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^ CORNELL UNIVERSITY. 
 
 53 
 
 A 42- THE . 
 
 llostueU p. bonier ICifcrsiVg 
 
 THE GIFT OF 
 
 ROSWELL P. FLOWER 
 
 FOR THE USE OF 
 
 THE N. Y. STATE VETERINARY COLLEGE. 
 
 1897 
 
Cornell University Library 
 RB 53.A42 
 
 Chemistry of urine; a practical guide to 
 
 3 1924 000 886 519 
 
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/cu31924000886519 
 
e^ 
 
 \b 
 
 CHEMISTRY OP URINE 
 
CHEMISTRY OF URINE 
 
 A PRACTICAL GUIDE TO THE 
 
 ANALYTICAL EXAMINATION OF DIABETIC, 
 ALBUMINOUS, AND GOUTY URINE 
 
 ALFRED H. ALLEN, F.I.C., F.C.S. 
 
 PAST PRESIDENT OF THE SOCIETY OF PUBLIC ANALYSTS ; LATE LECTURER ON THEORETICAL 
 
 AND PRACTICAL CHEMISTRY IN THE SCHOOL OF MEDICINE, SHEFFIELD ; PUBLIC 
 
 ANALYST FOR THE WEST RIDING OF YORKSHIRE, THE 
 
 CITY OF SHEFFIELD, ETC. ; 
 
 AUTHOR OF "COMMERCIAL ORGANIC ANALYSIS." 
 
 LONDON 
 
 & A. CHURCHILL 
 
 11 NEW BURLINGTON STREET 
 1895 
 G 
 
PREFACE. 
 
 A considerable portion of the contents of this 
 book was designed to form part of the concluding 
 volume of my "Commercial Organic Analysis." 
 Vol. III., Part 3rd, of that work deals mainly with 
 substances of animal origin, and will complete the chief 
 literary work of my life. But, of late, circumstances 
 have led me to devote much attention to the chemical 
 examination of urine, especially in relation to certain 
 pathological conditions of great importance in Life 
 Assurance reports, as well as in clinical diagnosis and 
 prognosis. These examinations have caused me to 
 investigate critically a large number of the analytical 
 methods which are in vogue for the examination of 
 urine, especially for sugar and albumin, and to confirm 
 or disprove certain statements generally accepted as 
 facts. 
 
 The results of this extensive laboratory work may 
 be of assistance to many interested in Urinary 
 Analysis. Physicians who are called on to advise as 
 to the acceptance or rejection of candidates for Life 
 Assurance often find this duty very onerous. Prog- 
 nosis with regard to patients who may be suffering 
 from glycosuria or albuminuria is a difficult and 
 
VI PREFACE. 
 
 anxious task ