing 
 of the precipitate with weak ammonia water until all traces 
 of silver are washed out, as may be shown by allowing a 
 few drops of the filtering washings to fall into some dilute 
 hydrochloric acid in a test-tube. The washing is complete 
 when a cloudiness is no longer obtained in this test. 
 
 Now pour some pure dilute nitric acid into the beaker 
 in which the precipitation was made, and which was 
 washed free from silver by the ammonia, and shake it 
 around until any traces of the silver urate precipitate are 
 dissolved. Put the funnel or Gooch crucible over a clean 
 receptacle and pour this acid liquid on the precipitate. 
 Silver urate dissolves completely in dilute nitric acid, and 
 enough of this is added, a little at a time, to bring about 
 complete solution. It now remains to titrate the silver in 
 this solution. To this end add 5 Cc. of the ferric alum so- 
 lution, and if the mixture is not clear and colorless, about 
 2 Cc. of pure strong nitric acid. Then from a burette run 
 in the sulphocyanate, (b), a little at a time, shaking after 
 each addition until a faint red shade of ferric sulpho- 
 cyanate becomes permanent. Toward the end of the ti- 
 tration a red appears as each drop of liquid from the 
 burette falls into the silver solution below, but this color 
 fades out on shaking and does not persist until the last 
 particle of silver has been taken up by the sulphocyanate. 
 Supposing now that 15 Cc. of the latter solution are re- 
 quired to reach this point we have 15 X .00336 =.0504 Gm. 
 as the amount of uric acid in the 50 Cc. of urine taken. 
 A volume as large as this would seldom be required, 5 to 
 10 Cc, corresponding to 16.8 to 33.6 Mg., is usually sufficient. 
 
262 THE ANALYSIS OF URINE. 
 
 The method gives results which are a little too high as 
 the silver carries down traces of other bodies as well as 
 uric acid. But the error is not great enough to interfere 
 with the practical application of the process where even 
 the best results are desired. The washing of the precipi- 
 tate of silver urate is the point which requires the greatest 
 care. A little practice will show how this can be best 
 done. 
 
 Hippuric Acid. 
 
 This acid is found in very small amount normally in 
 human urine, and is the chief nitrogenous product in the 
 urine of the herbivora. It is increased in human urine by 
 a diet of aromatic vegetable substances, but is never 
 abundant enough to have clinical importance. 
 
Chapter XV. 
 
 UREA. 
 
 I TREAisthe important nitrogenous substance excreted 
 ^ in human urine. A large part of the nitrogen of our 
 food is normally converted into urea for elimination from 
 the body. How this conversion takes place or where is not 
 known. That a part, at least, may be formed in the liver 
 from ammonium carbonate has been shown to be probable 
 but the connection between it and the antecedent muscu- 
 lar tissue is still very obscure. Not far from 90 per cent 
 of the nitrogen consumed as food is excreted as urea, but 
 the absolute amount of the latter passed in a day is exceed- 
 ingly variable. In the urine of the average man it is between 
 30 and 40 Gm. while in the urine of women it is less. The 
 variations depend mainly on the diet, the urea being high- 
 est with a diet rich in meat, eggs, beans, peas and similar 
 vegetables, and low with a diet of fruits, bread and po- 
 tatoes. The percentage amount of urea depends further on 
 the volume of the urine passed in a day and may vary from 
 a change in the amount of water consumed and also from 
 different losses by perspiration. The percentage amount 
 of urea depends also on the time when the urine is voided. 
 A determination of value- should therefore be made on the 
 mixed urine of the 24 hours. 
 
 It is usually assumed that 2 per cent is the average 
 amount excreted in health, but this is probably low. While 
 the variations from this mean are great in health they are 
 much more marked in pathological conditions. 
 
 Urea is increased in total amount although it may be 
 
264 THE ANALYSIS OF URINE. 
 
 diminished in percentage in diabetes mellitus and insipidus 
 and also in fevers. It has been found to be increased in 
 cases of poisoning by heavy metals, but why is not clearly 
 demonstrated. 
 
 Clinically, the increase in diabetes and fevers is of the 
 greatest interest because we have here evidence of in- 
 creased consumption of the nitrogenous tissues of the 
 body. A diminished elimination of urea has been ob- 
 served in acute yellow atrophy of the liver and in other 
 diseases of that organ. This has been taken to indicate 
 that the liver may be the place of formation of urea. In 
 cases of malnutrition in general the absolute and percent- 
 age amount of urea may be greatly diminished. 
 
 A marked decrease has been observed, also,' in diseases 
 involving structural changes in the tubules of the kidney 
 as in parenchymatous nephritis. 
 
 Recognition of Urea. Because of its extreme solu- 
 bility urea cannot be easily obtained by evaporation of 
 urine. It has been shown, however, in an earlier chapter 
 that by concentrating the urine slowly to a small volume — 
 to one-third or one-fourth — cooling and adding strong 
 nitric acid, a crystalline precipitate of plates of urea 
 nitrate separates which is characteristic. From this pre- 
 cipitate pure urea can be obtained. 
 
 Clinically, this test has no importance as we are con- 
 cerned only with a measurement of the amount of urea. 
 This determination can be made in several ways, but in 
 actual practice we employ three essentially different 
 methods. The first depends on the fact that solutions of 
 urea precipitate solutions of certain metals in a definite 
 manner from which a volumetric process has been derived. 
 The second depends on the fact that solutions of certain 
 oxidizing agents decompose solutions of urea with the lib- 
 
7V/E ANALYSIS OF URINE. 265 
 
 eration of its nitrogen (and carbon dioxide) in gaseous 
 form. From the known relations between weight and vol- 
 ume of the gas, and weight of nitrogen and weight of urea 
 the absolute amount of the latter may be calculated. The 
 third method depends on the fact that when the urea of 
 urine is decomposed into water, carbon dioxide and nitro- 
 gen its specific gravity is decreased in a manner empiri- 
 cally determined. The loss in specific gravity bears a 
 certain relation to weight of urea present. 
 
 Determination of Urea. 
 
 Liebig's Method. We have here the oldest, and in 
 many respects the best of our processes for the titration 
 of urea. The principle involved in the method is this. 
 When a solution of mercuric nitrate is added to a solu- 
 tion of urea a white precipitate forms and settles out. By 
 working with solutions of a certain definite concentration 
 it has been found that the reaction between the mercury 
 and urea takes place in constant proportion and accord- 
 ing to this equation : 
 
 2CON 2 H 4 +4Hg(N0 3 ) 2 +3H 2 = 
 
 2CON 2 H 4 .Hg(N0 3 ) 2 .3HgO+6HNO 3 
 
 This precipitate contains 10 parts of urea for every "72 
 parts of HgO. 72 Gm. of HgO dissolved in HN0 3 
 should precipitate, therefore, 10 Gm. of urea. 
 
 The same solution of mercury gives a yellow precipi- 
 tate with solution of sodium carbonate which is used as an 
 indicator in a manner to be described. 
 
 The urea solution to be analyzed is poured into a 
 beaker and standard solution of the mercuric nitrate added 
 gradually, with constant stirring from a burette. From 
 time to time a drop of the liquid above the precipi- 
 tate is taken on the end of a glass rod and brought in con- 
 
266 THE ANALYSIS OF URINE. 
 
 tact with a few drops of a concentrated solution of sodium 
 carbonate on a plate of dark glass. A yellowish precipitate 
 forms here if the drop contains any excess of the mercury 
 compound beyond that necessary to precipitate the urea. 
 The end of the reaction is frequently determined in this 
 manner, as in the original process, but not with greatest 
 accuracy. A modified process as now to be explained is 
 preferable, and easily carried out. 
 
 As the equation above shows, nitric acid is set free in 
 the reaction between the urea and the mercuric nitrate. 
 This acid has a decomposing effect on the precipitate, 
 tending to form new nitrate and thus diminish the amount 
 which, theoretically, should be added for complete pre- 
 cipitation. The nitric acid must therefore be neutralized 
 from time to time as formed, or better, just before the 
 end reaction with the indicator is tried. 
 
 It has been found, also, that to precipitate exactly 10 
 Mg. of urea in this manner, not 72 Mg. of mercuric oxide 
 in solution, but a slightly greater amount must be used. 
 The experiments of Pflueger showed that 77.2 ' Mg. is 
 needed for the purpose and the standard solution should 
 be made to contain 11.2 Gm. per liter. 
 
 In the titration of urine certain modifications must be 
 made which are not necessary in the titration of pure urea 
 solutions. The phosphates, sulphates and chlorides of 
 urine interfere with the reaction and must be removed 
 before the test is begun. 
 
 The phosphates and sulphates may be removed by pre- 
 cipitation with barium solution, while the chlorides may 
 be thrown out by silver nitrate. It is also possible to 
 make a correction for the chlorides instead of precipitating 
 them. The following solutions are necessary in making 
 the test. 
 
THE ANALYSIS OF URINE. 267 
 
 (a). Mercuric Nitrate Solution. This is made of 
 definite strength and should contain the equiva- 
 lent of 77.2 Gm. of the oxide in one liter. In 
 making this solution we may start with pure 
 metallic mercury, with mercuric oxide or with the 
 commercial nitrate (mercurous). With mercury 
 it can be made in this manner: Weigh out a 
 quantity of pure mercury and heat it in a porce- 
 lain dish or casserole with two to three times its 
 weight of strong nitric acid of 1.42 Sp. Gr. When 
 the mercury is in solution evaporate to the con- 
 sistence of a thick syrup and add from time to 
 time a few drops of nitric acid to complete the 
 oxidation. When the addition of the acid is no 
 longer followed by the evolution of red fumes the 
 action is complete and the mercury exists as 
 mercuric salt. Now pour into the syrupy residue 
 ten times its volume of water with constant stir- 
 ring. In adding the water it always happens 
 that a little of the nitrate is decomposed and 
 thrown down as a basic salt. Allow the liquid to 
 settle thoroughly, pour off from the sediment and 
 dissolve the latter in a few drops of nitric acid. 
 Add this now to the main solution and dilute it 
 with distilled water to make 1 liter of each 71.5 
 Gm. of mercury. 
 
 When mercuric oxide is employed, weigh out the proper 
 amount, dissolve it in a slight excess of strong, pure nitric 
 acid, evaporate to a syrup and treat as above. Finally, 
 dilute with water to yield a solution with 77.2 Gm. to the 
 liter. 
 
 (£). Baryta Solution to* precipitate phosphates and 
 sulphates. To one volume of a cold saturated 
 
268 THE ANALYSIS OF URINE. 
 
 solution of barium nitrate add two volumes of a 
 cold saturated solution of barium hydroxide. 
 Keep in a well stoppered bottle. 
 
 (c). Sodium Carbonate Solution. This is best made 
 of the pure, dry carbonate readily obtained as a 
 commercial article. It must be remembered, 
 however, that the so-called dry carbonate contains 
 a little water, which may be removed by heating, 
 in a platinum dish, to low redness. Dissolve 53 
 Gm. of the salt thus dried, in water and dilute to 
 one liter. 
 
 A mercury solution made of pure material according to 
 the above directions should have the correct strength, but 
 for control it may be tested by means of a solution of pure 
 urea in water. 
 
 (</). Standard Urea Solution. Weigh out 2 Gm. of 
 pure urea, which is now readily obtained, and dis- 
 solve it in distilled water to make 100 Cc. 
 
 Before making the test proper it is necessary to deter- 
 mine the relation of the sodium carbonate solution to the 
 mercury solution in presence of urea. This may be done 
 by taking exactly 10 Cc. of the urea solution and adding to 
 it 19 Cc. of the solution of mercuric nitrate. Shake thor- 
 oughly, allow to stand a minute and filter. Wash the 
 precipitate with a little distilled water, and to the mixed 
 filtrate and washings add two drops of a weak solution of 
 methyl orange, or enough to give a pink color. Then from 
 a burette run in the solution of sodium carbonate, with 
 constant shaking, until the pink' color changes to yellow. 
 The reaction is sharp enough for the purpose. Not over 
 11.5 Cc. of the alkali solution should be required for this. 
 Calculate the amount needed for each Cc. of mercuric 
 nitrate. Proceed now with the actual test. 
 
THE ANALYSIS OF URINE. 269 
 
 Measure 10 Cc. of the standard urea solution again and 
 run in 19.5 Cc. of the mercuric nitrate. Add now the cor- 
 rect number of cubic centimeters of soda solution required 
 to neutralize the acid from the nitrate as calculated from 
 the results of the last experiment. Then by means of a 
 stirring rod bring a drop of the liquid in the beaker in con- 
 tact with a drop of a semifluid mixture of sodium bicar- 
 bonate and water on a dark glass plate. Stir the two 
 together and observe the color. It should be white. Run 
 in two or three drops more of the mercury solution, 
 stir well and repeat the test, and continue until a slight 
 yellow color is obtained on mixing the drops from the 
 beaker with the moist bicarbonate. If the mercury solu- 
 tion is correct just 20 Cc. should be used for this. 
 
 The sodium bicarbonate used as indicator must be pure, 
 especially as regards freedom from chloride. An excess of 
 it can be washed in a beaker several times with a little cold 
 water, which is poured off leaving the salt finally in a pasty 
 condition suitable for use. 
 
 We proceed now to the actual test of a sample of urine. 
 Measure off accurately a definite volume and add to it just 
 half its volume of the baryta solution. Convenient pro- 
 portions are 50 Cc. of urine and 25 Cc. of the baryta solu- 
 tion. Shake thoroughly and filter through a dry filter 
 into a flask. Measure out now exactly 15 Cc. of this 
 filtrate which contains the urea of 10 Cc. of the original 
 urine, the baryta solution having taken out only phos- 
 phates, sulphates and carbonates, with certain bases. 
 This filtrate still contains chlorides which are objection- 
 able and which could be separated by another precipi- 
 tation with the proper amount of silver nitrate. It will 
 be, however, more convenient and fully as accurate to 
 make a correction for them at the end of the test, as will be 
 explained. The 15 Cc. of filtrate has an alkaline reaction 
 
270 THE ANALYSIS OF URINE '. 
 
 and must be neutralized. (If not alkaline a new precipita- 
 tion must be made, taking equal volumes of urine and bary- 
 ta solution, and finally 20 Cc. of the nitrate.) The neutral- 
 ization can be effected by adding carefully, a drop at a 
 time, dilute nitric acid, testing with litmus paper. 
 
 The nitrate thus prepared is titrated with the mercury 
 solution. Begin by adding a Cc. at a time and after each 
 addition bring a drop of the mixture in contact with a drop 
 of the semifluid sodium bicarbonate on a plate of dark 
 glass. The drops should be placed side by side and mixed 
 at the edges. At first the mixture remains white, even 
 after stirring, but as the addition of mercury is continued a 
 point is reached where the drop from the beaker brought in 
 contact with the moist bicarbonate gives a light yellow 
 shade. On stirring the drops together this yellow should 
 disappear, but this shows that the end of the reaction is 
 nearly reached. Add now the mercury solution in drops 
 and test after each addition. When the point is reached 
 where a faint yellow shade persists after stirring together 
 the drop from the beaker and the sodium bicarbonate it is 
 time to neutralize with the normal sodium carbonate solu- 
 tion. Run in the right number of cubic centimeters cor- 
 responding to the mercury used and now make the test for 
 the final reaction again and continue until the yellow color 
 appears. 
 
 Regard this test as preliminary and make a new one 
 with 15 Cc. of the filtrate neutralized as before. Run in 
 directly within 1 Cc. of the amount of mercury required, as 
 shown by the first test, neutralize and complete as before. 
 For each cubic centimeter used, after deducting for chlorides, 
 calculate 10 Mg. of urea. The correction for the chlorides 
 is based on the following principle : In the presence of 
 sodium chloride, or other chloride, the reaction between 
 mercuric nitrate and urea does not begin until enough of 
 
THE ANALYSIS OF URINE. 271 
 
 the former has been added to form mercuric chloride with 
 all chlorine present, according to the following equation: 
 
 Hg(N0 3 ) 3 +2NaCl=2NaN0 3 + HgCl 2 
 
 For the nitrate corresponding to 216 parts of HgO we 
 use here 1 1*7 parts of salt, or for 117 milligrams of salt, 
 216 milligrams of mercuric oxide, or 2.79 Cc. of the stand- 
 ard mercuric nitrate solution. 
 
 One milligram of sodium chloride, therefore, combines 
 with the mercury compound in .0238 Cc. of the standard 
 solution. Mercuric chloride does not react with the 
 sodium bicarbonate, and the amount formed is beyond that 
 shown by the titration. Therefore, to apply the correc- 
 tion, determine the chlorides present in 10 Cc. of urine (by 
 a process to be given later) calculate to sodium chloride, 
 and for each milligram found deduct .0238 Cc. from the 
 volume of the mercuric nitrate solution used in the titra- 
 tion. As the amount of chlorine in the urine is about 
 equivalent to one per cent of salt in the mean, an approxi- 
 mate correction is often made by subtracting 2 Cc. from 
 the volume of the mercury solution. 
 
 The above calculations are based on the supposition 
 that the original urine contains 2 per cent of urea, and 
 that a volume of about 20 Cc. of mercuric nitrate is used in 
 the titration. If the per cent of urea is much greater or 
 less than this a correction on account of volume must be 
 made. This correction has been worked out empirically 
 by Pflueger, and, without discussing how it is derived, it 
 will be sufficient to explain its application. 
 
 If more than two per cent of urea is present it is neces- 
 sary to add more than 20 Cc. of the mercuric solution in 
 titration. If the volume of the latter solution is greater 
 than the sum of the volumes of the prepared urine and 
 soda solution used in neutralization, this sum must be sub- 
 
272 THE ANALYSIS OF URINE. 
 
 tracted from the volume of the mercury solution and the 
 result multiplied by 0.03. The product is added to the 
 number of cubic centimeters of mercuric nitrate used, to 
 give the corrected result. If, on the other hand, the 
 volume of -mercuric nitrate used in titration is less than the 
 sum of the volumes of prepared urine and soda solution 
 the difference is multiplied by 0.08 and the product taken 
 from the number of Cc. of mercuric solution used, to 
 give the corrected result. In these calculations the vol- 
 ume of mercuric nitrate taken up by the chlorides must be 
 considered as part of the diluting liquid. The same must 
 be remembered in adding sodium carbonate for neutraliza- 
 tion. 
 
 The correction may be expressed in this formula, ac- 
 cording to Pflueger: 
 
 C=— (V,— V 3 )X0.08, 
 in which 
 
 C = the correction to be added or subtracted. 
 
 Vj = the sum of the volumes of the urine, soda solu- 
 tion and mercuric nitrate combined with the 
 chlorides. 
 
 V a = the volume of mercuric nitrate taken by urea. 
 
 In illustration we may take an actual case: 
 
 15.0 Cc. = the prepared urine (neutralized). 
 14.8 Cc. = the sodium carbonate used. 
 1.8 Cc. = the mercuric solution used by chlorides. 
 
 V, = 32.6 Cc. 
 V„ = 26.0 Cc. 
 
 6.6 
 — (Vj — V 2 )X0.08 = — 0.528 = C. 
 
 Therefore, 26 — 0.5 = 25.5 is the corrected volume of 
 mercuric nitrate, indicating, with the latter solution of 
 standard strength, 25.5 Gm. of urea in a liter. 
 
THE ANALYSIS OF URINE. 273 
 
 Method by Liberation of Nitrogen. — A solution of 
 urea is decomposed by a solution of a hypochlorite or hy- 
 pobromite as illustrated by this equation. 
 
 CON 2 H 4 +3NaOCl = CO a +N g +2H 2 0+3NaCl. 
 
 That is, nitrogen and carbon dioxide gases are given 
 off. If the reaction is allowed to take place in an alkaline 
 medium the carbon dioxide will be held and the nitrogen 
 alone given off. The volume liberated is a measure of the 
 weight of urea decomposed. From the above equation it 
 is seen that 28 parts by weight of nitrogen correspond to 
 60 of urea, from which follows that one cubic centimeter of 
 pure nitrogen gas, measured at a temperature of 0°C and 
 under the normal pressure of 760 Mm. corresponds to 
 .00269 Gm. of urea. One Gm. of urea furnishes 371.4 Cc. 
 of nitrogen gas. 
 
 In employing these principles in practice it is simply 
 necessary to bring together a measured volume of the 
 urine or urea solution and the hypochlorite or hypobro- 
 mite reagent under such conditions that all of the nitrogen 
 liberated may be collected and accurately measured. 
 
 As a reagent a solution of sodium hypobromite is very 
 commonly employed. As it does not keep well it must be 
 made fresh for use, which is inconvenient unless many tests 
 have to be made at one time. The reagent may be pre- 
 pared in this manner: 
 
 Dissolve 100 Gm. of good sodium hydroxide in 250 Cc. 
 of water. When cold add 25 Cc. of bromine by means of 
 a funnel tube carried to the center of the solution. The 
 bromine must be poured into the funnel, a little at a time, 
 and the lower end moved around to act as a stirrer and 
 mix the liquids. During the reaction the bottle or flask 
 containing the alkali should be surrounded by cold water, 
 and the mixture should be made out of doors or in a good 
 
274 
 
 THE ANALYSIS OF URINE. 
 
 fume closet. The finished solution contains an excess of 
 alkali sufficient to hold the 
 carbon dioxide given off when 
 it reacts on urea. In place of 
 this solution a strong hypo- 
 chlorite solution may be used 
 with advantage. The U. S. 
 P. solution when properly made 
 answers very well. Its prepa- 
 ration is given in the appen- 
 dix. 
 
 Many forms of apparatus 
 have been devised for this 
 purpose, one of the oldest and 
 best known of which is that 
 of Huefner, shown in Fig. 42. 
 
 At A below is a small ves- 
 sel, the bottom of which rests 
 on the support, and which 
 holds the urine to be decom- 
 posed. The capacity of this 
 vessel is usually between 5 
 and 10 Cc, but must be ac- 
 curately determined by filling 
 with mercury, pouring this out 
 and weighing it. Above A, 
 and separated from it by a 
 ground glass stopcock, is the 
 much larger vessel B, which 
 holds the hypochlorite or hy- 
 pobromite reagent. On the 
 narrow neck of B rests a cup- 
 shaped receptacle, C, to hold 
 water. Finally, over B a measuring tube is supported in 
 
 PIG. 48. 
 
THE ANALYSIS OF URINE. 275 
 
 such a manner that it must receive any gas passing up 
 from B. At the beginning of the experiment this meas- 
 uring tube D is filled with water. 
 
 The apparatus is used in the following manner: Rinse 
 out A and B and pour into the latter more than enough 
 urine to fill A. Open the stopcock and allow the urine to 
 flow down, which may be assisted when slow by moving a 
 thin glass rod or bit of wire up and down through the open- 
 ing in the stopper. When A is quite full close the stopper, 
 rinse the urine from B and fill it with the hypobromite 
 reagent. Now fill the cup C with water so that its surface 
 is one or two centimeters above the opening into B, fill the 
 graduated tube with water, close the end with the thumb 
 and invert it in the cup. Finally fasten it in position over 
 B as shown. 
 
 The stopcock is now opened which permits the heavier 
 reagent to sink and slowly mix with the urine. A libera- 
 tion of gas soon begins and proceeds slowly. At the end 
 of twenty or thirty minutes the reaction is complete, which 
 can be noted by the disappearance of the gas bubbles. 
 Close the end of the gas tube with the thumb, remove it 
 and immerse in a jar of water, having the temperature of 
 the air, until the water levels inside the tube and in the 
 jar, are the same. Clamp the tube in position and allow it 
 to stand until the temperature of the gas becomes con- 
 stant, which may require fifteen minutes. Finally adjust 
 the tube level again if necessary, read the volume of the 
 gas, note the height of the barometer and the temperature 
 as given by a thermometer hanging near the tube and re- 
 duce this volume to standard conditions by the following 
 formula: 
 
 „ -., b — w 
 
 (l+.00366t)760 
 
276 
 
 THE ANALYSIS OF URINE. 
 
 In this formula 
 
 V = the corrected volume. 
 V„ = the observed volume, 
 b = the barometric height, 
 w = the tension of water vapor at the observed 
 
 temperature, in millimeters of mercury, 
 t = the temperature as observed. 
 
 The values for w at different temperatures are given in 
 this table. 
 
 10° 9.165 Mm. 
 
 11° 9.792 Mm. 
 
 12° 10.457 Mm. 
 
 13° 11.162 Mm. 
 
 14° 11.908 Mm. 
 
 15° 12.699 Mm. 
 
 16° 13.536 Mm. 
 
 17° 14.421 Mm. 
 
 18° 15.357 Mm. 
 
 19° 16 346 Mm. 
 
 20° 17.391 Mm. 
 
 21° 18.495 Mm. 
 
 22° 19.659 Mm. 
 
 23° 20.888 Mm. 
 
 24° 22.184 Mm. 
 
 25° 23 550 Mm. 
 
 Having thus the volume of the gas under normal con- 
 ditions, the weight of urea corresponding can be calculated 
 by data already given. 
 
 The results obtained by this method are too low and 
 must be corrected, as will be explained later. 
 
 Another form of urea apparatus which can be con- 
 structed by any one is shown in Fig. 43. 
 
 The tall jar is filled with water which must stand until 
 it has the air temperature. A 50 Cc. burette is inverted in 
 the jar, the delivery end being connected with a bottle 
 holding about 150 Cc. by means of a piece of firm rubber 
 tubing. The rubber tube is slipped over a short glass tube 
 passing through the hole in a rubber stopper which must 
 close the bottle accurately. In the bottle is a short stout 
 test-tube, or vial, holding about 10 Cc. and which contains 
 the urine to be tested. Into the bottle itself is poured the 
 reagent as above described, which must not reach to the 
 top of the test-tube. On mixing the liquids the urea 
 
THE ANALYSIS OF URINE. 
 
 377 
 
 decomposes, liberating the gas as before, which passes 
 through the rubber tube and displaces water in the burette 
 so that the volume can be readily determined. 
 
 The test is made practically in this manner: Pour 
 about 20 Cc. of the strong hy- 
 pobromite or 40 Cc.of the weaker 
 hypochlorite reagent into the 
 bottle. With a pipette measure 
 some exact volume of urine, 
 usually 5 Cc.into the small test- 
 tube, and by means of small iron 
 ki | forceps place the latter carefully 
 
 j ! in the bottle containing the re- 
 
 \ i agent. Next insert the stopper 
 
 | i which connects the bottle with 
 
 ■ j the burette standing in the jar 
 
 ; i of water. The level of the water 
 
 I in the burette is displaced by 
 
 ) this operation. Now allow the 
 
 apparatus to remain at rest ten 
 , minutes until the air in the mix- 
 
 | ing bottle and tube has the tem- 
 j !: .. i perature of the outside air. 
 
 | j Through handling it of course 
 
 IKJlL liliS becomes warmer. At the end of 
 \j**«m*~^*r t jj e time, by means of the at- 
 fig. 43. tached clamp and not by the 
 
 hand, lift the burette, or depress 
 it if necessary, until the water levels inside and outside 
 are the same. Note the position of the water on the . 
 burette graduation. Read the air temperature by a ther- 
 mometer, which should be suspended near the tube, and 
 observe the height of the barometer. 
 
 Incline the bottle to mix the urine and the reagent j 
 
278 THE ANALYSIS OF URINE. 
 
 shake gently and repeat these operations from time to time. 
 On the decomposition of the urea nitrogen gas or its 
 equivalent volume of air passes over into the burette and 
 forces out some of the water. When, after repeatedly 
 shaking the bottle, no increase of the gas volume in the 
 burette can be observed allow the whole apparatus to stand 
 until the contents of the bottle and burette have cooled 
 down to the air temperature again. Then lift the burette, 
 with the clamp as before, to restore the levels and read the 
 gas volume. From this substract the volume at the first 
 reading. The difference is the volume of nitrogen gas 
 liberated in the reaction, at the observed temperature and 
 atmospheric pressure. If, as sometimes happens, more 
 gas is liberated than the burette will hold, repeat the ex- 
 periment, using urine diluted with an equal volume of 
 water. 
 
 The calculations are made as in the case given above, 
 as the gas volume is finally measured under the same con- 
 ditions. It is assumed here that the air temperature and 
 barometric pressure remain constant during the experi- 
 ment. 
 
 Exact .investigations have shown that the whole of the 
 nitrogen is not liberated in the reaction, as at first assumed, 
 but falls short between seven and eight per cent. It ap- 
 pears that four or five per cent of the nitrogen of the urea 
 is oxidized to nitric acid in the reaction and escapes 
 measurement. Another small portion is left in the am- 
 moniacal condition. Attempts have been made to prevent 
 this abnormal oxidation by adding to the urine a reducing 
 agent to destroy nitric acid as fast as formed. Dextrose 
 has been used for the purpose, also cane sugar, and ap- 
 parently with success. But a great excess of sugar must be 
 added. 
 
 It has been shown, also, that the theoretical yield of 
 
THE ANALYSTS OF URINE. 279 
 
 nitrogen can be approached by a modified method of apply- 
 ing the reagent. J. R. Duggan, working in Remseris labor- 
 atory, found that by mixing the alkali with the urine and 
 then adding the bromine so as to form hypobromite in pres- 
 ence of the urea the yield of nitrogen is much increased, 
 reaching nearly the amount called for by theory. The 
 simple bottle and burette apparatus may be used in this 
 manner by measuring the alkali, 20 Cc. of 20 per cent 
 sodium hydroxide solution, and urine, 5 Cc. into the bottle, 
 and the bromine, about 1 Cc, into the test-tube. The mix- 
 ture is made and process completed as before. The modi- 
 fication has not come into general use, probably because 
 of the objection to working with free bromine, at each 
 test. As the deficiency by the usual method has been 
 shown to be nearly eight per cent, it is sufficient for most 
 purposes to assume that the volume of gas obtained is 92 
 per cent of the whole and correct by calculation. 
 
 It remains now to describe two forms of apparatus 
 which are used without any correction, for rapid clinical 
 tests. 
 
 The Squibb Apparatus is the first of these, and one 
 which can be very highly recommended. The construc- 
 tion of the apparatus is shown by Fig. 44. 
 
 The upright bottle to the left contains the reagent, 
 hypochlorite or hypobromite as before. By means of a 
 pair of small forceps a short test-tube, F, containing a 
 measured volume of the urine is dropped into the bottle, 
 but carefully so that the liquids will not mix until the bot- 
 tle is shaken. A bent glass tube and a rubber tube con- 
 nect this with the bottle B, which at the beginning of the 
 test is quite filled with water. Another glass tube con- 
 nects B with the bottle D, empty at the beginning of the 
 test. 
 
280 
 
 THE ANALYSIS OF URINE. 
 
 To make the test pour into the first bottle 20 Cc. of 
 strong hypobromite solution, or 40 Cc. of the hypochlo- 
 rite. Measure accurately 4 or 5 Cc. of urine into the tube, 
 drop this into the bottle and insert the stopper. Fill B 
 quite full of water, and insert its stopper which drives out 
 a little water through the short tube into D. Allow the 
 whole apparatus to stand ten minutes to take the tempera- 
 ture of the air, then empty D and replace it. Now tip the 
 first bottle so as to mix the contents of F with the reagent 
 
 iniryiinintr-nirnr-nini 
 
 FIG. 44. 
 
 and shake gently. Bubbles of gas escape and passing 
 over into B drive out a corresponding volume of water. 
 Repeat the shaking of the reagent bottle several times. In 
 a few minutes the reaction is complete, but the apparatus 
 must be allowed to stand to cool down to the air tempera- 
 ture. A part of the water in D may be drawn back into B. 
 Finally measure the volume of water left in D, and take this 
 as the volume of gas liberated. Make the calculation as 
 before on the assumption that each Cc. of gas corresponds 
 to .0027 Gm. of urea. In this there are two errors which 
 nearly compensate each other. In the first place not all 
 the gas is liberated for the reasons explained above, but in 
 
THE ANALYSIS OF URINE. 281 
 
 the second place the volume read off is higher than nor- 
 mal because of higher temperature and lower barometer. 
 The results' obtained are therefore nearly correct, suffi- 
 ciently so for all clinical purposes. 
 
 Squibb has constructed a table from which the percent- 
 age of urea corresponding to any volume of gas, for a given 
 volume of urine taken, can be read at a glance without any 
 calculation whatever. This is convenient, but not necessary. 
 
 
 r\ The Doremus Apparatus. This is shown 
 
 W in the annexed cut, and at the present time is 
 
 very widely used by physicians because of the 
 simplicity of the method of employing it. 
 
 The graduated tube is filled to an indicating 
 mark with strong hypobromite solution and 
 then water is added to fill the remainder of the 
 tube and the bulb. By means of the pipette, 
 graduated to hold 1 Cc, this quantity of urine 
 is forced into the liquid in the upright part of 
 the tube. The urea is immediately decom- 
 posed, and its nitrogen ascends to the top of 
 the graduated part, where it is read off. The 
 longer marks on the tube indicate the fraction of a gram of 
 urea in the one cubic centimeter of urine taken; the 
 shorter marks indicate tenths. Multiplying by 100 we 
 obtain the number of grams of urea in 100 Cc. The 
 results are apt to be low from a loss of nitrogen through the 
 bulb which can scarcely be avoided. The instrument is 
 said to be "good enough" for clinical purposes, but cannot 
 be compared with that of Squibb for accuracy. No instru- 
 ment in which the volume of urine taken for the test is as 
 small as one cubic centimeter should be expected to give 
 even approximately accurate results, unless very great 
 precautions are taken in the measurement and in subse- 
 quent parts of the work. 
 
Chapter XVI. 
 
 THE DETERniNATION OF PHOSPHATES AND 
 CHLORIDES. 
 
 Phosphates. 
 
 DHOSPHORIC ACID occurs normally in the urine com- 
 1 bined with alkali and alkali-earth metals of which 
 combinations the alkali phosphajtes are soluble in water, 
 while the earthy phosphates are insoluble. In the urine, 
 however, they are held in solution through several agen- 
 cies. The larger part of the earthy phosphates appear- to 
 be held here normally in the acid condition, that is as com- 
 pounds of the formulas CaH 4 (POj 2 and MgH 4 (P0 4 ) 2 . 
 The salts of the type CaHP0 4 are present, also, in small 
 amount. As long as the urine maintains its acid reaction 
 these bodies may be expected to remain in solution, but if 
 it becomes alkaline by fermentation, or by the addition of 
 the hydroxides or carbonates of ammonium, sodium or po- 
 tassium, the acid phosphates are converted into insoluble, 
 neutral phosphates and precipitated. Most urines contain 
 along with the acid phosphate traces of neutral phosphates 
 which precipitate on boiling. It has been suggested that 
 these phosphates are held by traces of ammonium com- 
 pounds or by carbonic acid, both of which are driven off 
 by heat, allowing the phosphates to precipitate. It is well 
 known, however, that some urines can be boiled without 
 showing any sign of precipitation. In such cases it is 
 probable that the neutral phosphates are not present. 
 
 Part of the phosphoric acid of the urine comes directly 
 
THE ANALYSIS OF URINE. 283 
 
 from the phosphates of the food and another portion 
 results from the oxidation of the phosphorus holding 
 tissues. In health the rate of such oxidation is practically 
 constant, or nearly so, but in disease it may be greatly in- 
 creased or diminished. Variations in the amount of ex- 
 creted phosphates may become, therefore, of considerable 
 clinical importance. Great care must be observed, how- 
 ever, in drawing conclusions regarding the destruction of 
 phosphatic tissues from the results obtained by analysis, 
 because of the uncertainty of the amount taken with the 
 food and of what must be considered a normal excretion of 
 phosphoric acid. 
 
 Wheat bread constitutes one of our important articles 
 of food containing a relatively high amount of phosphates. 
 Owing to changes in milling processes introduced and ex- 
 tended in the past twenty years very material reductions 
 have been made in the percentage of phosphates and other 
 mineral substances left in our fine flour. The resulting 
 diminution in the phosphates of the urine from this cause 
 is appreciable. It must be remembered also that a part of 
 the phosphoric acid taken with the food is eliminated in 
 insoluble form with the faces. The amount so disposed of 
 depends on the nature of the original condition of combi- 
 nation of the phosphoric acid and on the amount of alkali- 
 earth bases present. These tend to form insoluble phos- 
 phates. With an exclusively vegetable diet the phos- 
 phates would, for this reason, be low, while with a meat 
 diet more would be excreted, because here the alkali 
 phosphates, especially potassium phosphate, are in excess. 
 On the other hand the phosphates are greatly increased in 
 the urine of people who consume great quantities of the 
 various phosphatic beverages which have become popular 
 in the United States in the past few years. It is readily 
 seen how quite erroneous conclusions could be drawn from 
 
284 THE ANALYSIS OF URINE. 
 
 the tests of urine of such persons. Single analyses for 
 phosporic acid may be very misleading. When the amount 
 of phosphoric acid passed with a given diet is known, 
 variations observed may sometimes be traced to certain 
 pathological conditions briefly mentioned in the following 
 paragraph. 
 
 In disease phosphates are found increased in rickets 
 and osteomalacia, possibly from the failure to deposit the 
 earthy phosphates in the bones. Meningitis is accom- 
 panied by an increase of the phosphates. The same is 
 true of other disorders of the brain as this organ is espe- 
 cially rich in phosphatic substances. It is said that phos- 
 phates are increased after a period of severe nervous strain. 
 What has been termed phosphatic diabetes has been de- 
 scribed as a condition in which a persistent excretion of 
 relatively large amounts of the phosphates is observed. 
 The earthy phosphates so discharged ma)' amount to 20 
 grams or more in a day. The causes leading to this con- 
 dition are not clearly defined. 
 
 The phosphates have been found diminished in diseases 
 accompanied by diminished nutrition, and in some diseases 
 of the heart and kidney. In dilute urine, low specific gravity 
 and high volume, the percentage amount of phosphoric acid 
 is much decreased, but it does not follow that a decrease 
 in the amount excreted in the twenty-four hours must 
 also be small. A safe estimate can be made only with a 
 mixed specimen taken, from the urine of the whole day 
 with consideration of the volume passed. 
 
 Various statements are found in the books regarding 
 the mean excretion of the alkali and earthy phosphates. 
 Different observers have reported between 2 and 5 Gm. of 
 phosphoric anhydride (P 2 6 ), while 3 Gm. may be taken, 
 perhaps, as the mean. 
 
 The recognition of the phosphates is an extremely easy 
 
THE ANALYSIS OF URINE. 285 
 
 matter. The presence of earthy phosphates may be 
 shown by adding to the urine enough ammonia water to 
 give a faint alkaline reaction and then warming. A floccu- 
 lent precipitate, resembling albumin, appears and is usually 
 white, or nearly so. But sometimes coloring matters 
 come down with it in amount sufficient to give it a brown- 
 ish or reddish shade. It will be recalled that the color of 
 this precipitate was referred to under the head of blood 
 tests. 
 
 The alkali phosphates can be detected in the filtrate 
 after separation of the earthy phosphates. To this end 
 add to the clear alkaline liquid a little more ammonia and 
 some clear magnesia mixture. A fine crystalline precipitate 
 of ammonium-magnesium phosphate separates and settles 
 rapidly. This is very characteristic. The qualitative tests 
 for phosphates have, however, little value in examination 
 of the urine. We are chiefly concerned with the amount, 
 the measurement of which will now be described. 
 
 Determination of Phosphates. It is customary to 
 measure the total phosphoric acid, not the alkali or earthy 
 phosphates separately. We have at our disposal several 
 methods, gravimetric and volumetric, of which the latter are 
 accurate and most convenient. A volumetric process will 
 be described which serves for the measurement of the 
 phosphoric acid as a whole, and which can be used for the 
 separate measurement of the earthy and alkali phosphates 
 by dealing with the precipitate and filtrate described in the 
 qualitative test above. This method depends on the fact 
 that solutions of uranium nitrate or acetate precipitate 
 phosphates in greenish yellow colored, flocculent form, and 
 that in a solution holding in suspension a precipitate of 
 uranium phosphate any excess of soluble uranium com- 
 pound may be recognized by the.reddish brown precipitate 
 
286 THE ANALYSIS OF URINE. 
 
 which it gives with a solution of potassium ferrocyanide. 
 The latter substance serves, therefore, as an indicator. If 
 to a phosphate solution in a beaker a dilute uranium solu- 
 tion be added precipitation continues until the whole of 
 the phosphates have gone into combination with the ura- 
 nium. If, during the precipitation, drops of liquid from 
 the beaker are brought in contact with drops of fresh ferro- 
 cyanide solution on a glass plate no reddish brown precipi- 
 tate of uranium ferrocyanide appears until the last trace of 
 uranium phosphate has been formed. The production of 
 uranium ferrocyanide is the indication, therefore, of the 
 finished precipitation of the phosphates. 
 
 The reaction between uranium and phosphates in acetic 
 acid solution is illustrated by this equation: 
 
 UO s (N0 3 ) 2 +KH 2 P0 4 =U0 2 HP0 4 +KN0 3 + HN0 3 
 
 From this it appears that 239 parts of uranium are 
 required to precipitate VI parts of P 8 6 . In order to have 
 the reaction take place as represented above, it is necessary 
 to neutralize the liberated nitric acid as fast as formed or 
 dispose of it in some other manner. The best plan is to 
 add to the solution to be precipitated acetic acid and sodium 
 acetate, the first of which brings the phosphates into the 
 acid condition, while the second is decomposed with the 
 formation of sodium nitrate and free acetic acid. Mineral 
 acids interfere with the reaction, while moderate amounts 
 of acetic acid do not. 
 
 In order to carry out this method we prepare the fol- 
 lowing solutions: 
 
 (a). Standard Uranium Solution. This is made by 
 dissolving 36 Gm. of the pure crystallized nitrate, 
 UO s (N0 3 ) 2 6H 2 in distilled water to make 
 one liter. The strength of the solution is 
 adjusted by experiment as explained below. 
 
THE ANALYSIS OF URINE. ?87 
 
 (&). Standard Phosphate Solution. This is made 
 by dissolving 10.085 Gm. of pure crystals of so- 
 dium phosphate, HNa 2 P0 4 .12H 2 0, in distilled 
 water to make one liter. 50 Cc. of this solution 
 contains .100 Gm. of P 2 6 . Small fresh, unef- 
 floresced crystals of the phosphate must be used 
 for this solution. 
 
 (0- Sodium Acetate Solution. Dissolve loo Gm. 
 in 800 Cc. of distilled water, add 100 Cc. of 30 per 
 cent acetic acid and then water enough to make 
 one liter. 
 
 (</). Fresh Ferrocyanide Solution. Dissolve 10 
 Gm. of pure potassium ferrocyanide in 100 Cc. of 
 distilled water. The solution should be kept in 
 the dark. 
 
 The actual value of the uranium solution is determined 
 by the following experiment: Measure out 50 Cc. of the 
 phosphate solution, (b), add 5 Cc. of the acetate solution, 
 (c), and heat in a beaker in a water-bath to near the boiling 
 temperature. Place several drops of the ferrocyanide 
 solution on a white plate. Fill a burette with the uranium 
 solution and when the solution in the beaker has reached 
 the proper temperature run into it from the burette 18 Cc. 
 of the uranium standard. Warm again, and by means of a 
 glass rod bring a drop of the liquid in the beaker in con- 
 tact with one of the ferrocyanide drops on the plate. If 
 the uranium solution has been properly made no red color 
 should yet appear. Now run in a fifth of one Cc. more 
 from the burette, warm and test again, and repeat these 
 operations until the first faint reddish shade begins to show 
 on bringing the two drops in contact. With this test as a 
 preliminary one make a second, adding at first one-fifth of 
 
288 THE ANALYSIS OF URINE. 
 
 a cubic centimeter less than the final result of the prelimi- 
 nary, and finish as before. Something less than 20 Cc. 
 should be needed to complete the reaction. Supposing 
 19.8 Cc. are required for the purpose the whole solution 
 should be diluted in the proportion, 
 
 19.8 : 20 :: a : x 
 
 in which a represents the volume on hand. We have 
 nowa standard uranium solution, each cubic centimeter of 
 which' precipitates exactly 5 milligrams of P 3 6 , and with 
 this we are able to measure phosphoric acid in unknown 
 solutions. 
 
 It is sometimes recommended to use uranium acetate 
 instead of nitrate in making this standard solution, and so 
 avoid a disturbing element in the liberation of the nitric 
 acid. But there are several advantages in the use of the 
 nitrate which should be mentioned. In the first place its 
 solutions keep better, and secondly, it can be obtained in 
 commerce in almost (if not quite) chemically pure condi- 
 tion, so that it is possible to make up a solution of nearly 
 correct strength by simply weighing out and dissolving the 
 crystals. Assuming 239 as the atomic weight of uranium, 
 and 0=16, the relation of the crystallized pure nitrate to 
 P 2 5 is 
 
 1006 : 142, 
 
 from which it follows that a liter of the standard solution 
 should contain 35.4 Gm. of the uranium salt, if each cubic 
 centimeter is to indicate five milligrams of P 2 O s . 
 
 The solution of sodium acetate with acetic acid must 
 always be added in the proportion given above if uniform 
 results are expected, and the ferrocyanide indicator must 
 be fresh and as weak as given. 
 
 The test of urine is made exactly as given in the above. 
 Measure out 50 Cc. of urine, add 5 Cc. of the acetate 
 
THE ANALYSIS OF URINE. 289 
 
 mixture, and finish as before. The 50 Cc. of urine, in the 
 mean, contains about as much phosphoric acid as was 
 present in the same volume of standard phosphate solu- 
 tion. The titration must be made hot, because the reac- 
 tion is much quicker and sharper in hot solution than in 
 cold. Make always two tests; the first is an approxima- 
 tion, while the second gives a much closer result. 
 
 A separate test of the earthy phosphates may be made 
 by adding to 200 Cc. of urine enough ammonia to give an 
 alkaline reaction. The urine then must stand until the 
 precipitated phosphates settle out. The precipitate is col- 
 lected on a small filter, washed with water containing a 
 very little ammonia, and then allowed to drain. It is next 
 dissolved in a small amount of acetic acid, the solution 
 diluted to 50 Cc, mixed with 5 Cc. of the sodium acetate 
 solution and titrated as before. The reaction here is not 
 quite as accurate as with the alkali phosphate, but the 
 results are satisfactory for the purpose. 
 
 The difference between the total phosphates and the 
 earthy phosphates, expressed in terms of P 2 O e , is the 
 amount combined as alkali phosphates. 
 
 The Determination of the Chlorides. 
 
 Practically, all of the chlorine taken into the stomach 
 with the food is eliminated with the urine. The chlorine 
 entering the body is mostly in the form of sodium chloride, 
 although traces of other chlorides are found in some of our 
 foodstuffs. 
 
 The amount of salt excreted depends, therefore, closely 
 on that consumed, and varies within wide limits. It is 
 commonly said that 10 to 15 Gm. daily include the 
 amounts passed in the great majority of urines. It must 
 be remembered, however, that in individual cases the 
 
290 THE ANALYSIS OF URINE. 
 
 upper limit may be very greatly exceeded. In the urine of 
 men who eat a great deal of salt food from 20 to 25 Gm. is 
 frequently found. In such cases the volume of water 
 drunk is usually large, so that the percentage amount of 
 salt passed . does not follow the same variations. Ex- 
 pressed in this manner, about one per cent represents the 
 average excretion. 
 
 Pathologically, chlorides are increased in total amount 
 in diabetes insipidus, and temporarily, sometimes in inter- 
 mittent fevers. A decrease in the chlorides is more fre- 
 quently observed, and has greater clinical importance. 
 
 This decrease is noticed generally in febrile conditions, 
 and especially if salty exudations are being formed in any 
 part of the body- In the serous accumulations of pleurisy 
 common salt is abundant, and at the same time greatly 
 diminished in the urine. The expectorated fluid in cases 
 of acute pneumonia contains much salt, which in conse- 
 quence is decreased in the urine. In some cases the 
 chlorine is practically absent from the urine. In any 
 event its reappearance in normal amount during the 
 progress of disease is a favorable sign, as indicating the 
 approach of normal conditions of absorption and excretion 
 in the body. Quantitative tests for the chlorides of the 
 urine become, therefore, of great importance, as their 
 absence or marked decrease can only occur in disorders 
 of serious nature. Fortunately, such tests are very easily 
 made, and by simple volumetric processes. 
 
 Volumetric Determination of Chlorine. The best 
 processes by which chlorine is measured volumetrically in 
 the urine and elsewhere depend on the reaction between 
 chlorides and silver nitrate, 
 
 NaCl+AgN0 3 =AgCl+NaN0 3 , 
 from which it appears that 5.84 milligrams of salt require 
 
THE ANALYSIS OF URINE. 291 
 
 for precipitation 16.95 milligrams of silvernitrate. In many 
 cases if a weak solution of silver nitrate be added to a weak 
 solution of salt in a bottle, with frequent shaking, the 
 curdy precipitate of silver chloride formed will settle out 
 so rapidly that it is possible to determine just when the re- 
 action is complete from the formation of no further pre- 
 cipitate in the nearly clear liquid as drops of silver nitrate 
 mix with it. A chloride can be added to precipitate silver 
 from a solution of its nitrate in the same manner, and so 
 delicate is the reaction that a method based on it is still 
 employed in many mints for determining the amount of 
 silver in bullion, coin or other alloy. 
 
 For the determination of chlorides in solutions, es- 
 pecially in urine, other methods, more convenient and 
 fully as accurate, are employed. If a weak solution of 
 silver nitrate be added to a neutral solution of a chloride 
 containing enough potassium chromate to impart a slight 
 yellow color to it the silver combines with the chlorine first 
 and then, only after this has been completely precipitated, 
 with the chromic acid to form brick-red silver chromate. 
 In such a mixture the appearance of the first tinge of red 
 is the indication that the chlorine has been wholly 
 precipitated. The neutral chromate is used here as the 
 indicator. As each drop of silver nitrate solution falls into 
 the solution of chloride and chromate a transitory reddish 
 color may appear before the reaction is completed, but this 
 vanishes on shaking or stirring the liquid and becomes 
 permanent only when the chlorides are completely com- 
 bined with silver. In using this method the following 
 solutions are employed: 
 
 (a.) Standard Silver Nitrate Solution, T \. Dis- 
 solve 16.954 Gm. of pure fused silver nitrate in 
 distilled water and dilute to one liter. Silver ni- 
 
292 THE ANALYSIS OF URINE. 
 
 trate can usually be obtained of sufficient purity 
 for the purpose, from dealers in fine chemicals, 
 but should be fused in a porcelain crucible at a 
 low temperature before being weighed out. The 
 correctness of the solution may be tested by. the 
 following: 
 
 (£.) Standard Sodium Chloride Solution, r Y Dis- 
 solve 5. 83? Gm. of pure, dry, recrystallized sodium 
 chloride in distilled water and dilute to one liter. 
 
 (c.) Potassium Chromate Indicator. Dissolve 10 
 Gm. of pure crystals, free from traces of chlorine, 
 in 100 Cc. of distilled water. 
 
 To test the accuracy of the standard silver solution fill 
 a burette with the same and then measure into a beaker 25 
 Cc. of the salt solution and add a few. drops of the indica- 
 tor. Now slowly run solution from the burette into the 
 beaker, shaking the latter meanwhile, and continue until 
 the curdy white precipitate shows a tinge of red from pres- 
 ence of a little chromate formed. Exactly 25 Cc. of the 
 silver solution should be needed for this. One Cc. of the 
 silver solution so made precipitates 3. 537 Mg. of chlorine 
 or shows 5.837 Mg. of sodium chloride. 
 
 This method cannot be applied directly to the urine 
 because of its yellow color which obscures the end reaction 
 and because, also, of the presence of certain organic mat- 
 ters which interfere to some extent. Therefore proceed as 
 follows: Measure out accurately into a platinum or porce- 
 lain dish 10 Cc. of the urine and add about 2 Gm. of potas- 
 sium nitrate and 1 Gm. of dry sodium carbonate, both free 
 from chlorides. Evaporate to dryness on the water- bath and 
 then heat over the free flame, at first gently and finally to 
 a higher temperature until the mass fuses. The organic 
 matter is destroyed by the nitrate leaving finally a white 
 
THE ANALYSfS OF URINE. 293 
 
 molten residue. Allow it to cool, dissolve in water and add 
 enough pure nitric acid to give a faint acid reaction. This 
 destroys the carbonate. The slight excess of nitric acid 
 must in turn be neutralized and this is done by adding a little 
 precipitated and thoroughly washed (chlorine free) calcium 
 carbonate. Pour the solution now into a flask, rinse the dish 
 thoroughly and add the rinsings to the liquid in the flask. 
 Next add 2 drops of the chromate indicator and then the T \ 
 silver solution, until the faint red of silver chromate mixed 
 with the chloride appears showing the end of the titration- 
 If the urine contains sugar or albumin in more than traces 
 evaporate and heat with the sodium carbonate first and then 
 add the nitrate, a little at a time, to the charred mass to 
 avoid too explosive an oxidation. The titration of the 
 residue from the urine is therefore similar to the process by 
 which the correctness of the silver solution was determined 
 above. If in the titration 22 Cc. of the silver solution were 
 used we have 22X3.537 = 7'7.81 Mg. of chlorine in the 10 
 Cc. of urine, corresponding to 128.4 Mg. of sodium chlo- 
 ride. The sodium carbonate should not be omitted in this 
 method as without it there is danger of loss of chlorine by 
 volatilization. With care it gives excellent results but at 
 present is not as generally employed as is the next one. 
 
 Volhard's Method. We have here a method by which 
 the chlorine in urine can be quickly and accurately deter- 
 mined without fusion. The principle involved in the proc- 
 ess is this. If to a chloride solution a definite volume of 
 standard silver solution be added, and this in excess of 
 that necessary to precipitate the chloride, the amount of 
 this excess can be found by another reaction, subtracted 
 and leave as the difference the volume actually needed for 
 the chloride. The reaction for the excess depends on these 
 facts. A sulphocyanate solution gives with silver nitrate 
 
394 THE ANALYSIS OF URINE. 
 
 solution a white precipitate of silver sulphocyanate, 
 AgSCN. It also gives with a ferric solution a deep red 
 color due to the formation of soluble ferric sulphocyanate, 
 Fe 2 S 6 (CN) 6 . If the silver and ferric solutions are mixed 
 and the sulphocyanate added the second reaction does not 
 begin until the first is completed, that is, the silver must be 
 first thrown down as white sulphocyanate before a perma- 
 nent red shade of ferric sulphocyanate appears. The pres- 
 ence of silver chloride interferes but slightly with these 
 reactions. Therefore, if we have a sulphocyanate solution 
 of definite strength we can use it with the ferric indicator 
 to measure the excess of silver used after precipitating the 
 chlorine of a solution. 
 
 The reaction between silver nitrate and a sulphocyanate 
 is expressed by the following equation: 
 
 AgN0 3 +NH 4 SCN = Ag SCN+NH 4 N0 3 . 
 
 For 16.954 Mg. of the silver nitrate we use 1. 591 Mg. 
 of the sulphocyanate. In this method the standard solu- 
 tions required are 
 
 (a.) Standard Silver Nitrate Solution, T V Made 
 as before with 16.954 Gm. of the fused salt to the 
 liter. As the solutions are used with nitric acid 
 present the standard can also be made by weigh- 
 ing out accurately 10.766 Gm. of pure silver and 
 dissolving it, in a flask, in pure nitric acid. Most 
 of the excess of nitric acid is removed by evapo- 
 ration and air is blown through to drive out nitrous 
 fumes. The solution is cooled and diluted to 1 
 liter. 
 
 (b.) Standard Sulphocyanate Solution, T \. This 
 may be made of the potassium or ammonium salt, 
 but the latter is more commonly used. . Weigh 
 
THE ANALYSIS OF URINE. 205 
 
 out about 7.7 Gm. of the pure crystals, dis- 
 solve in water and make up to 1 liter. Determine 
 the exact strength as explained below. The true 
 weight cannot be weighed out directly because 
 the otherwise pure salt is frequently a little moist, 
 and because further a salt, pure to begin with, 
 undergoes frequently a slight change on standing. 
 
 (£.) Ferric Solution as Indicator. Use for this a 
 nearly saturated solution of ammonium ferric sul- 
 phate (ferric alum) free from chlorine. 
 
 To find the exact strength of the sulphocyanate solu- 
 tion proceed as follows: Measure into a flask or beaker 
 25 Cc. of the T \ silver solution and add to it 2 or 3 Cc. of 
 the ferric indicator. This gives some color and a slight 
 opalescence. Now add about 2 Cc. of pure strong nitric acid, 
 which removes the color and clears the mixture. Into this, 
 from a burette, let the sulphocyanate solution flow, a little 
 at a time, shaking after each addition. A red color ap- 
 pears temporarily, but vanishes on shaking. After a time 
 this red disappears more slowly, which shows that the end 
 of the reaction is near. The burette solution is therefore 
 added more carefully, best by drops, until at last a single 
 drop is sufficient to give a permanent reddish tinge. 
 Something less than 25 Cc. should be used for this. Re- 
 peat the test and if the same result is found dilute the 
 sulphocyanate solution so as to make 25 Cc. of the volume 
 used in the titration. For instance, if 24.2 Cc. were re- 
 quired 900 Cc. of the solution may be diluted in this pro- 
 portion : 
 
 24.2 : 25 :: 900 : x. ■. x = 929.8. 
 
 We have now a standard sulphocyanate solution corre- 
 sponding exactly to the silver solution. To test it further 
 
296 THE ANALYSIS OF URINE. 
 
 and illustrate its use with chlorides measure out 25 Cc. of 
 the T ^ sodium chloride solution described a few pages 
 back, and add to it, from a burette, exactly 30 Cc. of the 
 silver nitrate solution, then the ferric indicator and the 
 nitric acid as given above. Shake the mixture and filter 
 it through a small filter into a clean flask or beaker. 
 Wash out the vessel in which the precipitate was made with 
 about 20 Cc. of pure water, pouring the washings through 
 the filter. Then wash the filter with about 20 Cc. more of 
 water allowing the washings to mix with the first filtrate. 
 This mixed filtrate contains all the silver used in excess of 
 the chloride. Now bring it under the sulphocyanate bu- 
 rette and add this solution until a reddish tinge becomes 
 permanent. Exactly 5 Cc. should be necessary for this. 
 
 The chlorides of the urine may be treated in about the 
 same manner. To a measured volume of the urine, usual- 
 ly 10 Cc. , an excess of silver nitrate solution is added, 25 
 Cc. with most urines is enough, and then the indicator and 
 acid. The mixture is filtered, the precipitate washed and 
 in the filtrate the excess of silver is found by sulphocyanate 
 as above. But it occasionally happens that the addition of 
 nitric acid to the urine develops a red color which obscures 
 the end reaction. To avoid this the urine titration should 
 always be made in the following manner: 
 
 To 10 Cc. of urine add 2 to 3 Cc. of pure strong nitric 
 acid, then the ferric indicator and three drops of a saturated, 
 chlorine free solution of potassium permanganate. This 
 gives at first a very deep red color, but it soon fades and 
 with it the urine colors, by oxidation. It is not well to add 
 more of the permanganate than here given. 
 
 To the yellow solution add now 25 Cc. of the standard 
 silver solution, shake well and filter. Wash the beaker 
 and filter thoroughly as above described, and in the mixed 
 filtrate and washings find the excess of silver. If in doing 
 
THE ANALYSIS OF URINE. 297 
 
 this the first drops of the sulphocyanate solution added 
 produce a deep red color it shows that too little silver had 
 been used in the first place. Make therefore, a new test, 
 using more silver nitrate solution, 30 to 50 Cc. 
 
 To illustrate, if we use for 10 Cc. of urine, 25 Cc. of 
 silver nitrate, then 3.4 Cc. of the sulphocyanate, 25 — 3.4 = 
 21.6, the amount of silver nitrate solution actually needed 
 for the chlorides. 21.6x3.537 = 76.40 Mg. of chlorine in the 
 10 Cc. of urine, which corresponds to 126.07 Mg. of NaCl 
 in the 10 Cc. or to 12.607 Gm. per liter. If the urine 
 tested had a specific gravity of 1.020 this would equal 1.24 
 per cent. 
 
 Other Mineral Substances. 
 
 Several other groups of compounds occur in the urine 
 which may be briefly referred to. The sulphates of sodium 
 and potassium are found to the extent of about 2 Gm. 
 daily in average urine, and in traces certain ethereal sul- 
 phates also occur. The clinical significance of these bodies 
 is not very clear, and besides we have no volumetric 
 method accurate enough for their determination. The 
 gravimetric methods by which they may be measured can 
 not be described in this place. 
 
 In normal urine several carbonates are often found. 
 The carbonate of ammonium is probably always present in 
 small amount, but in fermented urine, either before or after 
 voiding, its amount may be very great, imparting a strong 
 alkaline reaction. Nitrates, nitrites, sulphides, bromides, 
 and iodides after taking certain remedies, and several other 
 salts have been found in the urine in small amount, but no 
 great clinical importance attaches to them. 
 
Chapter XVII. 
 
 THE SEDinENT FROM URINE. 
 
 T TRINE is frequently cloudy when passed and on stand- 
 *— ' ing deposits a sediment of the substances imparting 
 the cloudiness. Other urines which may appear perfectly 
 clear at first also throw down deposits after a time. This 
 is always the case with urine allowed to stand long enough 
 to undergo alkaline fermentation, when a precipitate of 
 phosphates forms. The deposit is frequently caused by a 
 change of temperature. Warm voided urine holding an 
 excess of urates may be perfectly clear, but becomes 
 cloudy as its temperature goes down with the formation of 
 a light reddish sediment. This is a perfectly normal ac- 
 tion, and indeed most sediments may be considered in the 
 same light. Urine containing a deposit is not necessarily 
 pathological. 
 
 There are conditions, however, in which the sediment 
 is an indication of abnormality, and its examination be- 
 comes important clinically. Certain sediments are patho- 
 logical because of their origin, others because of their 
 amount. For instance, blood and pus corpuscles, casts of 
 the uriniferous tubules of the kidney and a few other forms 
 are not found normally in urine, and their presence is of 
 importance, whether observed in large or small quantity. 
 Sediments containing phosphates, uric acid and urates, cal- 
 cium oxalate and other salts are common enough and usu- 
 ally attract no attention, but if the amount of these de- 
 posits is very large there may be attached to them clinical 
 significance and the)' deserve study. 
 
THE ANALYSIS OF URINE. 299 
 
 In the examination of a sediment it is necessary to 
 allow the urine to stand long enough to deposit the im- 
 portant forms it may contain, which may require twenty- 
 four hours or more. For the deposition of a sediment the 
 urine should be left in a place with an even temperature, 
 preferably not above 15° C. A low temperature favors the 
 precipitation of urates, while decomposition may begin 
 if the temperature be allowed to go up. Some of the light 
 organic forms have a specific gravity so little above that 
 of the urine that they may remain a long time in suspen- 
 sion. It is important, therefore, to allow plenty of time for 
 these to settle. If the weather is warm and there is no good 
 means at hand for keeping the temperature of the urine 
 down until the examination can be made, or if for any 
 reason this must be delayed for some days, it is well to add 
 some preservative to the urine, i. e., something to prevent 
 fermentation. Many substances have been suggested for 
 this purpose, some of which are very objectionable inas- 
 much as they form precipitates which often obscure what 
 is sought for. Chloroform is the simplest and at the same 
 time one of the best substances which can be added. 
 
 To 100 Cc. of the urine to be set aside for tests add 3 
 or 4 drops of chloroform and dissolve by shaking. It is 
 not well to add more than this as there is danger of leaving 
 minute droplets undissolved, and these are confusing in 
 the subsequent examination. The chloroform may be ap- 
 plied in the form of aqueous solution. Add about 10 Gm. of 
 chloroform to a liter of distilled water and shake thoroughly; 
 about three-fourths will dissolve at the ordinary tempera- 
 ture; 25 Cc. of this saturated solution may be added to 
 100 Cc. of the urine to be examined, which is then allowed 
 to stand as before. 
 
 After the deposit has settled pour off the supernatant 
 liquid very carefully and by means of a small pipette with 
 
300 
 
 THE ANALYSIS OF UKINE. 
 
 a coarse opening transfer one or two drops to a perfectly 
 clean glass slide. Clean a cover glass with great care and 
 by means of small brass forceps lower it on the drops of 
 liquid in such a manner as to exclude air bubbles. This 
 can be done by lowering it inclined to the slide, not paral- 
 
 FIG. 46. 
 
 Sargent's centrifugal machine. 
 
 lei with it, so as to touch the liquid on one side first. In 
 settling down the cover now pushes the air in front of it 
 and gives a field generally free from bubbles. The slide is 
 then examined under a microscope with a magnifying 
 power of 250 to 300 diameters. Either natural or artificial 
 light may be used, but it must not be very bright. A very 
 common mistake in the examination of urinary sediments 
 by the microscope is to employ so high a degree of illumi- 
 
THE ANALYSIS OF URINE. 301 
 
 nation that the lighter and nearly transparent bodies are 
 completely overlooked. 
 
 Recently centrifugal machines have been introduced 
 which may be employed to settle the urine. The latter for 
 this purpose is placed in strong test-tubes which are 
 caused to rotate so rapidly that all suspended matters are 
 thrown to the bottom of the tubes, these being hung by 
 the neck in such a manner that in rotation the bot- 
 tom flies up and outward. Some of these rotating ma- 
 chines are operated by hand, others by water power or 
 electricity. A very convenient form run by a small water 
 motor is now to be had from dealers. It is simply neces- 
 sary to attach the motor by means of a rubber tube to a 
 faucet delivering water under ordinary city pressure to ob- 
 tain all the power necessary. 
 
 Where the current furnished for electric lighting by the 
 incandescent system is available, centrifugal machines 
 operated by small electric motors are even more convenient. 
 The cut above shows a small machine which has given ex- 
 cellent results. The wires from the motor in the base of 
 the machine are attached by a socket in place of the com- 
 mon incandescent lamps now everywhere used. 
 
 A very high velocity is attained in these machines by 
 aid of which the deposit may be secured in minutes instead 
 of hours. The sediment is left in a very concentrated con- 
 dition in the bottom of the tube, and from it the super- 
 natant urine can be poured much better than when it pre- 
 cipitates in a beaker or bottle in the usual manner. 
 
 Sediments from urine are commonly classed as organ- 
 ized and unorganized, these divisions being then subdivided 
 according to various plans. The important forms under 
 each division are shown in the following schemes : 
 
302 
 
 THE ANALYSIS OF URINE. 
 
 Organized 
 Sediments. 
 
 Blood corpuscles. 
 
 Mucus and pus corpuscles. 
 
 Epithelium from various locations. 
 
 Mucin bands, or threads. 
 
 Casts of the uriniferous tubules. 
 
 Spermatozoa. 
 
 Fragments of cancer tissue. 
 
 Fungi. 
 
 Certain other parasites. 
 
 Uric acid. 
 Various urates. 
 Leucin and tyrosin. 
 Cystin. 
 Cholesterin. 
 Fat globules. 
 Hippuric acid. 
 Calcium carbonate. 
 Calcium phosphate. 
 Calcium oxalate. 
 Magnesium phosphates. 
 
 In addition to these there are often found in the urine 
 certain bodies whose presence must be called accidental, 
 for instance hairs, fibers of cotton, silk or wool, starch 
 granules, bits of wood, mineral dust, etc. Some of these 
 will be referred to later. 
 
 Unorganized 
 Sediments. 
 
 Organized Sediments. 
 
 Blood Corpuscles. 
 Urine containing blood presents a characterestic appear- 
 ance easily recognized, unless it be present in very small 
 quantity. If the reaction of the urine is acid the color is 
 generally dark; but if alkaline the shade is inclined to 
 
THE ANALYSIS OF URINE. 303 
 
 reddish. Blood corpuscles enter the urine from several 
 different sources and their presence is usually a pathological 
 indication, but not always, as they may come, for instance, 
 from menstruation. The kidneys, or their pelves, the 
 ureters, the bladder* the urethra, the vagina or the uterus 
 may be the seat of the lesion from which the blood starts, 
 and its appearance sometimes gives a clue to its origin. 
 
 Fresh blood corpuscles are clear in outline and show 
 distinctly their bi-concavity. But corpuscles which have 
 
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 • *• * • •• * o • 
 
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 FIG. 47. 
 Human blood corpuscles, 400 diameters. 
 
 been long in contact with the urine become much swollen, 
 less distinct in outline, often biconvex, or nearly spherical 
 even, and lighter in color. As long as the reaction of the 
 urine is acid the corpuscles remain comparatively fresh 
 in appearance but with the beginning of the alkaline 
 reaction disintegration and loss of color soon set in. 
 
 The microscopic recognition of blood in urine is easy 
 enough if it is not too old. The fresh, red corpuscles of 
 human blood have a mean diameter of about .OOV? Mm., 
 but when swollen by absorption of water they are some- 
 what larger. When seen on edge they appear as shown at 
 
 1,. 
 
 f 1 
 
 ' 1 
 
 1 
 
 1 \ 
 
 I % 
 
 l «.l 
 
 u 
 
 1*1 
 
 t 
 
 < e , 
 
 
 *P as 
 
 * 
 
 
 % 
 
 I % 
 
 
 jit 
 
 <l 
 
 t % 
 
304 THE ANALYSIS OF URINE. 
 
 the left in the figure above. If presenting the fiat side to 
 the eye they appear as discs whose centers grow alternately 
 light and dark by changing the focus of the instrument. 
 In old urine, especially with alkaline reaction they appear 
 as granulated spheres, shown in the center of the figure. 
 In all cases the color is more or less yellowish. It is gen- 
 erally assumed that the paler washed out corpuscles come 
 from lesions higher up, from the pelvis, or kidney even, 
 while the brighter fresh blood points to a lesion nearer the 
 point of discharge, that is from the bladder or urethra. 
 This is pretty certain to be the case if the blood is dis- 
 charged but little mixed with the urine and settles rapidly 
 as a distinct mass. 
 
 Mucus and Pus Corpuscles. 
 
 These are white corpuscles somewhat larger than the 
 red blood corpuscles and spherical in outline. The term 
 leucocyte is frequently applied to these as well as to the so- 
 called white corpuscles of blood. Their size varies greatly 
 but the average diameter may be given as .009 Mm. All 
 these corpuscles present when fresh a slightly granular 
 appearance and occasionally show one or more nuclei. 
 The addition of a little acetic acid to the sediment brings 
 the nucleus out distinctly so that it may be seen under the 
 microscope as a characteristic appearance. 
 
 Mucus corpuscles in small number are normally present 
 in urine, but pus corpuscles enter the urine as a constituent 
 of pus itself which is an albuminous product discharged 
 from suppurating surfaces and not normal. In a former 
 chapter it was shown that the reactions of mucus and 
 albumin are distinct, but urine containing pus always 
 affords reactions for albumin. Pus in urine tends to form 
 a sediment at the bottom of the containing vessel, and may 
 be recognized by the following method : 
 
THE ANALYSIS OF URINE. 305 
 
 Donne's Test. Pour the urine from the sediment and 
 add to the latter an equal volume of thick potassium 
 hydroxide solution, or a small piece of the solid potassa. 
 Stir with a glass rod. The strong alkali converts the pus 
 into a thick viscid mass closely resembling white of egg. 
 Sometimes this is so thick that the test-tube containing it 
 can be inverted without spilling it. In. alkaline urine this 
 glairy mass is sometimes spontaneously formed. 
 
 '.?•' 
 
 FIG. 48. 
 
 Pus corpuscles, 400 diameters. 
 
 The appearance of the mucus or pus corpuscles in 
 urine depends largely on the concentration of the latter. 
 In urine of low. specific gravity the corpuscles absorb water 
 and swell to larger size than normal, while in a highly con- 
 centrated urine they may give out water and become re- 
 duced in size and shrunken in appearance. 
 
 To recognize them under the microscope transfer a 
 few drops of the sediment to a slide and cover as usual. 
 If the nuclei are not distinct place a drop of diluted acetic 
 acid on the slide at the edge of the cover glass. Part of 
 the acid will flow under the cover and mix with the urine. 
 As it does this the clearing up of the corpuscles with ap- 
 pearance of the nuclei can be very easily followed. Urine 
 
306 
 
 THE ANALYSIS OF URINE. 
 
 containing much pus is white and milky. The same ap- 
 pearance is often noticed with an excess of earthy phos- 
 phates, but the latter clear up with acids while the pus 
 does not. 
 
 Epithelium Cells. 
 
 Epithelium cells from different sources may appear 
 normally in the urine, and the light cloud which separates 
 from normal urine on standing consists chiefly of these 
 cells. When present in small amount this epithelium has 
 usually no clinical importance, as it easily finds its way 
 into the urine from the bladder, vagina or urethra. An 
 
 FIG. 49. 
 
 Scaly and spherical epithelium. 
 
 abundance of cells from these organs would, however, be 
 considered pathological, pointing to a catarrhal condition. 
 Unfortunately, it is not possible in all cases to deter- 
 mine the source of the cells, as found in urine, partly be- 
 cause cells from different localities have frequently the 
 same general appearance, and partly because, owing to 
 immersion in the urine, they become greatly changed from 
 what they are in the tissue as shown by the microscopic 
 
THE ANALYSIS OF URINE. 307 
 
 study of sections. It is customary to make three rough 
 divisions of the cells as found in the urine : 
 
 1. Spherical cells. 2. Columnar or conical cells. 3. 
 Flat or scaly cells. 
 
 The spherical cells are probably normally much flat- 
 tened but by absorption of water they become swollen 
 and globular. These cells may be derived from several 
 sources, as from the uriniferous tubules or from the deeper 
 layers of the lining membrane of the pelvis of the kidney, 
 or the bladder, or the male urethra. 
 
 These cells have a well-defined nucleus resembling that 
 of a pus cell. But they are much larger, and besides show 
 
 FIG. SO. 
 
 Conical epithelium. 
 
 the nucleus without addition of acid. In nephritis, or 
 other structural diseases of the kidney, these round cells 
 are found along with albumin, and their recognition is 
 then a matter of importance as indicating a breaking down 
 of the tubular walls. Sometimes these cells form a variety 
 of tube cast, to be described later. But it must be remem- 
 bered that we cannot distinguish with certainty between 
 the cells from the tubules and those from the other locali- 
 ties mentioned. 
 
 Conical cells come generally from the pelvis of the kid- 
 
308 THE ANALYSIS OF URINE. 
 
 ney, from the ureters and urethra. Some of these cells are 
 furnished with one or two processes, and are broad in 
 the middle and taper toward each end, while the others 
 are broad at the base and taper to a point. 
 
 The large flat cells come from the vagina or bladder, 
 and it is generally impossible to distinguish between them. 
 Sometimes they are very nearly circular, sometimes irregu- 
 larly polygonal in outline. Sometimes the vaginal epi- 
 thelium is found in layers of scales, which appear thicker 
 and tougher than the cells from the bladder, which occur 
 singly. 
 
 What was said about the decomposition of blood or pus 
 cells in urine obtains also for the various epithelium cells. 
 In acid urine they may maintain their distinct outlines 
 many days, but in alkaline secretion they soon undergo 
 disintegration, which makes their recognition practically 
 impossible. In general the greatest importance attaches 
 to the cells from the tubules of the kidney. The presence 
 of albumin in more than minute traces in the urine would 
 suggest that any smaller spherical cells present may have 
 had their origin in the kidney rather than in the bladder 
 or male urethra. In general it may be said that urine con- 
 taining large numbers of the smaller, round tubule cells 
 with albumin will also show casts. 
 
 Mucin Bands 
 
 Urine containing much mucus sometimes exhibits a de- 
 posit consisting of long threads or bands, curved and bent 
 in every direction. These bands are important because 
 they are sometimes confounded with the tube casts to be 
 described next. They can be produced in urine highly 
 charged with mucus by the addition of acids, and appear 
 therefore sometimes spontaneously when the urine becomes 
 acid. These threads are sometimes covered with a fine 
 
THE ANALYSIS OF URINE. 
 
 309 
 
 deposit of granular urates and these bear some resem- 
 blance to granular casts. In general, however, they are 
 relatively longer and narrower than the true casts of the 
 uriniferous tubules. The mucin threads can occur and 
 frequently do occur in urine entirely free from albumin, 
 while true tube casts are usually associated with albumin, 
 although not always, as will be explained below. The 
 
 1^: 
 
 FIG. 51. 
 
 Mucin bands or threads. 
 
 length and shape of the mucin threads may generally be 
 relied upon to distinguish them from true casts. 
 
 Casts. 
 
 The structures properly termed casts are seldom found 
 in urine which does not contain albumin. They are 
 formed in the uriniferous tubules, and to a certain extent, 
 are "casts" of portions of the same. Their specific 
 gravity differs but little from that of the urine, for which 
 reason they remain long in suspension. It is therefore 
 necessary to allow the urine to stand some hours at rest, 
 over night or longer, before attempting an examination. 
 
 Casts of the uriniferous tubules rarely appear in normal 
 
310 THE ANALYSIS OF URINE. 
 
 urine and their recognition is therefore a matter of the high- 
 est importance in diagnosis. Much has been written on 
 the subject of the origin of these bodies in the kidney and 
 several theories have been advanced to account for their 
 formation and chemical constitution. Most of this discus- 
 sion would be out of place in a work like the present deal- 
 ing mainly with questions of analysis, but enough will 
 be given to aid the student in his examinations. It must 
 be said that few subjects are more perplexing to the begin- 
 ner than that of their certain recognition, because of the 
 fact that some varieties are so transparent as to be almost 
 invisible, while others are closely resembled by formations 
 
 0) 
 
 *-■' 
 
 FIG. 53. 
 
 Epithelium casts. To the right a bunch of urates or false cast. 
 
 of entirely different nature not pathological. With prac- 
 tice, however, these difficulties can be surmounted. 
 
 Most of the bodies termed casts are formed of organ- 
 ized structures or the remains of such, but another and 
 rather common form consists of crystalline matter, usually 
 uric acid or fine granular urates. 
 
 These bunches of urates have no pathological signifi- 
 cance and are of frequent occurrence. Urine containing 
 them clears up by heat and the deposits themselves are 
 
THE ANALYSIS OF URINE. 
 
 311 
 
 dissipated by weak alkali. While it is true that they re- 
 semble to some degree the so-called granular casts referred 
 to below there are certain well-defined points of difference. 
 The bunches of urates lack the coherence which can be ob- 
 served in the true casts, and besides the granulation is 
 finer and more clearly defined. 
 
 The fact that mucin bands occasionally appear cov- 
 ered with a precipitate of granular urates has been re- 
 ferred to. These aggregations are more compact than the 
 loose bunches of urates just mentioned and much longer 
 
 
 FIG. 53. 
 
 Blood casts and granular casts. 
 
 generally. They are also darker and therefore more easily 
 seen than are the casts proper or the urates. 
 
 The true casts are made up of matter in which evidence 
 of cell structure or transformation is visible. An accurate 
 classification of these bodies cannot yet be made, and, as 
 said, authors differ regarding the importance of several 
 forms and their origin. But for our purpose it will be suf- 
 ficient to make the following rough division, which accords 
 in the main with what is found in the text-books of urine 
 analysis: 
 
312 THE ANALYSIS OF URINE. 
 
 J, Blood casts; 2, Epithelium casts; 3, Granular casts; 
 4, Fatty casts; 5, Waxy casts; 6, Hyaline casts. 
 
 What are termed blood casts consist of or contain co- 
 agulated blood recognized by the corpuscles. Plugs of 
 this coagulated matter are forced out from the tubules by 
 pressure from behind, and form one of the most character- 
 istic varieties of casts. They are generally very dark in 
 color, and easily distinguished from other matter. A repre- 
 sentation of blood casts is given in the preceding cut. 
 
 In epithelium casts the characteristic substance is the 
 lining epithelium of the tubule. Sometimes this lining 
 epithelium becomes detached in the form of a hollow cyl- 
 inder, the walls consisting of the united cells. Again, the 
 coagulated contents of the tubule in passing out may carry 
 the epithelium with it as a coating. In either case a grave 
 disorder of the kidneys is indicated, as acute nephritis, or 
 other disease in which a profound alteration of the internal 
 structure of the organ is involved. 
 
 What are termed granular casts, proper, appear in a 
 variety of forms, produced probably by the disintegration 
 of blood or epithelium casts. 
 
 There is no uniformity in the fineness of the granulation; 
 sometimes a high amplification is necessary to disclose the 
 structure. Occasionally blood corpuscles, epithelium, fat 
 globules and crystals can be detected in them, and when 
 derived from blood cast disintegration they usually have 
 a yellowish red color, which makes their recognition com- 
 paratively easy. In outline they are generally regular, 
 with rounded ends, one of which is somewhat pointed. 
 Frequently, however, they appear to be broken, the ends 
 showing irregular fracture. 
 
 Fatty casts contain oil drops produced by some variety 
 of fatty degeneration of the tissues of the kidney. These 
 oil drops may form coherent bunches, or they may be held 
 
THE ANALYSIS OF URINE. 
 
 313 
 
 by patches of epithelium. It also happens that epithelium 
 or granular casts may be partially covered by oil drops. 
 The name fatty cast is applied to those in which the fat 
 globules predominate. Along with these globules the 
 microscope sometimes shows crystals of free fatty acids, 
 and probably also of soaps containing calcium and mag- 
 nesium. 
 
 Waxy casts consist of the peculiar matter produced by 
 amyloid degeneration of the kidney. They have a glisten- 
 ing waxlike or vitreous appearance, and refract light very 
 
 i'i%/ 
 
 fi-j 
 
 FIG. 54. 
 
 Waxy and hyaline casts. 
 
 strongly. Sometimes they reach a great length, and they 
 frequently are found with blood corpuscles or oil drops on 
 the surface. They have been detected in several renal 
 disorders. Illustrations are given to the left above. 
 
 True hyaline casts are nearly transparent and hard to 
 see unless the illumination is very carefully managed. To 
 detect them it is often necessary to add a few drops of a 
 dilute solution of iodine in potassium iodide to the sedi- 
 ment. This imparts a slight color which renders them 
 visible. 
 
 The hyaline casts seem to be formed by the passage of 
 
314 THE ANALYSIS OF URINE. 
 
 homogeneous matter from the tubules, leaving the epithe- 
 lium behind. A cast is rarely perfectly hyaline, as at least 
 an occasional blood corpuscle, fat globule or epithelium cell 
 will usually be found attached to it. Waxy casts may be 
 looked upon as a special form of hyaline casts. Very im- 
 perfect representations are given in the above cut. 
 
 In general, it must be said that the representation of 
 these casts on paper is a very difficult matter. Ordinarily 
 they are drawn and printed much too heavy and dark. 
 Hyaline casts do not necessarily indicate kidney disease, 
 although this is usually the case. They have been found in 
 urine free from albumin and under circumstances not con- 
 nected with renal disorders. 
 
 The preservation of sediments containing casts is un- 
 usually difficult because of the nature of the material to be 
 preserved. In urine of the slightest alkalinity their dis- 
 integration soon begins so that the outlines are rendered 
 indistinct, often making identification impossible. 
 
 For temporary preservation the addition of chloroform 
 renders as good service as anything else. Many other sedi- 
 ments can be permanently mounted and kept for future 
 comparison but with casts this can rarely be done. 
 
 Beginners are apt to overlook casts in their first examin- 
 ations. It must be remembered that some of them are 
 nearly transparent and unless brought into proper focus 
 they may not be seen at all. At the outset students usu- 
 ally employ too bright a light in looking for casts. While 
 no specific directions can be given regarding the inten- 
 sity of illumination best suited to the purpose, this may be 
 said that the light commonly found necessary in studying 
 ordinary histological slides is far too bright to use in the 
 search for casts. 
 
 Practice alone, first under the direction of the instruc- 
 tor, will indicate what is proper here. 
 
THE ANALYSIS OF URINE. 315 
 
 Spermatozoa. 
 
 These minute bodies, as found in the semen of man, 
 have a mean length of about .050 Mm. Nearly one-tenth 
 of this is in the head portion. When observed in recently 
 discharged semen they have a characteristic spontaneous 
 movement by which they are propelled forward rapidly. 
 This motion is soon lost if the semen is diluted with water 
 or similar liquid. Hence, as usually seen in urine they are 
 entirely motionless. They are found abundantly in the 
 urine of men after coitus or nocturnal emissions, and also 
 
 FIG. 55. 
 
 Human spermatozoa. 
 
 in spermatorrhoea, when their presence is continuous and 
 characteristic. 
 
 In the urine of women they are likewise found after con- 
 nection, and their detection here has often interest from 
 the medico-legal standpoint. The proof of rape can often 
 be established by the recognition of spermatozoa. 
 
 Although their motion is soon lost in foreign liquids the 
 substance itself of the spermatozoa is not readily destroyed. 
 In this respect they are more permanent, probably, than all 
 other organized structures found in the urine and can be 
 readily distinguished after many days or months even in 
 urine, or in sediments which have become dry. 
 
 For their recognition a power of 250 diameters is suffi- 
 cient, but 350 to 400 diameters is preferable. With this 
 
316 THE ANALYSIS OF URINE. 
 
 higher amplification it is possible to readily distinguish 
 between spermatozoa and certain fungus growths bearing 
 some resemblance to them. 
 
 Cancer Tissue. 
 
 Fragments of cancerous and other morbid growths are 
 occasionally met with in the urine, and when certainly 
 recognized are an important aid in diagnosis. 
 
 The recognition of cancer cells along with the several 
 kinds of epithelium cells found in urine is difficult and lies 
 somewhat beyond the usual line of urine examinations. 
 
 FIG. 56. 
 
 Cancer cells which have been seen in urine. 
 
 Sometimes the identification is comparatively simple 
 because of the unusual shape of the cells, or when they are 
 present in great number, but this is not often the case. 
 
 The cut shows some of these cells figured by Beale. 
 
 Fungi. 
 
 The urine sometimes contains certain fungus growths 
 the recognition of which is important. These may have 
 entered the urine after voiding or they may have come 
 from the bladder. 
 
 Normal urine when passed is probably free from fungi 
 of all kinds, but in a short time certain organisms enter it 
 
7WE ANALYSIS OF URINE. 317 
 
 from the air or from other sources and become active in 
 producing in it characteristic changes. 
 
 The three important groups of fungi, the schizomycetes or 
 bacteria, the hyphomycetes or moulds and the blastomycetes or 
 yeasts are represented in the organisms sometimes found in 
 the urine. The conditions under which they are found will 
 be briefly explained. 
 
 Of the bacteria the following have been observed : 
 
 Micrococcus Ureae. This is the exceedingly common 
 form found on urine undergoing alkaline fermentation by 
 which urea is converted into ammonium carbonate. It is 
 usually introduced from the air and multiplies very rapidly 
 under ordinary conditions. Nearly all old specimens of 
 urine, unless containing some active preservative are 
 found infected with this small organism. The micrococci 
 are minute spherical bodies belonging to the suborder 
 sphmrobacteria, and are found separate or in chains. They 
 are the smallest of the organized forms occurring in urine 
 and appear under a power of 250 diameters but little more 
 than points. While generally finding their way into urine 
 after it has been voided they are occasionally present in 
 the bladder. It is usually held that under such circum- 
 stances they have been introduced by a dirty catheter or 
 sound, although cases are on record where this has not 
 been proven. 
 
 In the bladder they give rise to alkaline fermentation, 
 so that the voided urine may show ammonium carbonate 
 directly. 
 
 Streptococcus Pyogenes is a pathogenic form some- 
 times found in the urine in cases of infectious diseases. 
 
 Sarcinae. The genus sarcina is frequently classed with 
 the sphaerobacteria and several species have been found in 
 
318 THE ANALYSIS OF URINE. 
 
 urine. The cells are larger than those of micrococcus 
 ureae, and are arranged in groups of two or four usually. 
 They are not pathogenic. 
 
 Bacilli. Several species of the genus bacillus are found 
 in urine in disease. The most important of these are the 
 typhoid bacillus, bacillus typhi abdominalis, the tubercle 
 bacillus, bacillus tuberculosis and the bacillus of glanders, 
 bacillus mallei. These bacilli occur in urine only during 
 the progress of the corresponding diseases and their detec- 
 tion is of the highest interest. A description of the 
 
 m >."«» 
 
 %& \\\h 
 
 M 
 
 «i« 
 
 * 
 
 tfS 
 
 ♦ * 
 
 FIG. 57. 
 
 Micrococci and other bacteria. 
 
 methods to be followed for the certain demonstration of 
 these bodies is not within the scope of this book, but must 
 be looked for in the laboratory manuals of bacteriology. 
 
 Spirilla. Certain species of the genus spirillum have 
 been found in urine. The best known of these is the 
 spirillum of relapsing fever, spirillum Obermeieri. This is 
 only found rarely and as its habitat is the blood of relaps- 
 ing fever patients it must enter the urine through a haemor- 
 rhage into the kidney. Its form is that of a long, wavy 
 spiral, which makes its detection somewhat easy. 
 
THE ANALYSIS OF URINE. 
 
 319 
 
 Although not pathogenic it is well to call attention to 
 certain moulds which may sometimes be seen in urine. The 
 common blue-green mould, penicillium glaucum, is the best 
 known of these, and is occasionally found in urine along 
 with yeast cells. Another mould which has been found in 
 urine is the oidium lactis, commonly occurring in milk and 
 butter. It has been observed in fermenting diabetic urine. 
 Both of these fungi enter the urine after voiding. In 
 urine which has stood sometime in a cool place the penicil- 
 lium glaucum sometimes becomes covered with an incrusta- 
 tion of urates or minute crystals of uric acid. 
 
 QO(f 
 
 FIG. 58. 
 
 Yeast cells and common mould. 
 
 Finally we have yeast cells in urine and sometimes in 
 great numbers. Like other fungi they enter the urine 
 from the air and when not very abundant have no signifi- 
 cance. In great numbers the yeast cells suggest presence 
 of sugar. The ordinary yeast plant, saccharomyces cerevisice, 
 is shown, isolated and budding, in the accompanying 
 
 figure. 
 
 Other Parasites. 
 
 Besides fungi the urine in rare cases contains other 
 parasites of animal as well as vegetable nature. Some of 
 
320 THE ANALYSIS OF URINE. 
 
 these enter it accidentally from the air and have no inter- 
 est, but a few are present in the urine when passed, and 
 are of importance. Among these are certain thread worms, 
 the eggs of worms and hooklets of a species of tape- 
 worm, the taenia echinoeoccus. 
 
 These forms can appear in the urine only through rup- 
 ture from some other organ, and while rare here are com- 
 mon enough in Egypt and other tropical countries. Urine 
 containing eggs of worms, the worms themselves or frag- 
 ments usually contains blood or other evidence of rupture. 
 
Chapter XVIII. 
 
 UNORGANIZED SEDIHENTS AND CALCULI. 
 Unorganized Sediments. 
 
 Uric Acid. 
 
 A MONG the more common of the unorganized sediments 
 **• found in urine this must be mentioned first. As was 
 explained in the last chapter uric acid occurs normally in 
 combination in all human urine. 
 
 Some time after its passage urine often undergoes what 
 has been spoken of as the acid fermentation by which a pre- 
 cipitate of urates and even free uric acid may appear. This 
 reaction is in no sense due to a ferment process in the ordi- 
 nary sense of the term, but is probably brought about by a 
 purely chemical double decomposition. Urine contains 
 acid sodium phosphate and neutral sodium urate and it has 
 been suggested that these react on each other according to 
 the following equation: 
 
 Na 2 C 6 H 2 N 4 3 +NaH 2 P0 4 =NaC 5 H 3 N 4 3 + Na g HP0 4 
 
 The precipitate of acid urate settles out and forms a 
 light reddish deposit. If the amount of acid phosphates 
 present is excessive the reaction may go still further, re- 
 sulting in the precipitation of free uric acid. The well char- 
 acterized crystals of uric acid are often found with the 
 sediment of fine urates. Sometimes this liberation and pre- 
 cipitation of the acid takes place in the bladder and the 
 urine as passed shows the crystals or "gravel." If they are 
 relatively large, which is sometimes the case, their passage 
 through the urethra may cause severe pain. 
 
322 
 
 THE ANALYSIS OF URINE. 
 
 As the illustrations show uric acid occurs in a great 
 variety of forms. The rosettes and whetstone shaped crys- 
 
 tals are probably the most common, while long spiculated 
 forms are frequently seen. Pure uric acid is colorless but 
 
 as deposited from urine it is always reddish yellow, be- 
 cause of its property of carrying down coloring matters. 
 
THE ANALYSIS OF URINE. 323 
 
 The crystals are often so large that their general form can 
 be seen by the naked eye; usually, however, they are 
 minute. 
 
 Uric acid crystals when once deposited are not readily 
 redissolved by heat, but they go into solution by the addi- 
 tion of alkali. If the urine contains extraneous matter, as 
 specks of dusts, bits of hair, cotton or wool fibers the crys- 
 tals are very apt to deposit on them. 
 
 Urates. 
 
 The common fine sediments of urine are usually urates 
 or amorphous phosphates. They can be most readily dis- 
 
 A 
 
 B 
 
 FIG. 61. 
 
 Common crystalline and granular urates. 
 
 tinguished by their behavior with acids and on application 
 of heat. Urates disappear on warming the urine contain- 
 ing them, while a phosphate sediment is rendered more 
 abundant. A urate sediment is little changed by acids, 
 while the phosphates dissolve completely if the urine is 
 made acid in reaction with hydrochloric or nitric acid. The 
 acid urates of sodium and ammonium are the must abun- 
 dant and are shown in the cut. Acid ammonium urate may 
 
324 THE ANALYSIS OF URINE. 
 
 exist in urine which has become alkaline from the decom- 
 position of urea and formation of ammonium carbonate, 
 and may therefore be seen in company with the phosphate 
 sediments. The other urates dissolve in alkaline urine. 
 Like uric acid the urates appear in a great variety of forms, 
 and there is still some uncertainty about the composition 
 of some of their crystals which have been found in urine. 
 
 Leucin and Tyrosin. 
 
 These two substances are of rare occurrence in urine 
 and appear only under pathological conditions. Urine con- 
 taining them shows usually strong indications of the pres- 
 ence of biliary matters as they generally are found in con- 
 sequence of some grave disorder of the liver in which de- 
 struction of its tissue is involved. They have been most 
 frequently found, and associated, in acute yellow atrophy 
 of the liver and in severe cases of phosphorus poisoning. 
 In general they must be considered as products of disinte- 
 gration and are produced in the intestine in large quantity 
 by bacterial agency in the last stages of the digestion of 
 albuminoids, as was pointed out in an earlier chapter. 
 
 As both bodies are slightly soluble they may not be seen 
 directly, but only after partial concentration of the urine. 
 In pure condition leucin crystallizes in thin plates but 
 from urine it separates in spherical bunches made up of 
 fine plates or needles. These bunches are sometimes so 
 compact that it is hard to distinguish between them and 
 other substances, particularly lime soaps and oil drops. 
 Chemical tests must therefore be applied. If mercurous 
 nitrate is added to a leucin solution and the mixture is 
 warmed metallic mercury precipitates. This test can be 
 carried out only when the substance is abundant enough to 
 be purified by crystallization from hot water. Pure leucin 
 when strongly heated with nitric acid on platinum forms a 
 
THE ANALYSIS OF URINE. 
 
 325 
 
 colorless residue, which when heated with potassium hy- 
 droxide leaves an oil-like drop that does not wet the plati- 
 num. 
 
 Tyrosin is usually seen in long needles, which some- 
 times are bunched in the form of sheaves and more readily 
 recognized than is leucin. Tyrosin heated with nitric 
 acid on platinum turns orange yellow, and leaves a dark 
 residue which becomes reddish yellow by addition of 
 caustic alkali. Solutions containing tyrosin when treated 
 
 FIG. 63. 
 
 Leucin spheres, tyrosin needles and cystin plates. 
 
 hot with mercuric nitrate and potassium nitrite turn red 
 and finally throw down a red precipitate. 
 
 Leucin and tyrosin may be present in the urine yet not 
 show as a sediment. For their detection under these cir- 
 cumstances precipitate the urine with basic lead acetate 
 and filter. Separate the excess of lead from the filtrate by 
 hydrogen sulphide and filter again. Concentrate the fil- 
 trate on a water-bath to a syrup and treat it with a little 
 absolute alcohol to remove urea. Some leucin may dissolve 
 
326 THE ANALYSIS OF URINE. 
 
 with the urea in this treatment. Next boil the residue with 
 60 to 70 per cent alcohol, filter, concentrate the filtrate to a 
 small volume and allow it to stand in a cool place for crys- 
 tallization. If crystals appear their form indicates whether 
 the\' consist of pure tyrosin or a mixture of tyrosin and 
 leucin. The latter, being more soluble in strong alcohol, 
 can be separated by washing with this liquid. The final 
 tyrosin residue can be used for the tests given above. 
 
 The alcoholic solutions may contain leucin, which can 
 be recognized after evaporation. Both leucin and tyrosin 
 decompose readily in urine undergoing putrefactive 
 changes; it is therefore necessary to apply the test to urine 
 as fresh as possible. 
 
 Cystin. 
 
 This is a rare sediment, although it is found constantly 
 in the urine of certain individuals. It crystallizes in thin 
 hexagonal plates, small ones sometimes resting upon or 
 overlapping large ones. The crystals are regular in form 
 but variable in size and readily recognized. A rare form 
 of uric acid crystallizes in a somewhat similar manner but 
 the two substances differ in their behavior toward am- 
 monia. To distinguish between them in the microscopic 
 test place a drop of ammonia water on the slide and allow 
 it to pass under the cover glass. Cystin dissolves, but, 
 unless heated, uric acid does not. When the ammonia 
 evaporates cystin reprecipitates. 
 
 Cystin is precipitated from urine by addition of acetic 
 acid. Mucin and uric acid may come down at the same 
 time. The precipitate is collected on a filter, washed with 
 water and finally dissolved in ammonia. By neutralizing 
 the ammoniacal filtrate with acetic acid and concentrating 
 a little, it comes down in the characteristic form suitable 
 for microscopic recognition. 
 
THE ANALYSIS OF URINE. 
 
 Cholesterin. 
 
 327 
 
 This substance occurs occasionally in urine, and pos- 
 sibly only in cases of cystitis. It is recognized by its 
 characteristic crystalline form, large, thin plates, shown 
 in the following cut. These plates are nearly transparent 
 but from their size cannot be overlooked. 
 
 FIG. 63. 
 
 Cholesterin plates and fat globules. 
 
 Fat Globules. 
 
 These are often seen in urine, but in most cases have 
 not been voided with it. They can come from several 
 extraneous sources, as from a catheter, from vessels in 
 which the urine is collected or sent for examination, from 
 admixed sputum, etc., which facts should be borne in mind. 
 
 Fat has been found in cases of fatty degeneration of the 
 kidney and more abundantly in chyluria where communi- 
 cation seems to be formed between the lymphatics and the 
 urinary tract by the invasion of the small thread worms 
 referred to above. 
 
 Hippuric Acid. 
 
 This acid is found normally in human urine in small 
 amount. It may be found in large quantity after taking 
 
328 THE ANALYSIS OF URINE. 
 
 benzoic acid and may even appear in crystalline form in the 
 sediment. It has no pathological importance, ordinarily. 
 
 Calcium Carbonate. 
 
 This is sometimes observed as a coarse, granular sedi- 
 ment which dissolves with effervescence in acetic acid. It 
 occasionally forms dumb-bell crystals, and is devoid of path- 
 ological importance. 
 
 Calcium Sulphate. 
 
 Crystals of this substance are rarely found in urine. 
 They form long colorless needles or narrow, thin plates. 
 
 Calcium Oxalate. 
 
 We have here one of the commoner of the crystalline 
 bodies observed in urine. 
 
 This may be found in neutral or alkaline urine, but 
 more commonly in that of acid reaction. It occurs nor- 
 mally and sometimes is very abundant, especially after the 
 consumption of vegetables containing oxalic acid. 
 
 Two principal forms of the crystals are found, the octa- 
 hedral and dumb-bell crystals. 
 
 The octahedra have one very short axis which gives 
 the crystals a flat appearance. When seen with the short 
 axis perpendicular to the plane of the cover glass, which is 
 the common position, they appear as squares crossed by 
 two bright lines. Sometimes they are seen on edge, and 
 then present a rhomb in section with one diameter very 
 much shorter than the other. 
 
 A form of triple phosphate bears a slight resemblance 
 to calcium oxalate, but it is soluble in acetic acid, while 
 the oxalate is not. 
 
 The dumb-bells are much less common than the octa- 
 hedra, and are found in several modified forms, as shown 
 in one of the figures. 
 
THE ANALYSIS OF URINE. 329 
 
 The clinical significance of the oxalate is not clearly 
 understood; It does not seem to be characteristic of any 
 disease even when occurring in quantity. It has been 
 found considerably increased in dyspeptic conditions, but 
 not always, and many of the statements found concerning 
 its significance seem to have been based on insufficient 
 observations. 
 
 Urine may contain a large amount of oxalic acid which 
 does not show as a sediment, but must be found by pre- 
 
 FIG. 64. 
 
 Calcium oxalate. 
 
 cipitation by calcium chloride in presence of ammonium 
 hydroxide. Acetic acid is then added in very slight excess 
 and the mixture is allowed to stand for precipitation. 
 
 The constant or prolonged excretion of large amounts 
 of oxalic acid is spoken of as oxaluria. 
 
 The Phosphates. 
 
 It was explained in Chapter XVI. that phosphates of 
 alkali and alkali-earth metals occur normally in the urine, 
 and a method was given for their measurement. As sedi- 
 
330 
 
 THE ANALYSIS OF URINE. 
 
 ment we know several forms of calcium and magnesium 
 phosphates and the microscopic detection of these will be 
 here explained. In normal fresh urine of acid reaction these 
 phosphates are held in solution, but if the urine as passed 
 is alkaline it is often turbid from the presence of basic 
 phosphates held in suspension. Urine which has stood 
 long enough to undergo the alkaline fermentation always 
 contains phosphates in the sediment. Finally, it must be 
 remembered that a neutral or very slightly acid urine, con- 
 
 FIG. 65. 
 
 Triple phosphate. 
 
 taining ammonium salts in abundance, may also deposit 
 a crystalline precipitate of ammonium magnesium phos- 
 phate. The common phosphate sediments are those con- 
 sisting of ammonium magnesium phosphate (triple phos- 
 phate), basic magnesium phosphate, neutral calcium 
 phosphate and mixed amorphous phosphates of calcium 
 and magnesium. 
 
 Triple Phosphate. Of the crystalline phosphate de- 
 posits this is the most abundant and at the same time the 
 most characteristic. 
 
THE ANALYSIS OF URINE. 
 
 331 
 
 The crystals are the largest found in urine, and from 
 their shape are sometimes spoken of as coffin lid crystals. 
 Ordinarily they are not found in perfectly fresh urine, but 
 after it has undergone the alkaline fermentation they are 
 generally present in profusion. 
 
 Basic Magnesium Phosphates. Crystals having the 
 composition Mg 3 (P0 4 ) 2 .22H 3 are sometimes found in 
 urine of nearly neutral reaction. They consist of thin, 
 
 FIG. 66. 
 
 Neutral calcium phosphate and amorphous phosphate. 
 
 transparent rhombic plates with angles of approximately 
 60° and 120°. If urine containing this sediment becomes 
 alkaline triple phosphate forms. 
 
 Neutral Calcium Phosphate. This has the compo- 
 sition CaHP0 4 .2H z O and is found in urine of neutral or 
 slightly acid reaction. It crystallizes frequently in rosettes 
 formed of wedge-shaped single crystals uniting at their 
 apices. The cut above shows some variations in the form. 
 
 Amorphous Phosphates. Finally we have the very 
 common, finely granular earthy phosphates in amorphous 
 condition. This sediment dissolves readily in weak acetic 
 
332 THE ANALYSIS OF VRINE. 
 
 acid and is colorless. The common amorphous urate sedi- 
 ment is colored and does not dissolve in acetic acid. On 
 addition of sodium carbonate or hydroxide to urine the pre- 
 cipitate which forms consists mainly of this phosphate. 
 
 These several phosphates can be produced artificially 
 and should be made for study and comparison. The 
 neutral (basic) magnesium phosphate can be made by dis- 
 solving 15 Gm. of crystallized common sodium phosphate 
 in 200 Cc. of water and mixing this with 3.7 Gm. of crystal- 
 lized magnesium sulphate in 2000 Cc. of water. Enough 
 sodium bicarbonate is added to give an amphoteric reac- 
 tion and then the mixture is allowed to stand a day or 
 more for precipitation. 
 
 Crystals of triple phosphate of peculiar form are often 
 obtained by adding ammonia to urine, and sometimes a 
 trace of ammonia is sufficient to throw down the crystals 
 of neutral calcium phosphate. The latter can also be ob- 
 tained by adding to a weak solution of crystallized sodium 
 phosphate a trace of acid and then a very little calcium 
 chloride solution. 
 
 Foreign Matters. 
 
 The sediment of urine often contains foreign sub- 
 stances which have become mixed with it accidentally. 
 The most common of these are hairs, woolen, cotton or 
 silk fibers, granules of starch, fat globules, dust and sand 
 granules, bits of woody fiber and remains of articles of 
 food. Some of these are represented in the cuts on the 
 following pages. 
 
 Urinary Calculi. 
 
 Calculi like the sediments just described are formed by 
 the precipitation of certain substances from the urine, but 
 in compact form. Occasionally a calculus consists of a 
 single substance, as calcium oxalate or cystin, but in the 
 
THE ANALYSIS OF URINE. 
 
 333 
 
 great majority of cases a mixture of bodies is present, 
 these being deposited usually in layers around a nucleus 
 which serves as the foundation of the concretion. Calculi 
 are built up much as certain forms of crystals are by suc- 
 cessive depositions on a nucleus. Uric acid is a very 
 common nucleus on which may be deposited urates, 
 phosphates, organic matters, etc. 
 
 Calculi are sometimes distinguished as primary or sec- 
 ondary. Primary calculi may be traced to an alteration of 
 
 Z, linen fibers; H, hemp fibers; J, jute fibers; B, cotton fibers; S, silk 
 fibers; A, alpaca wool; E, fine wool; W, common wool. 
 
 the urine of such a nature that its reaction is constantly 
 acid. The foundation for the concretions in this case is 
 found in the kidney and they are built up of such sub- 
 stances as most easily deposit from acid urine. Secondary 
 calculi are generally formed in the bladder and have as 
 nuclei matters precipitated from alkaline urine, coagulated 
 blood or other organic substances. Sometimes fragments 
 introduced into the bladder from without serve as the foun- 
 dation for these secondary formations. Bits of catheters, 
 remains of bougies, and other things have been found as 
 
334 
 
 THE ANALYSIS OF URINE. 
 
 the nuclei around which concretions have formed. The 
 recognition of the nucleus is a matter of the first impor- 
 tance as this gives a clew to the determining cause active 
 in the formation of the calculus. 
 
 In making an examination, then, of a calculus, it is 
 first cut in two by means of a very sharp thin saw. This 
 exposes the nucleus which may often be recognized by the 
 eye alone. If one of the halves be polished it is often 
 possible to discern distinctly the various layers grouped 
 around the center. 
 
 FIG. 68. 
 
 Left, pubic hair with spermatozoa; center, hair of woman's head; 
 right, cat's fur. 
 
 In a large number of cases examined by Ultzmann 
 about 80 per cent were found to contain uric acid as the 
 nucleus. 
 
 Chemical Examination. 
 
 In the chemical examination of a calculus several 
 methods may be employed. We may begin by applying 
 certain preliminary tests designed to show the general na- 
 ture of the stone. 
 
 Heat Test. Reduce some of the calculus to a pow- 
 der and heat to bright redness on platinum foil. Two cases 
 may arise: (a), the powder is completely consumed; (b), 
 the powder is only partially consumed or not at all. 
 
THE ANALYSIS OF URINE. 335 
 
 Case (a.) If this is the result of the incineration the 
 following substances may be suspected: 
 
 Uric Acid, which may be recognized by dissolving a 
 little of the powder in weak alkali, precipitating by hydro- 
 chloric acid and examining the precipitate by the micro, 
 scope. 
 
 Ammonium Urate. This gives the above reaction 
 under the microscope, and is further recognized by the lib- 
 eration of ammonia when heated with a little pure sodium 
 hydroxide solution. 
 
 Cystin. Dissolve some of the powder in ammonia, fil- 
 ter if necessary and allow drops of the filtrate to evaporate 
 spontaneously on a slide. Cystin is then recognized by the 
 microscope as already explained. Cystin contains sulphur 
 which on burning on the platinum foil gives rise to a disa- 
 greeable sharp odor. If a little of the powder be heated 
 with a mixture of potassium nitrate and sodium carbonate 
 the sulphur is oxidized to sulphate, which may be recog- 
 nized by the usual tests. 
 
 Xanthin. This is a rare substance in calculi. Those 
 consisting wholly of xanthin are brown in color and take a 
 wax-like polish. A method of recognition will be given 
 below. 
 
 Organized Matter. Parts of blood cells, epithelium, 
 precipitated mucin, pus corpuscles and similar substances 
 may become entangled with the growing stone and even 
 form a large part of it. On burning, these bodies are rec- 
 ognized by the characteristic odor of nitrogenous matter. 
 
 Case (i.) When an incombustible residue is left on the 
 platinum foil the stone may contain the following constitu- 
 ents: 
 
336 THE ANALYSIS OF URINE. 
 
 Calcium Oxalate. Stones of this substance are very 
 hard and break with a crystalline fracture. They are often 
 called "mulberry calculi." When the powder is heated it 
 decomposes, leaving carbonate, which may be recognized 
 by its effervescence with acids. 
 
 Calcium and Magnesium Phosphates. These leave 
 a residue in which the metals and phosphoric acid may be 
 detected by simple tests of qualitative analysis. The ig- 
 nited powder is soluble in hydrochloric acid without effer- 
 vescence. When ammonia is added to this solution in 
 quantity sufficient to give an alkaline reaction a precipitate 
 of triple phosphate or calcium phosphate appears, which 
 may be recognized by the microscope. 
 
 The above tests are generally sufficient to tell all that 
 is practically necessary about the calculus. If more de- 
 tailed information is desired a systematic analysis may be 
 made according to the following scheme. 
 
 Systematic Analysis. 1. Reduce the calculus to a 
 fine powder and pour over it some water and finally dilute 
 hydrochloric acid in a beaker. Warm gently half an hour, 
 or longer, on the water-bath. Then allow to cool and 
 filter. 
 
 2. Treatment of the residue. It seldom happens that 
 the calculus is completely soluble in the weak acid. A 
 residue usually remains which may contain uric acid, xan- 
 thin, calcium sulphate, and remains of organized matter. 
 To prove the xanthin treat the residue with warm dilute 
 ammonia and filter. The filtrate contains the xanthin if 
 it is present. Acidify it with nitric acid and add a small 
 amount of silver nitrate solution. This produces a floccu- 
 lent precipitate which dissolves by warming, and crystal- 
 lizes on cooling in bunches of fine needles. 
 
 In the residue free from the xanthin look for calcium 
 
THE ANALYSIS OF URINE. 337 
 
 sulphate by extracting with water and applying the usual 
 tests. This solution may contain uric acid which is recog- 
 nized by evaporation and crystallization after adding a lit- 
 tle hydrochloric acid. In the final residue some uric acid 
 may be also present. Dissolve in alkali, reprecipitate with 
 hydrochloric acid, and examine any crystals which may 
 form under the microscope. 
 
 3. Treatment of the hydrochloric acid solution. This 
 may contain calcium oxalate, cystin, the phosphates, and 
 possibly some xanthin. Look for the last in a small por- 
 tion of the solution. Make this portion alkaline with am- 
 monia, add a few drops of calcium chloride solution, filter 
 if a precipitate forms and treat the filtrate with ammoni- 
 acal silver nitrate solution. In presence of xanthin a floc- 
 culent precipitate forms. 
 
 Dilute the remaining and larger portion of the hydro- 
 chloric acid solution with twice its volume of water, add 
 enough ammonia to give a strong alkaline reaction and 
 then acetic acid to restore a weak acid reaction. By this 
 treatment phosphates are held in solution while calcium 
 oxalate, if present, precipitates. Therefore allow the mix- 
 ture to stand half an hour and then filter off any precipi- 
 tate which appears. This precipitate may contain cystin 
 as well as calcium oxalate. Cystin may be dissolved 
 by pouring ammonia on the filter, and on evaporating the 
 ammoniacal solution is obtained in form suitable for mi- 
 croscopic examination. 
 
 The residue free from cystin is dried and heated to red- 
 ness on platinum foil. This treatment converts calcium 
 oxalate into carbonate. Place the foil in a beaker and add 
 some dilute acetic acid; an effervescence shows the car- 
 bonate. To the clear solution add next some ammonium 
 oxalate which gives a white precipitate of calcium oxalate, 
 if the latter metal is present. 
 
338 THE ANALYSIS OF URINE. 
 
 We have next to look for the phosphates and bases in 
 the acetic acid solution obtained after filtering off cyst in 
 and calcium oxalate. More calcium may be present, in 
 excess of that combined as oxalate, which may be recog- 
 nized by adding a little solution of ammonium oxalate. If 
 a precipitate forms treat the whole of the liquid with the 
 ammonium oxalate, after warming on the water-bath, allow 
 to stand an hour and filter. Concentrate the filtrate in 
 platinum to a small volume, transfer to a large test-tube and 
 add enough ammonia to produce an alkaline reaction. If 
 a precipitate appears now it must consist of magnesium 
 phosphate, showing both magnesium and phosphoric acid 
 as present in the original. If no precipitate appears mag- 
 nesium is absent but phosphoric acid may still be present. 
 To find it divide the ammoniacal liquid into two portions. 
 To one add a few drops of magnesia mixture, and to the 
 other add nitric acid in slight excess and then ammonium 
 molybdate reagent. Both tests should yield the reactions 
 characteristic of phosphates, if present. 
 
 This procedure serves for the recognition of the im- 
 portant constituents of calculi. But ammonium, potassium 
 and sodium compounds are sometimes present and may be 
 recognized readily. To detect ammonium salts the original 
 calculus powder may be heated with pure potassium 
 hydroxide solution, or the hydrochloric acid solution of the 
 calculus may be neutralized and heated with the same 
 solution. The ammonia is recognized by the odor or by 
 its reaction on moistened red litmus paper. 
 
 To recognize the alkali metals a solution of the 
 powdered calculus in hydrochloric acid is treated with 
 pure ammonia and a little ammonium carbonate in excess. 
 The precipitate formed is allowed to settle and filtered off. 
 The filtrate is then evaporated to dryness in a platinum 
 dish and the residue strongly heated to drive off all am- 
 
THE ANALYSIS OF URINE. 339 
 
 monium salts. What is now left contains sodium and 
 potassium if they were present in the original. Moisten 
 this final residue with water and a drop of hydrochloric 
 acid and test with a platinum wire in the flame of a Bun- 
 sen burner, using a deep blue glass when looking for 
 potassium. Only a very intense yellow color can be taken 
 as indicative of sodium. 
 
APPENDIX. 
 
 Test Solutions and Tables. 
 
APPENDIX. 
 
 343 
 
 TABLE OF ATOMIC WEIGHTS. 
 
 (ACCORDING TO MEYER AND SEUBERT.) 
 
 Aluminum [ Al 
 
 Antimony Sb 
 
 Arsenic i As 
 
 Barium Ba 
 
 Beryllium . . . Be 
 
 Bismuth Bi 
 
 Boron i B 
 
 Bromine Br 
 
 Cadmium Cd 
 
 Caesium | Cs 
 
 Calcium Ca 
 
 Carbon C 
 
 Cerium j Ce 
 
 Chlorine CI 
 
 Chromium Cr 
 
 Cobalt ; Co 
 
 Columbium Cb 
 
 Copper Cu 
 
 Didymium Di 
 
 Erbium Er 
 
 Fluorine F 
 
 Gallium Ga 
 
 Germanium Ge 
 
 Gold Au 
 
 Hydrogen ; H 
 
 Indium In 
 
 Iodine I 
 
 Iridium Ir 
 
 Iron \ Fe 
 
 Lanthanum ! La 
 
 Lead j Pb 
 
 Lithium Li 
 
 Magnesium Mg 
 
 Manganese I Mn 
 
 Mercury. I Hg 
 
 Atomic 
 Weight. 
 
 27.04 
 
 119.6 
 74.9 
 136.9 
 9.03 
 208.9 
 10.9 
 79.76 
 111.5 
 132.7 
 39.91 
 11.97 
 139.9 
 35.37 
 52.0 
 58.6 
 93.7 
 63.18 
 142.0 
 166.0 
 19.0 
 69.9 
 72.3 
 196.7 
 1.0 
 113.6 
 126.53 
 192.5 
 55.88 
 138.2 
 206.4 
 7.01 
 24.3 
 54.8 
 199.8 
 
 Name. 
 
 Symbol. 
 
 Atomic 
 Weight. 
 
 Molybdenum . . 
 
 Mo 
 
 95.9 
 
 Nickel 
 
 Ni 
 
 58.6 
 
 Nitrogen 
 
 N 
 
 14.01 
 
 
 Os 
 
 190.3 
 
 
 O 
 
 15.96 
 
 
 Pd 
 
 106.35 
 
 Phosphorus . . . 
 
 P 
 
 30.96 
 
 
 Pt 
 
 194.3 
 
 Potassium 
 
 K 
 
 39.03 
 
 Rhodium 
 
 Rh 
 
 102.9 
 
 
 Rb 
 
 85.2 
 
 Ruthenium 
 
 Ru 
 
 101.4 
 
 
 Sm 
 
 149.62 
 
 Scandium 
 
 Sc 
 
 43.97 
 
 
 Se 
 
 78.87 
 
 Silicon 
 
 Si 
 
 28.3 
 
 Silver 
 
 Ag 
 
 107.66 
 
 i Sodium 
 
 Na 
 
 23.0 
 
 Strontium .... 
 
 Sr 
 
 87.3 
 
 
 S 
 
 31.98 
 
 Tantalum 
 
 Ta 
 
 182.0 
 
 Tellurium .... 
 
 Te 
 
 125.0 
 
 Terbium 
 
 Tb 
 
 159.1 
 
 Thallium 
 
 Tl 
 
 203.7 
 
 Thorium 
 
 Th 
 
 231.9 
 
 Tin 
 
 Sn 
 Ti 
 
 118 8 
 
 Titanium 
 
 48.0 
 
 Tungsten 
 
 W 
 
 183.6 
 
 Uranium ..... 
 
 U 
 
 239.0 
 
 Vanadium 
 
 V 
 
 51.1 
 
 Ytterbium 
 
 Yb' 
 
 172.6 
 
 Yttrium 
 
 Yt 
 
 88.9 
 
 
 Zn 
 Zr 
 
 65 1 
 
 Zirconium .... 
 
 90.4 
 
344 APPENDIX. 
 
 List of General Reagents and Test Solutions. 
 
 Acid, sulphuric (strong). The commercial acid is sufficient for. 
 most purposes, where a strong acid is called for. Where the pure acid 
 is required it is mentioned in the text. 
 
 Acid, sulphuric (dilute). Add one part of the above acid to four 
 parts of distilled water, mix thoroughly, and allow to stand twenty-four 
 hours. Then siphon, or pour off the clear liquid, which is ready for use. 
 The strong acid must be poured into the water very slowly, and with 
 constant stirring, to avoid a too sudden elevation of temperature. 
 
 Acid, nitric (strong). The strong commercial acid can be employed 
 in most cases where this acid is called for. A pure strong acid is also 
 employed, occasionally. 
 
 Acid, nitric (dilute). Where this acid is called for as a reagent it 
 should be made by mixing one part of the pure strong acid with four 
 parts of distilled water It should be free from traces of chlorine and 
 sulphates. 
 
 Acid, hydrochloric (strong). The yellow commercial acid is large- 
 ly used in the laboratory in the preparation of other substances It is 
 seldom pure enough to be employed as a test reagent. 
 
 -A colorless acid, free from organic matter, iron and traces of sul- 
 phates, must be used when the pure strong acid is called for. 
 
 Acid, hydrochloric (dilute.) This is frequently used in liberat- 
 ing hydrogen sulphide, carbon dioxide, and hydrogen, and for other pur- 
 poses, and need not be pure. Where the dilute acid is called for as a 
 reagent it must be made by mixing one part of the pure strong acid 
 with four parts of distilled water. 
 
 Acid, acetic. Mix one part of the pure "glacial " acid with four 
 parts of water. 
 
 Ammonia water. The strong solution is seldom used in analysis. 
 The solution usually employed is made by mixing one volume of the 
 stronger ammonia water (containing about 28 per cent of the gas) with 
 three volumes of distilled water. 
 
 The solution should be free from carbonic acid, as presence of this 
 would interfere with several of the tests where it is employed. 
 
 Ammonium carbonate. Dissolve one part of the pure powdered 
 crystals in five parts of dilute ammonia water. 
 
 Ammonium chloride. Dissolve one part of the pure salt in ten 
 parts of water. 
 
 Ammonium molybdate. A solution of this salt is chiefly used as a 
 test for phosphoric acid, and should be prepared in this way : Dissolve 
 3 Gm of the crystals in 20 Cc. of water, and pour this solution in 20 
 Cc. of strong nitric acid. Warm the mixture to about 40° C. (not 
 above), and allow to settle. 
 
APPENDIX. 345 
 
 As the reagent does not keep well, it should be made only in small 
 quantities. 
 
 Ammonium oxalate. Dissolve one part of the pure crystals in 
 twenty parts of water. 
 
 Ammonium sulphide. Dilute a quantity o£ strong ammonia water 
 with an equal volume of water. Take three-fifths of this mixture and 
 saturate it with hydrogen sulphide. Then add the remaining two-fifths 
 of the diluted ammonia water and mix thoroughly. 
 
 Barium chloride. Dissolve one part of the crystals in ten parts of 
 water. 
 
 Calcium hydroxide. Slake pure white lijne and pour over it a 
 large excess of water. Allow to settle and throw away the clear liquid. 
 Again add pure water, shake thoroughly, allow to settle as before, and 
 decant the clear liquid into bottles for use. These bottles must be 
 tightly stoppered. The portion rejected contains small amounts of 
 impurities, possibly present in the lime. 
 
 Lead acetate. One part of the pure crystals to ten parts of water. 
 It may be necessary to add a few drops of acetic acid to secure a clear 
 solution. 
 
 Lead acetate, basic. To make a liter of solution of this reagent 
 weigh put 170 Gm. of lead acetate and 120 Gm. of yellow lead oxide. 
 
 Dissolve the lead acetate in 800 Cc. of boiling, distilled water, in a 
 glass or porcelain vessel. Then add the oxide of lead and boil for half 
 an hour, occasionally adding enough hot distilled water to make up the 
 loss by evaporation. Remove the heat, allow the liquid to cool, and add 
 enough distilled water, previously boiled and cooled, to make the prod- 
 uct measure 1,000 Cc. Finally, filter the liquid in a covered funnel. 
 
 Solution of basic lead acetate should be kept in well-stoppered 
 bottles. 
 
 Magnesia mixture. Dissolve 100 Gm. of magnesium sulphate and 
 100 Gm. of ammonium chloride in 800 Cc. of water, and add 100 Cc. of 
 strong ammonia water. Allow the mixture to stand twenty-four hours, 
 and filter. 
 
 Mercuric chloride. One part of the pure crystals to twenty parts 
 of water. 
 
 Millon's reagent. Dissolve one part of mercury in two parts of 
 strong nitric acid, by aid of heat finally, and after cooling dilute the 
 solution with twice its volume of water. 
 
 Potassium bichromate. Dissolve one part of the pure crystals in 
 ten parts of water. The dry powdered crystals are also used. 
 
 Potassium chromate. Dissolve one part of the crystals, free 
 from chlorine, in ten parts of water. 
 
 Potassium ferrocyanide. Dissolve one part of the crystals in 
 twenty parts of water. 
 
346 APPENDIX. 
 
 Potassium hydroxide. Several solutions are employed in an- 
 alytical chemistry. For most purposes one containing ten per cent of 
 the "stick'' hydroxide is sufficient. 
 
 Sodium hydroxide. Dissolve one part of the best "stick" hydrox- 
 ide in ten pans of water, allow to settle, and decant the clear solution. 
 
 This solution acts on glass bottles, and soon deposits a sediment. 
 Hence, a great deal of it should not be made at one time. The glass 
 stoppers of bottles containing sodium hydroxide, and many other sub- 
 stances, should be covered with a thin layer of paraffine, which prevents 
 their sticking fast. 
 
 Sodium hypochlorite. The "chlorinated soda" of the U. S. P. is 
 to be used here, and the solution may be made in this manner: Weigh 
 out 75 Gm. of good commercial "chloride of lime" and rub it up with 
 200 Cc. of water to a thin cream. Allow this to settle and pour the 
 liquid through a filter. Stir the residue with a second 200 Cc. of water, 
 pour the whole on the filter and wash the insoluble residue with 100 Cc. 
 of water, allowing this to mix with the 400 Cc. Now dissolve 150 
 Gm. of sodium carbonate (crystals) in 300 Cc. of hot water and 
 pour this into the other solution. Warm the mixed solution and stir it 
 well. Pour the mixture on a filter and when the liquid has run through 
 pour on water enough to bring the filtrate up to 1000 Cc. The solution 
 so obtained should be kept in the dark. 
 
 Special Reagents. 
 
 Solutions for Sugar Tests. 
 
 Fehling solution. This has been referred to at length in Chapters 
 II. and XII., and its preparation will now be given. In presence of 
 alkali copper solutions are reduced by dextrose approximately according 
 to this proportion: 
 
 0(CuSO 4 .5H a O) : C.HhO,. 
 1244.00 : 179.58. 
 
 from which it follows that 34.64 Mg. of the crystallized sulphate in so- 
 lution oxidizes 5 Mg. of dextrose in solution. 
 
 The Fehling solution proper consists of a mixture of copper sul- 
 phate, an alkali and a tartrate. Investigation has shown that as 
 alkali sodium hydroxide is preferable and that Rochelle salt is the best 
 tartrate for the purpose. It has also been found that the best results are 
 obtained if the copper and tartrate are mixed just before needed for use. 
 Therefore, prepare solutions separately as follows: 
 
 1. Dissolve 69.28 Gm. of pure, recrystallized copper sulphate in dis- 
 tilled water to make 1 liter of solution. Much of the copper sulphate 
 sold by druggists contains ferrous sulphate and is not suitable for the pur- 
 pose. 
 
 2. Dissolve 100 Gm. of sodium hydroxide in sticks in 500 Cc. of 
 water, heat to boiling and add gradually 350 Gm. of pure recrystallized 
 Rochelle salt. Stir until all is dissolved. Allow the solution to stand 24 
 hours in a covered vessel, then filter through asbestos into a liter flask 
 and add water enough to make the solution 1 liter. The sodium hydrox- 
 ide for this purpose should be the grade designated ' precipitated by al- 
 cohol" and the Rochelle salt should be practically pure. 
 
APPENDIX. 347 
 
 3. By mixing equal volumes of these two solutions the Fehling liquid 
 is prepared, containing 34.04 Mg. of the copper sulphate in each cubic 
 centimeter. This mixture is made when required for use. 
 
 The Loewe solution. According to Loewe copper solutions pre- 
 pared with glycerol are more stable than are those with a tartrate, while 
 the oxidizing power of the copper hydroxide is practically the same. 
 For quantitative tests Loewe prepares a solution by mixing 
 
 Crystallized copper sulphate 34.64 Gm. 
 
 Pure glycerol 26.00 Gm. 
 
 Sodium hydroxide solution, 1.34 Sp. Gr. 70.00 Cc. 
 with a small amount of water and heating to dissolve, after which the 
 solution is diluted to 1 liter 
 
 When used as a qualitative test this solution is diluted by adding 
 glycerol. In place of the crystallized copper sulphate Loewe recom- 
 mends to weigh out the corresponding amount of precipitated, washed 
 and dried copper hydroxide, wliich can be kept indefinitely in perfectly 
 stable form. 
 
 Pavy's solution. The use of the Pavy liquid as a sugar test was 
 explained in Chapter XII. It is prepared as follows: 
 
 Dissolve 34.64 Gm. of crystallized copper sulphate, 170 Gm. of Ro- 
 chelle salt and 170 Gm. of good stick potassium hydroxide in distilled 
 water to make 1 liter. Mix 120 Cc. of this solution with 400 Cc. of 
 strong ammonia water, Sp. Gr. 0.88, and dilute with distilled water to 1 
 liter. The oxidizing power of this solution is assumed to be just one- 
 tenth of that of the Fehling solution, which would follow if the reaction 
 takes place in the proportion, 6 (CuSCvoHaO) : CeH u O e . 
 
 Loewe-Pavy solution, as recommended by Dr. Purdy. 
 This is made by dissolving 
 
 Crystallized copper sulphate 4.74 Gm. 
 
 Potassium hydroxide 23. 50 ' ' 
 
 Ammonia water, Sp. Gr. 0.90 450.00 Cc. 
 
 Glycerol 38.00 " 
 
 in enough water to make 1000 Cc. 
 
 Dissolve the copper sulphate and glycerol in 200 Cc. of the water. 
 In another 200 Cc. dissolve the alkali. Mix the two solutions, cool, add 
 the ammonia and make up to the 1000 Cc. Pure chemicals must be em- 
 ployed 
 
 * According to Dr. Purdy 3.") Cc. of this solution oxidizes 20 Mg. of 
 dextrose. 
 
 Schmiedeberg's solution. Dissolve 34.64 Gm. of pure crystallized 
 copper sulphate in 200 Cc. of water and 16 Gm. of mannitol in 100 Cc. 
 of water. Mix the two solutions and add 480 Cc. of sodium hydroxide 
 solution, having a specific gravity of 1.145. Dilute to 1 liter. This solu- 
 tion is assumed to have the oxidizing power of the Fehling solution. 
 
 Knapp's solution. This is made by dissolving 10 Gm of dry mer- 
 curic cyanide in 600 Cc. of distilled water. To this solution is added 
 100 Cc. of a solution of sodium hydroxide, having a specific gravity of 
 1.145 and the mixture is diluted to 1 liter. 
 
 Sachsse's solution. Dissolve 18 Gm. of pure mercuric iodide and 
 
348 APPENDIX. 
 
 25 Gm. of potassium iodide in water. Add a solution of 80 Gm. of good 
 potassium hydroxide and dilute to 1 liter. These solutions are nearly 
 equal in oxidizing power. It has been shown that 50 Cc. of Sachsse's 
 solution is reduced by 168 Mg. of dextrose. 
 
 Solutions for Water Tests. 
 
 Nessler's reagent. Dissolve 13 Gm. of mercuric chloride and 35 
 Gm. of potassium iodide in 600 Cc. of water by aid of heat. When all 
 is dissolved add a little more mercuric chloride until a trifling red pre- 
 cipitate remains. Dissolve 160 Gm. of potassium hydroxide in 200 Cc. 
 of water, and when cold add this solution to the other. Allow the mix- 
 ture to stand twenty-four hours and pour the clear solution from the 
 brownish precipitate. Sensitize this liquid poured off by adding, a drop 
 at the time, solution of mercuric chloride as long as a clear solution is 
 left on shaking. 
 
 Alkaline permanganate solution. Dissolve 8 Gm. of potassium 
 permanganate crystals in 800 Cc. of water, add 200 Gm. of potassium 
 hydroxide in sticks and water to make 1200 Cc. Boil this solution down 
 to 1000 Cc. in a porcelain dish, cool and pour into a bottle which close 
 with a paraffined glass stopper. 
 
 Other solutions used in water tests are described in Chapter VIII. 
 
 Indicators. 
 
 The indicators most commonly employed in the titration of acids 
 and alkalies are aqueous or alcoholic solutions of lilmtts, cochineal, phenol 
 phthalein, methyl orange and rosolic acid. In addition to these certain 
 others are employed for special purposes, and among these there may be 
 mentioned, tongo red, btnzopurpurin, methyl violet and the iropccolins . A 
 few explanations will be given on the preparation of the first. 
 
 Litmus. Crude litmus comes in commerce in the form of small 
 blue cubes. These are powdered and extracted by hot water. The 
 aqueous solution is concentrated and acidified with acetic acid, after 
 which it is evaporated to a paste. Treat this with an excess of 85 per 
 cent alcohol which dissolves foreign matters but leaves the true color. 
 Throw the mixture on a filter and wash the residue with strong alcohol. 
 Then dissolve the precipitated color on the filter by means of hot water 
 and keep this aqueous solution for use in a bottle loosely stoppered as ac- 
 cess of air is necessary for its preservation. 
 
 A neutral litmus solution is used for certain purposes and may be pre- 
 pared by dividing the aqueous solution just described into two portions, 
 one of which is rendered faintly acid by nitric acid, while the other is 
 made alkaline by potassium hydroxide, added in drops of very dilute 
 solution. On mixing these two liquids the product will be found prac- 
 tically neutral. 
 
 Cochineal. A solution is made by extracting 1 part of the crushed 
 cochineal with 10 parts of weak alcohol. This indicator is valuable in 
 titrating in presence of carbonic acid or ammonia. 
 
APPENDIX. 
 
 349 
 
 Phenol phthalein. Dissolve 1 part of the pure commercial prod- 
 uct in 200 parts of 50 per cent alcohol. This indicator cannot be well 
 used with ammonia or in presence of free carbonic acid. 
 
 Methyl orange. Dissolve 1 part of the color in 1,000 parts of 
 distilled water. Although this solution is very weak a single drop is 
 sufficient for an ordinary titration; with more the change of color is less 
 characteristic or sharp The indicator is valuable in the titration of 
 carbonates or ammoniacal liquids. Carbonic acid does not act on it. 
 
 Rosolic acid. Dissolve 1 part in 500 parts of 50 per cent alcohol. 
 This is a sensitive indicator for the mineral acids. 
 
 Tables of Weights and Measures. 
 
 The Metric System. 
 
 1 Meter = 100 Centimeters (Cm.) = 1,000 Millimeters (Mm. 
 1 Liter = 1,000 Cubic Centimeters (Cc.) 
 
 1 Kilogram = 1,000 Grams (Gm.) 
 1 Gram = 1.000 Milligrams (Mg.) 
 
 American Weights and Measures. 
 
 1 Gallon 
 
 1 Pint 
 
 1 Fluidounce 
 
 1 Fluidrachm 
 1 Avoirdupois pound 
 1 Avoirdupois ounce 
 
 1 Apothecaries' ounce 
 1 Apothecaries' drachm 
 
 8 Pints. 
 16 Fluidounces. 
 
 8 Fluidrachms. 
 60 Minims. 
 
 16 Avoirdupois ounces. 
 437# Grains. 
 
 8 Drachms. 
 60 Grains. 
 
 Equivalents. 
 
 1 Meter 
 
 1 Liter 
 
 1 Liter 
 
 1 Cubic Centimeter 
 
 1 Kilogram 
 
 1 Kilogram 
 
 1 Gram 
 
 1 U. S. fluidounce 
 
 1 Imperial fluidounce 
 
 1 U. S. Apoih. ounce 
 
 1 Avoird. ounce 
 
 1 Grain 
 
 39 370 
 
 33.815 
 
 35.219 
 
 16.231 
 
 32.151 
 
 35.274 
 
 15.432 
 
 29.57 
 
 28.39 
 
 31.103 
 
 28.349 
 
 64.798 
 
 Inches. 
 
 U. S Fluidounces. 
 
 Imp. Fluidounces. 
 
 U. S. Minims. 
 
 U. S. Apoth. ounces. 
 
 Avoirdupois ounces. 
 
 Grains. 
 
 Cc. 
 
 Cc. 
 
 Gm. 
 
 Gm. 
 
 Milligrams. 
 
 fj S Fluidounce of Water weighs 0.95 U. S. Apoth. ounce. 
 96 U S Fluidounces of Water weigh 100 Avoirdupois ounces. 
 1 Imperial Fluidounce of Water weighs 1 Avoirdupois ounce. 
 
 Determination of the Specific Gravity of Liquids. 
 
 The specific gravity of liquids may be found by several methods, 
 most accurately by means of the pycnometer or small weighing bottle. 
 
350 
 
 APPENDIX. 
 
 For approximate tests specific gravity bulbs, illustrated by the urinom- 
 eter already described, are very commonly used. 
 
 A very convenient and accurate apparatus for these determinations 
 is the Mohr-Westphal balance, shown in the figure below. From the 
 outer end of the beam is suspended a small weight, A, usually in the 
 form of a thermometer, which hung in the air, holds the beam in equilib- 
 
 FIG. 69. 
 
 num. If the weight is immersed in pure water in the jar G, a counter- 
 poise, B, must be hung on the end of the beam to restore the equilibrium. 
 This weight B, when hung on the beam, is therefore able to hold the 
 latter in horizontal position when the liquid below has unit density. 
 The beam is decimally divided and other specific gravities are found by 
 the use of the riders shown. C has the same weight as B, D=^^C, and 
 -£=tV D- With the weights hung as in the figure the specific gravity of 
 the liquid in the jar is 1.025. The apparatus gives a correct result for* 
 some definite temperature only, usually 15° C. 
 
APPENDIX. 
 
 351 
 
 Approximate Specific Gravity Tables. 
 
 Hydrochloric Acid. 
 
 Nitric Acid. 
 
 Sp. Gr. 
 
 15° 
 4° 
 
 Parts of HC1 
 
 by weight in 
 
 100 parts. 
 
 Sp. Gr. 
 
 4° 
 
 Parts of HNO3 
 
 by weight in 
 
 100 parts. 
 
 1.000 
 
 0.16 
 1.15 
 3.14 
 3.12 
 4.13 
 5.15 
 6.15 
 7.15 
 8 16 
 9.16 
 10.17 
 11.18 
 13.19 
 13.19 
 14.17 
 15.16 
 16.15 
 17.13 
 18.11 
 19.06 
 20.01 
 20.97 
 21.92 
 22.86 
 23.82 
 24.78 
 25.75 
 26.70 
 37.66 
 28.61 
 29.57 
 30.55 
 31.52 
 32.49 
 33.46 
 34.42 
 35.39 
 36.31 
 37.23 
 38.16 
 39.11 
 
 1.00 ... 
 
 10 
 
 1.005 
 
 1.01. 
 
 1 90 
 
 1.010 
 
 1.02 . 
 
 3 70 
 
 1.015 
 
 1.030 
 
 1.03 
 
 1.04 
 
 1.05 . 
 
 5.50 
 7 26 
 
 1.025 
 
 8 99 
 
 1.030 
 
 1.06 
 
 1.07 
 
 10.68 
 
 1.035 
 
 12.33 
 
 1.040 
 
 1.08 
 
 13.95 
 
 1.045 
 
 1.09 
 
 15.53 
 
 
 1.10 
 
 17.11 
 
 1 . 055 
 
 1.11 
 
 18.69 
 
 1.060 
 
 1.12 
 
 20.23 
 
 1.065 
 
 1 070 
 
 1.13 
 
 1.14 
 
 21.77 
 23.31 
 
 1 075 
 
 1.15 
 
 24.84 
 
 1 080 
 
 1.16 
 
 26.36 
 
 1 085 
 
 1.17 
 
 27.88 
 
 1 090 . 
 
 1.18 
 
 29.38 
 
 1 095 
 
 1.19 
 
 30.88 
 
 1.100 
 
 1.20 
 
 1.21 
 
 32.36 
 
 1 105 
 
 33 82 
 
 1 no 
 
 1.23 
 
 1.23 
 
 35.28 
 
 1 120 
 
 36.78 
 
 1.24 
 
 38.29 
 
 1 125 
 
 
 39.82 
 
 1 130 
 
 1.26... 
 
 41.34 
 
 1 135 
 
 1.27 
 
 42.87 
 
 1 140 
 
 1.38 
 
 44.41 
 
 
 1.29 
 
 45.95 
 
 1.30 
 
 47.49 
 
 
 1.31 
 
 1.32 
 
 1.33 
 
 1.34 
 
 49.07 
 
 
 50.71 
 
 
 52.37 
 
 
 54.07 
 
 
 1.35 
 
 55.79 
 
 
 1.36 
 
 1.37 
 
 1.38 
 
 57.57 
 
 
 59.39 
 61.27 
 
 
 1.39 
 
 63.23 
 
 1.40 
 
 65 30 
 
 
 1.41 
 
 67.50 
 
 
 
 1.42 
 
 69.80 
 
 
 
 1.43 
 
 72.17 
 
 
 
 1.44 
 
 74.68 
 
 
 
 1.45 
 
 77.28 
 
352 
 
 APPENDIX. 
 
 Sulphuric Acid. 
 
 Sp. Gr. 
 
 15° 
 4° 
 
 Parts of 
 H a SO»bywt. 
 in 100 parts. 
 
 Sp. Gr. 
 
 '5° 
 4° 
 
 Parts of 
 H a SO«bywt. 
 in 100 parts. 
 
 1.00 
 
 0.09 
 
 1.57 
 3.03 
 4.49 
 5.96 
 7.37 
 8.77 
 10.19 
 11.60 
 12.99 
 14.35 
 15.71 
 17.01 
 18.31 
 19.6L 
 20.91 
 22.19 
 23.47 
 Z4.76 
 26.04 
 27.32 
 28.58 
 29.84 
 31.11 
 32.28 
 33.43 
 34.57 
 35.70 
 36.87 
 38.03 
 39.19 
 40.35 
 41.50 
 42.66 
 43.74 
 44.82 
 45 88 
 46.94 
 48.00 
 49.00 
 50.11 
 51.15 
 52.15 
 53.11 
 54.07 
 55.03 
 
 
 55.97 
 
 1 01 
 
 1 47 
 
 56.90 
 
 1.02 
 
 1.48 
 
 57.8i 
 
 1.03 . 
 
 1 49 
 
 58.74 
 
 1.04.. 
 
 1 50 
 
 59.70 
 
 1.05 
 
 1 51 
 
 60.65 
 
 1.06 
 
 1.52 
 
 61.59 
 
 1 07 
 
 1 53 
 
 62 53 
 
 1.08 
 
 1.54 
 
 63 43 
 
 1.09 
 
 1.55 
 
 1.56 
 
 64 26 
 
 1.10 
 
 65 01 
 
 1.11 
 
 1.57 
 
 65 90 
 
 1.12 
 
 1.58 
 
 1 59 
 
 66 71 
 
 1.13 
 
 67 59 
 
 1.14 
 
 1.15 
 
 1.60 
 
 1.61 
 
 68.51 
 69 43 
 
 1.16 
 
 1.62 
 
 1.63 
 
 1.64 
 
 70 32 
 
 1.17 
 
 1.18 
 
 71.16 
 71 99 
 
 1.19 
 
 1.65 
 
 72 82 
 
 1.20 
 
 1.66 
 
 73.64 
 74 51 
 
 1.21 
 
 1.67 
 
 1.22 
 
 1.68 
 
 1.69 
 
 1.70 
 
 75.42 
 76.30 
 77 17 
 
 1.23 
 
 1.24 
 
 1.25 
 
 1.71 
 
 78.04 
 78.92 
 79.80 
 80.68 
 81.56 
 82.44 
 83.32 
 84.50 
 85.70 
 86 90 
 88.30 
 90.05 
 92.10 
 93.00 
 94.00 
 95.00 
 96.00 
 97.00 
 98.00 
 99.00 
 100.00 
 
 1.26 
 
 1.72 
 
 1.27 
 
 1.73 
 
 1.28 
 
 1.29 ". 
 
 1.74 
 
 1.75 
 
 1.30 
 
 1.76 
 
 1.31 
 
 1.77 
 
 1.32 
 
 1.78 
 
 1.33 
 
 1.79 
 
 1.34 
 
 1.80 
 
 1.81 
 
 1.35 
 
 1.36 
 
 1 37 
 
 1.82... . 
 1.83.. 
 
 1.38 
 
 1 8372... 
 
 1.39 
 
 1.40 
 
 1.8390... . 
 1.8406... 
 1.8410 
 
 1.41 
 
 1.42 
 
 1.43 
 
 1 8412 
 
 1.44 
 
 1.8403 
 
 1.45 
 
 1.8384 
 
 

 
 
 APPENDIX. 
 
 
 
 353 
 
 Potassium 
 
 Hydroxide. 
 
 Sodium Hydroxide. 
 
 
 ffi 2 
 
 
 *s 
 
 
 xs 
 
 
 X2 
 
 
 s«- 
 
 
 OSj 
 
 
 oSj 
 
 
 OSj 
 
 
 i£ ~— 
 
 
 u sua 
 
 
 rt Ckfl 
 
 
 rt O.J3 
 
 Sp. Gr. 
 
 15 ■ 
 
 so 
 
 "S8| 
 
 Sp. Gr. 
 
 15'- 
 
 bO 
 
 °§1 
 
 Sp. Gr. 
 15 • 
 
 Z bo 
 
 ! § 1 
 
 Sp. Gr. 
 
 '5 • 
 
 Z M 
 
 
 S-9* 
 
 
 
 
 U* 
 
 
 S-2-° 
 
 
 a. 
 
 
 O. 
 
 
 a. 
 
 
 a. 
 
 1.009 
 
 1 
 
 1.361 
 
 36 
 
 1.012 
 
 i 
 
 1 395 
 
 36 
 
 1.017 
 
 2 
 
 1.374 
 
 37 
 
 1.023 
 
 2 
 
 1.405 
 
 37 
 
 1.025 
 
 3 
 
 1.387 
 
 •38 
 
 1.035 
 
 3 
 
 1.415 
 
 38 
 
 1.033 
 
 ■1 
 
 1.400 
 
 39 
 
 1.046 
 
 4 
 
 1.426 
 
 39 
 
 1.041 
 
 5 
 
 1.412 
 
 40 
 
 1.058 
 
 5 
 
 1.437 
 
 40 
 
 1.049 
 
 6 
 
 - 1.425 
 
 41 
 
 1.070 
 
 6 
 
 1.447 
 
 41 
 
 1.058 
 
 7 
 
 1.438 
 
 42 
 
 1.081 
 
 7 
 
 1.457 
 
 42 
 
 1.065 
 
 8 
 
 1.450 
 
 43 
 
 1.092 
 
 8 
 
 1.468 
 
 43 
 
 1.074 
 
 9 
 
 1.462 
 
 44 
 
 1.103 
 
 9 
 
 1.478 
 
 44 
 
 1.083 
 
 10 
 
 1.475 
 
 45 
 
 1.115 
 
 10 
 
 1,488 
 
 45 
 
 1.092 
 
 11 
 
 1.488 
 
 46 
 
 1 126 
 
 11 
 
 1.499 
 
 46 
 
 1.101 
 
 13 
 
 1.499 
 
 47 
 
 1.137 
 
 12 
 
 1.509 
 
 47 
 
 1.110 
 
 13 
 
 1.511 
 
 48 
 
 1.148 
 
 13 
 
 1.519 
 
 48 
 
 1.119 
 
 14 
 
 1.525 
 
 49 
 
 1.159 
 
 14 
 
 1.529 
 
 49 
 
 1.128 
 
 15 
 
 1.539 
 
 50 
 
 1.170 
 
 15 
 
 1.540 
 
 50 
 
 1.137 
 
 16 
 
 1.552 
 
 51 
 
 1.181 
 
 16 
 
 1.550 
 
 51 
 
 1.146 
 
 17 
 
 1.565 
 
 52 
 
 1.192 
 
 17 
 
 1.560 
 
 52 
 
 1.155 
 
 18 
 
 1.578 
 
 53 
 
 1.202 
 
 18 
 
 1.570 
 
 53 
 
 1.166 
 
 19 
 
 1.590 
 
 54 
 
 1.213 
 
 19 . 
 
 1.580 
 
 54 
 
 1.177 
 
 20 
 
 1.604 
 
 55 
 
 1.225 
 
 20 
 
 1.59J 
 
 55 
 
 1.188 
 
 21 
 
 1.618 
 
 56 
 
 1.236 
 
 21 
 
 1.601 
 
 56 
 
 1.198 
 
 22 
 
 1.630 
 
 57 
 
 1.247 
 
 22 
 
 1.611 
 
 57 
 
 1.209 
 
 23 
 
 1.642 
 
 58 
 
 1.258 
 
 23 
 
 1.622 
 
 58 
 
 1.220 
 
 24 
 
 1.655 
 
 59 
 
 1.269 
 
 24 
 
 1.633 
 
 59 
 
 1.230 
 
 25 
 
 1.667 
 
 60 
 
 1.279 
 
 25 
 
 1.643 
 
 60 
 
 1.241 
 
 26 
 
 1.681 
 
 61 
 
 1.290 
 
 26 
 
 1.654 
 
 61 
 
 1.252 
 
 27 
 
 1.695 
 
 62 
 
 1.300 
 
 27 
 
 1.664 
 
 62 
 
 1.264 
 
 28 
 
 1.705 
 
 63 
 
 1.310 
 
 28 
 
 1.674 
 
 63 
 
 1.276 
 
 29 
 
 1.718 
 
 64 
 
 1.321 
 
 29 
 
 1.684 
 
 64 
 
 1.288 
 
 30 
 
 1.729 
 
 65 
 
 1.332 
 
 30 
 
 1.695 
 
 65 
 
 1.300 
 
 31 
 
 1.740 
 
 66 
 
 1.343 
 
 31 
 
 1.705 
 
 66 
 
 1.311 
 
 32 
 
 1.754 
 
 67 
 
 1.353 
 
 32 
 
 1.715 
 
 67 
 
 1.324 
 
 33 
 
 1.768 
 
 68 
 
 1.363 
 
 33 
 
 1.726 
 
 68 
 
 1.336 
 
 34 
 
 1.780 
 
 69 
 
 1.374 
 
 34 
 
 1.737 
 
 69 
 
 1.349 
 
 35 
 
 1.790 
 
 70 
 
 1.384 
 
 35 
 
 1.748 
 
 70 
 
354 
 
 APPENDIX. 
 
 Ammonia Water. 
 
 Sp. Gr. 
 
 15° 
 4° 
 
 Parts of NH, 
 in loo parts 
 by weight. 
 
 Sp. Gr. 
 
 15° 
 4° 
 
 Parts of NH, 
 in zoo parts . 
 by weight. 
 
 1.000 
 
 0.00 
 
 0.45 
 
 0.91 
 
 1.37 
 
 1.84 
 
 2.31 
 
 2.80 
 
 3.30 
 
 3.80 
 
 4.30 
 
 4.80 
 
 5.00 
 
 5.30 
 
 5.80 
 
 6.30 
 
 6.80 
 
 7.31 
 
 7.82 
 
 8.33 
 
 8.84 
 
 9.35 
 
 9.91 
 
 10.00 
 
 10.47 
 
 11.03 
 
 11.60 
 
 12.17 
 
 12.74 
 
 13.31 
 
 13.88 
 
 14.46 
 
 15.00 
 
 15.04 
 
 0.940 
 
 15.63 
 
 0.998 
 
 0.938 
 
 16.22 
 
 0.996 
 
 0.936 
 
 16.82 
 
 0.994 
 
 0.934 
 
 17.42 
 
 0.992 
 
 0.932 
 
 18.03 
 
 0.990 
 
 0.930 
 
 18.64 
 
 0.988 
 
 0.928 
 
 19.25 
 
 0.986 
 
 0.926 
 
 19.87 
 
 0.984 
 
 0.9256 
 
 20.00 
 
 0.982 
 
 0.922 
 
 0.920 
 
 20.49 
 
 0.980 
 
 21.12 
 
 0.9792 
 
 21.75 
 
 0.978 
 
 0.918 
 
 22.39 
 
 0.076...., 
 
 0.916 
 
 23.03 
 
 0.974 
 
 914 . . 
 
 23.68 
 
 0.972 
 
 0.912 
 
 24.33 
 
 0.970 
 
 0.910 
 
 24.99 
 
 0.968 
 
 0.9099 
 
 . 25.00 
 
 0.966 
 
 0.908 
 
 0.906 .. 
 
 35 65 
 
 0.964 
 
 26.31 
 
 0.962 
 
 0.904 
 
 26.98 
 
 0.960 
 
 0.902 
 
 27.65 
 
 0.9597 
 
 0.900 
 
 0.898 .. 
 
 28 33 
 
 0.958 '..... 
 
 29.01 
 
 0.956 
 
 896 . 
 
 29 69 
 
 0.954 
 
 0.8951 .. 
 
 30.00 
 
 0.952 
 
 0.894 
 
 30.37 
 
 0.950 
 
 0.892 
 
 31 05 
 
 0.948 
 
 0.890 
 
 31.75 
 
 0.946 
 
 0.888 
 
 32 50 
 
 0.944 
 
 886 . . 
 
 33 25 
 
 0.9421 
 
 884 
 
 34 10 
 
 0.942 
 
 882 
 
 34 95 
 
 
 
 
APPENDIX. 
 
 355 
 
 Alcohol. 
 
 Per cent by 
 volume. 
 
 Sp. Gr. 
 15-56° 
 15.56° 
 
 Per cent by 
 volume. 
 
 Sp. Gr. 
 15.56° 
 15.56° 
 
 Per cent by 
 volume. 
 
 Sp. Gr. 
 
 15-56° 
 15.56° 
 
 
 
 1.00000 
 0.99847 
 699 
 555 
 415 
 279 
 147 
 019 
 0.98895 
 774 
 657 
 543 
 432 
 324 
 218 
 114 
 
 on 
 
 0.97909 
 808 
 708 
 608 
 507 
 406 
 304 
 201 
 097 
 
 0.96991 
 883 
 772 
 658 
 541 
 421 
 298 
 172 
 
 34 
 
 0.96043 
 
 0.95910 
 773 
 632 
 487 
 338 
 185 
 029 
 
 0.94868 
 704 
 536 
 364 
 188 
 008 
 
 0.93824 
 636 
 445 
 250 
 052 
 
 0.92850 
 646 
 439 
 229 
 015 
 
 0.91799 
 580 
 358 
 134 
 
 0.90907 
 678 
 447 
 214 
 
 0.89978 
 740 
 
 68 
 
 0.89499 
 
 1 
 
 35 
 
 69 
 
 70 
 
 256 
 
 3 
 
 36 
 
 010 
 
 3 
 
 37 : 
 
 71 
 
 0.88762 
 
 4 
 
 38 
 
 72 
 
 511 
 
 5 
 
 39. 
 40 
 
 73 
 
 257 
 
 6 
 
 74 
 
 000 
 
 7 
 
 41.. 
 
 75 
 
 0.87740 
 
 8 
 
 42 
 
 76 
 
 477 
 
 9 
 
 43.. 
 
 77 
 
 211 
 
 10 
 
 44 
 
 78 
 
 0.86943 
 
 11 
 
 45 
 
 79 
 
 80 
 
 670 
 
 12 
 
 46 
 
 395 
 
 13 
 
 47 
 
 81 
 
 116 
 
 14 ., 
 
 48 
 
 82 
 
 0.85833 
 
 15 
 
 49 
 
 50 
 
 83 
 
 547 
 
 16 
 
 84 
 
 256 
 
 17 . 
 
 51 
 
 85 
 
 0.84961 
 
 18 . 
 
 52... 
 
 86 
 
 660 
 
 19 
 
 53. . 
 
 87 
 
 355 
 
 20 . 
 
 54... 
 
 88 
 
 044 
 
 21 
 
 55 
 
 89 
 
 90 
 
 0.83726 
 
 22 
 
 56.. 
 
 400 
 
 23 
 
 57. .. 
 
 91 
 
 065 
 
 24 
 
 58 
 
 92 
 
 0.82721 
 
 25 
 
 59 
 
 93 
 
 365 
 
 26 
 
 60 
 
 61 
 
 94 
 
 0.81997 
 
 27 
 
 95 
 
 616 
 
 28 
 
 62 
 
 96 
 
 217 
 
 29 
 
 63. 
 
 97 
 
 0.80800 
 
 30 
 
 31 
 
 64. 
 
 98 
 
 359 
 
 65 
 
 99 
 
 0.79891 
 
 32 
 
 66 
 
 100 
 
 0.79391 
 
 33 
 
 67 
 
 
 
 
 
 
356 
 
 APPENDIX. 
 
 Volume and Specific Gravity of Water of Different 
 Temperatures. 
 
 (Calculations of Volkmann from results of Kopp, Hagen, Matthiesen, 
 Jolly and Pierre.) 
 
 Temperature C. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 14 
 
 15 
 
 16 
 
 17 
 
 18 
 
 19 
 
 20 
 
 21 
 
 22 
 
 23. 
 
 24, 
 
 25 
 
 30, 
 
 Specific gravity, or 
 weight of i Cc. 
 
 of water in 
 vacuo in grams. 
 
 Differences for i°, 
 Sp. Gr. and volume. 
 
 99988 
 99993 
 99997 
 
 00000 
 99999 
 99997 
 
 99982 
 99974 
 99965 
 99954 
 99943 
 99930 
 99915 
 99900 
 99884 
 
 99847 
 99827 
 99807 
 99785 
 99762 
 99739 
 99714 
 99577 
 
 0.00005 
 .00004 
 .00002 
 .00001 
 .00001 
 .00002 
 .00004 
 .00005 
 .00006 
 .00008 
 .00009 
 .00011 
 .00011 
 .00013 
 .00015 
 .00015 
 .00016 
 .00018 
 .00019 
 .00020 
 .00021 
 .00022 
 .00023 
 .00023 
 .00025 
 .00027 
 
 Volumes of one 
 
 gram of water 
 
 in Cc. 
 
 1.00012 
 1.00007 
 1.00003 
 1.00001 
 1.00000 
 1.00001 
 1.00003 
 1.00007 
 1.00012 
 1.00018 
 1.00026 
 1.00035 
 1.00046 
 •1.00057 
 1.00070 
 1.00085 
 1.00100 
 1.00116 
 1.00134 
 1.00153 
 1.00173 
 1.00194 
 1.00216 
 1.00238 
 1.00262 
 1.00287 
 1.00425 
 
APPENDIX. 
 
 357 
 
 Specific Gravity and Volume of Mercury for Certain 
 Temperatures. 
 
 Temp. 
 C. 
 
 Sp. Gr. or wt. of 
 i Cc. in grams. 
 
 Log. 
 
 Vol. of i Gm. in 
 Cc. 
 
 Log. 
 
 
 
 13.5953 
 
 1.13339 
 
 0.073555 
 
 — 2.86661 
 
 4 
 
 13.5854 
 
 1.13307 
 
 0.073608 
 
 2.86693 
 
 5 
 
 13.5830 
 
 1 . 13299 
 
 0.073622 
 
 2.86701 
 
 10 
 
 13.5707 
 
 1.13260 
 
 0.073688 
 
 2.86740 
 
 15 
 
 13.5584 
 
 1.13221 
 
 0.073755 
 
 2.86779 
 
 20 
 
 13.5461 
 
 1.13182 
 
 0.073822 
 
 2.86818 
 
 25 
 
 13.5339 
 
 1.13142 
 
 0.073888 
 
 2.86858 
 
 30 
 
 13.5217 
 
 1.13103 
 
 0.073955 
 
 2.86897 
 
 Tension of Aqueous Vapor at Different Temperatures, Ex= 
 
 pressed in Millimeters of Mercury Pressure 
 
 Equivalent. 
 
 Temp. C. 
 
 Hg. in Mm. 
 
 Temp. C. 
 
 Hg. in Mm. 
 
 Temp. C. 
 
 Hg. in Mm. 
 
 
 
 4.57 
 
 11 
 
 9.77 
 
 22... J .'.... 
 
 19.63 
 
 1 
 
 4.91 
 
 12 
 
 10.43 
 
 23 
 
 20.86 
 
 2 
 
 5.27 
 
 13 
 
 11.14 
 
 24 
 
 22.15 
 
 3 
 
 5.66 
 
 14 
 
 11.88 
 
 25 
 
 23.52 
 
 4 
 
 6.07 
 6.51 
 
 15 
 
 16 
 
 12.07 
 13.51 
 
 26 
 
 24.96 
 
 5 
 
 27 
 
 26.47 
 
 6 
 
 6.97 
 
 17 
 
 14.40 
 
 28 
 
 28.06 
 
 
 7.47 
 
 18 
 
 15.33 
 
 29 
 
 29.74 
 
 8 
 
 7.99 
 
 19 
 
 16.32 
 
 30 
 
 31.51 
 
 9 
 
 8.55 
 
 20 
 
 17.36 
 
 31 
 
 33.37 
 
 10 
 
 9.14 
 
 21 
 
 i 
 
 '.8.47 
 
 32 
 
 35.32 
 
358 
 
 APPENDIX. 
 
 The Relation Between the Barometric Pressure and Boiling 
 Point of Water. 
 
 (For Correction of Thermometers. ) 
 
 Barometric 
 
 Boiling 
 
 Barometric 
 
 Boiling 
 
 Barometric 
 
 Boiling 
 
 height in 
 
 Point 
 
 height in 
 
 Point 
 
 height in 
 
 Point 
 
 Mm. 
 
 C. 
 
 Mm. 
 
 C. 
 
 Mm. 
 
 C. 
 
 710 
 
 98.11 
 
 731 
 
 98.92 
 
 ?52 
 
 99.70 
 
 711 
 
 98.15 
 
 732 
 
 98.96 
 
 753 
 
 99.74 
 
 712 
 
 98.19 
 
 733 
 
 98.99 
 
 754 
 
 99.78 
 
 713 
 
 98.23 
 
 734 
 
 99.03 
 
 755 
 
 99.82 
 
 714 
 
 98.27 
 
 735 
 
 99.07 
 
 756 
 
 99.85 
 
 715 
 
 98.30 
 
 736 
 
 99.11 
 
 757 
 
 99.89 
 
 716 
 
 98.34 
 
 737 
 
 99.14 
 
 758 
 
 99.92 
 
 717 
 
 98.38 
 
 738 
 
 99.18 
 
 759 
 
 99.96 
 
 718 
 
 98.42 
 
 739 
 
 99.22 
 
 760 
 
 100.00 
 
 '719 
 
 98.46 
 
 740 
 
 99.26 
 
 761 
 
 100.03 
 
 720 
 
 98.50 
 
 741 
 
 99.30 
 
 762 
 
 100.07 
 
 721 
 
 98.54 
 
 742 
 
 99.33 
 
 763 
 
 100.11 
 
 722 
 
 98.57 
 
 743 
 
 99.37 
 
 764 
 
 100.14 
 
 723 
 
 98.61 
 
 744 
 
 99.41 
 
 765 
 
 100.18 
 
 724 
 
 98.65 
 
 745 
 
 99.44 
 
 766 
 
 100.22 
 
 725 
 
 98.69 
 
 746 
 
 99.48 
 
 767 
 
 100.26 
 
 726 
 
 98.73 
 
 747 
 
 99.52 
 
 768 
 
 100.29 
 
 727 
 
 98.77 
 
 748 
 
 99.55 
 
 769 
 
 100.33 
 
 728 
 
 98.80 
 
 749 
 
 99.59 
 
 770 
 
 100.36 
 
 729 
 
 98.84 
 
 750 
 
 99.63 
 
 771 
 
 100.40 
 
 730 
 
 98.88 
 
 751 
 
 99.67 
 
 772 
 
 1.00.44 
 
 Formula for Correction of Thermometer Reading on Account of 
 Projecting Thread. 
 
 T= the corrected temperature. 
 
 x = the thermometer reading observed. 
 
 x'= the mean temperature of projecting thread. 
 
 p= the length of the projecting thread in degrees. 
 
 0.000143 is a factor determined by Thorpe empirically. 
 
 T=*-|-0.000143/ 0— *'). 
 For example: If 150° is the observed reading, and 70 is the number 
 of degrees outside the hot vapor or liquid in which the thermometer is 
 suspended, while the mean temperature of this part of the thread is 30° 
 (as shown by a second small thermometer hanging against the first) the 
 correction, 0.000143^ (x— x'), is 1.20 and the corrected reading 151.2.° 
 
APPK.\ D1X. 
 
 359 
 
 Table Showing the Average Composition of Several Vegetable 
 and Animal Food Stuffs. 
 
 Wheat 
 
 Rye 
 
 Barley 
 
 Oats 
 
 Corn 
 
 Buckwheat, with hulls 
 
 Rice 
 
 Peas, ripe 
 
 Beans, ripe 
 
 Peas, green 
 
 Beans, green 
 
 Potatoes 
 
 Beets 
 
 Fat steer 
 
 Medium steer 
 
 Fat sheep 
 
 Medium sheep 
 
 Fat hog 
 
 Lean bog 
 
 Horse flesh 
 
 Water. 
 
 13.65 
 
 15.06 
 
 13.77 
 
 12.37 
 
 13.12 
 
 11. 
 
 13.11 
 
 14.99 
 
 14.76 
 
 78.44 
 
 84.0' 
 
 75.48 
 
 87.71 
 
 Pro- 
 
 teids. 
 
 12.35 
 
 11.52 
 
 11.14 
 
 10.41 
 
 9.85 
 
 10.30 
 
 7.85 
 
 22.85 
 
 24.27 
 
 6.35 
 
 5.43 
 
 1.95 
 
 1.09 
 
 Fat. 
 
 1.75 
 1.79 
 2.16 
 5.23 
 4.62 
 2.81 
 0.88 
 1.79 
 1.61 
 0.53 
 0.33 
 0.15 
 0.11 
 
 Carbo- Cellu 
 hydrate lose. 
 
 67.91 
 67.81 
 64.93 
 57.78 
 08.41 
 55.81 
 76.52 
 52.36 
 49.01 
 12.00 
 
 7.35 
 20.69 
 
 9.26 
 
 2.53 
 2.01 
 5.31 
 
 11.19 
 2.49 
 
 16.43 
 0.63 
 5.43 
 7.09 
 1.87 
 2.08 
 0.75 
 0.98 
 
 Ash. 
 
 1.81 
 1.81 
 2.69 
 3.02 
 1.51 
 2.72 
 1.01 
 2.58 
 3.26 
 0.81 
 0.74 
 0.98 
 0.95 
 
 55.42 
 72.25 
 47.91 
 75.99 
 47.40 
 72.57 
 74.27 
 
 17.19 
 20.91 
 14.80 
 17.11 
 14.54 
 20.25 
 21.71 
 
 26.38 
 5.19 
 
 36.39 
 5.77 
 
 37.34 
 6.81 
 2.55 
 
 1.08 
 1.17 
 0.85 
 1.33 
 0.72 
 1.10 
 1.01 
 
 animal. 
 
 The values given for the animal products represent the whole 
 
360 APPENDIX. 
 
 List of Apparatus Required in Performing the Experiments of 
 This Book. 
 
 The following list embraces the important apparatus called for in 
 the experiments of the Chemical Physiology and Urine Analysis. When 
 thought necessary some of the pieces may be used in common by several 
 students. The apparatus can be purchased by students at the attached 
 prices, kindly furnished the author by Messrs. E. H. Sargent & Co., of 
 Chicago: 
 
 3 beakers, 100 Cc., each : 10 
 
 2 beakers, 250 Cc, each 18 
 
 2 beakers, 400 Cc, each 25 
 
 2 flasks, 100 Cc, each 15 
 
 2 flasks, 250 Cc, each 20 
 
 2 flasks, 500 Cc, each 25 
 
 1 rack and 10 6-inch test-tubes 75 
 
 1 Bunsen burner and rubber tube 75 
 
 1 porcelain dish, 6 inches 45 
 
 2 porcelain dishes, 3 inches, each 20 
 
 1 test-tube holder, wooden 10 
 
 1 test-tube brush, sponge end. 10 
 
 1 50 Cc. burette, g. s 2.00 
 
 1 50 Cc. pipette 40 
 
 1 25 Cc. pipette 35 
 
 1 5 Cc. pipette 15 
 
 1 100 Cc. graduate 50 
 
 1 100° thermometer 85 
 
 1 funnel tube 15 
 
 2 funnels, 70 Mm., each 15 
 
 1 horn spatula, 6 inches 15 
 
 1 retort stand with 3 rings 70 
 
 1 6-inch copper water-bath with rings 1.50 
 
 1 piece of wire gauze, 4 inches square 10 
 
 In addition to these items the student will need an assortment of 
 corks, glass tubing, rubber connections, files and a few other small arti- 
 cles. 
 
INDEX. 
 
 Abnormal coloring matters 246 
 
 Absorption analysis 156-165 
 
 Acetic acid fermentation 55 
 
 Aceto-acetic acid 240 
 
 Acetone in urine 238 
 
 Acetonuria 238 
 
 Acid albumin 74 
 
 Air tests 142 
 
 Albumins 67 
 
 classification of 70 
 
 composition of 67 
 
 in urine 187 
 
 reactions for 68 
 
 Albumose 81 and 204 
 
 amount of 206 
 
 tests for 83 
 
 Alcohol tables 355 
 
 Alcoholic fermentation 54 
 
 Alkali albumin 73 
 
 Alkapton 251 
 
 Almen's test 250 
 
 Alpha-naphthol test 224 
 
 Ammonia water 354 
 
 Amorphous phosphates 331 
 
 urates 323 
 
 Amount of albumin 198 
 
 albumose 206 
 
 phosphates ■. 285 
 
 sugar 227 
 
 uric acid 257 
 
 Amyloid substance 89 
 
 Analysis of calculi 332-339 
 
 of meat extract 169-173 
 
 by the spectroscope. . . .156-165 
 
 Animal starch 57 
 
 Antialbumose 82 
 
 Antipeptone 82 
 
 Aqueous vapor tension 357 
 
 Atomic weights 343 
 
 Bacilli 318 
 
 Baryta solution 267 
 
 Beef extract 127-131 
 
 Bile 117 
 
 Bile acid 117 and 252 
 
 action on fats 119 
 
 pigments 118 
 
 colors in urine 246 
 
 Bilirubin 117 
 
 Biliverdin 117 
 
 Bismuth test 220 
 
 Biuret test 69 and 208 
 
 Blood 91-101 
 
 coagulation of 94 
 
 coloring matters 248 
 
 composition of 91 and 92 
 
 corpuscles 108 and 302 
 
 corpuscle counters. 109 and 110 
 
 spectra 105 and 106 
 
 tests 92 
 
 Boettger's bismuth test 220 
 
 Bone constituents Ill 
 
 Bright's disease 188 
 
 British gum 29 
 
 Bruecke's solution 221 
 
 Burettes 11 
 
 Burette apparatus 277 
 
 Butyric fermentation 56 
 
 Calcium carbonate 328 
 
 oxalate 328 
 
 phosphate 331 
 
 sulphate 328 
 
362 
 
 INDEX. 
 
 Calculi 332 
 
 Cancer tissue 316 
 
 Carbohydrates 19-59 
 
 Casein 75 
 
 Casts 309-314 
 
 Centrifugal machine 300 
 
 Chlorides in urine 289 
 
 Chlorides in water 135 
 
 Cholesterin , 327 
 
 Chrysophanic acid 250 
 
 Classification of albumins 70 
 
 of sediments 302 
 
 Coagulated blood 94 
 
 proteids 80 
 
 Coagulation test 189 
 
 Colloids 16 
 
 Coloring matter in urine 242 
 
 Color of urine 185 
 
 Composition of bone Ill 
 
 of milk 121 
 
 Corpuscle counters 109 
 
 Crystallin 78 
 
 Crystalloids 16 
 
 Cystin 326 
 
 Determination of albumin 198 
 
 of chlorides 289 
 
 of nitrogen 165 
 
 of phosphates 285 
 
 of sugars 35 
 
 of urea 265 
 
 of uric acid 254 
 
 Derived albumins 73 
 
 Dextrin 29 
 
 Dextrose 30 
 
 tests for 31-33 
 
 Diabetes mellitus 182 
 
 Diacetic acid 240 
 
 Dialysis 15 
 
 Dialyzer 17 
 
 Diffusion of peptones 89 
 
 Digestion of proteids 82 
 
 Doremus' apparatus 281 
 
 Double iodide test 194 
 
 Egg albumin 71 
 
 Emulsions 62 
 
 Epithelium 306 
 
 Esbach's albuminometer. . . . . . 200 
 
 Expired air 141 
 
 Extract of meat 127 
 
 of pancreas 28 
 
 Fats 59-66 
 
 crystallization of 63 
 
 crystals 64 and 65 
 
 in milk 122 
 
 origin of 59 
 
 saponification 60 
 
 Fatty acids 61 
 
 Fehling's solution 35 
 
 test 217 
 
 Fermentation of sugar 54 
 
 test 225 
 
 Ferrocyanide test 196 
 
 Fibrin 80 
 
 Fibrinogin 79 
 
 Fleischl's hamiometer 100 
 
 Flour 131 
 
 Foodstuffs 359 
 
 Foreign matters in urine 332 
 
 Fungi 316 
 
 Gastric juice 114 
 
 tests of 114 
 
 Globin 80 
 
 Globulins 77 
 
 Glycerol 62 
 
 Glycocholic acid 148 
 
 Glycocoll 146 
 
 Glycogen 57 
 
 Gmelin's test 246 
 
 Gowers' hasmoglobinometer. . . 98 
 
INDEX. 
 
 Gravimetric method 199 
 
 Guaiacum test 92 
 
 Gunning's method 160 
 
 Haemometer 100 
 
 Haeser's coefficient 181 
 
 Hsemin crystals 93 
 
 Haycraft's method 259 
 
 Heat test 189 
 
 Heller's test 247 and 249 
 
 Haemoglobin 94 
 
 amount of 97-101 
 
 Hasmoglobinometer 98 
 
 Hemipeptone 82 
 
 Hippuric acid 262 
 
 Hofmeister's test 208 
 
 Huef ner's apparatus 274 
 
 Hydrochloric acid 351 
 
 Indican 244 
 
 Indicators 348 and 349 
 
 Inosite in urine 237 
 
 Introduction 1 
 
 Invert sugar 36 
 
 Kjeldahl process 165-169 
 
 Knapp's solution 232 
 
 Lactic fermentation 56 
 
 Lactose in urine 237 
 
 Laevulose in urine 236 
 
 Lardacein 89 
 
 Legal's test 239 
 
 Leucin 82 
 
 and tyrosin... 149 and 324-326 
 
 preparation of 149 and 140 
 
 Liberation of nitrogen 273 
 
 Lieben's test 239 
 
 Lieberkuehn's jelly 74 
 
 Liebig's method 265 
 
 Loewe's test 220 
 
 Magnesium phosphate 331 
 
 Maltose 36 
 
 Meal 131 
 
 Measuring apparatus 11-12 
 
 Meat extract 127 and 169 
 
 Mercuric nitrate solution 267 
 
 Mercuric-potassium iodide test 194 
 
 Mercury, specific gravity 357 
 
 Methaemoglobin 105 
 
 Micrococcus ureae 317 
 
 Milk 121 
 
 peptonization of 126 
 
 sugar 34 
 
 Millon's reagent 69 
 
 Myosin 79 
 
 Molisch's test 33 
 
 Moore's test 215 
 
 Mucin 211-212 
 
 bands 308-309 
 
 Mucus corpuscles 304-305 
 
 Murexid test 255 
 
 Native albumirs 79 
 
 Nicol's prism 40-44 
 
 Nitric acid 351 
 
 Nitric acid test 191 
 
 Nitrates and nitrites 139 
 
 Nitrogen, determination of 1 65 
 
 Normal colors 242 
 
 Oder of urine 184 
 
 Organic compounds 3 
 
 classification of 5 
 
 Organized sediments 301-302 
 
 Outline of urine tests 178 
 
 Oxidation tests 137 
 
 Pancreatic digestion 87 
 
 Paraglobulin 78 
 
 Pavy's solution 230 
 
 Pepsin, tests of 84 
 
 Peptic digestion 83 
 
 Peptones 81 and 206 
 
364 
 
 INDEX. 
 
 Peptones, diffusion of 89 
 
 tests for 83 
 
 Peptonized milk 136 
 
 Peptonuria 206 
 
 Phenols in urine 251 
 
 Phenylhydrazinetest 223 
 
 Phosphates 329 
 
 in urine 282 
 
 Picric acid test 197 
 
 Pigments of bile 118 
 
 Polarimetry 233 
 
 Polariscopes 39 
 
 Polarization of light 40 
 
 methods 39-53 
 
 Potassium hydroxide 353 
 
 Preliminary tests 17? 
 
 Preparation of urea 143 
 
 Preservation of sediments 299 
 
 Proteids 67-90 
 
 in milk 124 
 
 separation of 169 
 
 Pus corpuscles 304 
 
 Reaction of blood 93 
 
 of urine 1 83 
 
 Reagents 344-348 
 
 Rennet 125 
 
 Saccharoses 33 
 
 Sachsse's solution 232 
 
 Salicylic acid 251 
 
 Saliva -* . . 113 
 
 Salkowski-Ludwig method 256 
 
 Santonin 250 
 
 Schmiedeberg's test 220 
 
 Sediment of urine 298 
 
 Serum albumin 71 and 188 
 
 globulin 202 
 
 Silver nitrate solution. .291 and 294 
 
 Sodium chloride solution 292 
 
 Sodium hydroxide 353 
 
 Special problems 143 
 
 Specific gravity 17 * nd 34 j> 
 
 of urine 
 
 tables 351-357 
 
 Specific rotation 
 
 Spectra of blood 105 
 
 Spectro-photometer 150 
 
 Spectro-photometry 150 165 
 
 Spectroscope 1"1 
 
 Spermatozoa """ 
 
 Squibb's apparatus 279 
 
 Standard solutions 8 
 
 Starch 19-29 
 
 and acids 22 
 
 and malt extract 26 
 
 and pancreatic extract.... 27 
 
 and saliva 25 
 
 preparation 19 
 
 properties 20 
 
 Struve's test 249 
 
 Stutzer's method 169 
 
 Sugars. 29-58 
 
 determination of 35 
 
 in milk 123 
 
 in urine 213 
 
 reactions 226 
 
 Sulphocyanate solution 294 
 
 Sulphuric acid 352 
 
 Syntonin 77 
 
 Synthesis '. 146 
 
 Tanret's solution 194 
 
 Tension of aqueous vapor. .... 357 
 
 Test solutions 344-348 
 
 Tests, 
 
 Almen's 250 
 
 alpha-naphthol .' 224 
 
 bismuth ; . 220 
 
 biuret 69 and 208 
 
 Boettger's 220 
 
 coagulation 189 
 
 congo red ijg 
 
INDEX. 
 
 365 
 
 Tests, 
 
 Donne's '305 
 
 double iodide 194 
 
 emerald green 115 
 
 Esbach's 200 
 
 Fehling 31 and 217 
 
 fermentation 225 
 
 ferrocyanide 196 
 
 Gmelin's 118 and 246 
 
 guaiacum 92 
 
 Haycraft's 259 
 
 Heller's 247 and 249 
 
 Hofmeister's 208 
 
 Knapp's 232 
 
 Legal' s 239 
 
 Lieben's 239 
 
 Loewe's 220 
 
 methyl violet 116 
 
 Molisch's 33 
 
 Moore's 215 
 
 murexid 255 
 
 Nessler 136 
 
 oxidation 137 
 
 Pavy's 230 
 
 permanganate 138 
 
 Pettenkofer's 117 
 
 phenyl hydrazine ... 33 and 223 
 
 phloroglucin and vanillin. . 116 
 
 picric acid 197 
 
 polarization 39 
 
 Sachsse's 23S 
 
 Schmiedeberg's 220 
 
 specific gravity 179 
 
 Struve's 249 
 
 sulphate 203 
 
 Tanret's 194 
 
 Trommer's 30 and 215 
 
 tropaeolin 116 
 
 Trousseau's . . .' 247 
 
 Weyl's 130 
 
 xantho-proteic 69 
 
 Thermometers 13 
 
 correction 358 
 
 Triple phosphate 330 
 
 Trommer's test 215 
 
 Trousseau's test 247 
 
 Tyrosin 82 and 149 
 
 preparation of 149 and 150 
 
 Unorganized sediment. .301 and 321 
 
 Uranium solution 286 
 
 Urates 323 
 
 Urea 263 
 
 preparation of 143-145 
 
 solution 268 
 
 Uric acid 245 and 321 
 
 synthesis of 14G 
 
 Urinary calculi 332 
 
 Urine, aceto-acetic acid in 240 
 
 acetone in 238 
 
 albumins in 187 
 
 albumose in 204 
 
 alkapton in 251 
 
 analysis of 177 
 
 bile acids in 252 
 
 biliary colors in 246 
 
 blood colors in 248 
 
 blood corpuscles in 302 
 
 calcium oxalate in 328 
 
 cancer tissue in 316 
 
 casts in 309 
 
 chlorides in 289 
 
 cholesterin in 327 
 
 chrysophanic acid in 250 
 
 color 185 
 
 colors in 242 
 
 epithelium in 306 
 
 fungi in 816 
 
 hippuric acid in 262 
 
 indican in 244 
 
 lactose in 237 
 
 lsevulose in 236 
 
36C 
 
 INDEX. 
 
 Urine, 
 
 leucin and tyrosin in 324 
 
 mucin in 211 
 
 mucin bands in 308 
 
 mucus and pus in 304 
 
 oder 184 
 
 peptone in 206 
 
 phenols in 251 
 
 phosphates in 282 and 329 
 
 reaction of 183 
 
 salicylic acid in 251 
 
 santonin in 250 
 
 sediment of 298 
 
 serum globulin in 202 
 
 specific gravity of 179 
 
 spermatozoa in 315 
 
 sugar in 213 
 
 urea in 263 
 
 uric acid in 254 and 321 
 
 urobilin in 242 
 
 Urine, 
 
 urohaematin in 244 
 
 urophain in 244 
 
 Urinometer 180 
 
 Urobilin 242 
 
 Urohaematin 244 
 
 Urophain 244 
 
 Vitellin 78 
 
 Volhard's method. 293 
 
 Volume method 200 
 
 Water 134 
 
 analysis of 135-144 
 
 boiling point 358 
 
 specific gravity of 358 
 
 Weights and measures 349 
 
 Weyl's reaction 130 
 
 Xanthoproteic reaction 69 
 
 Yeast 132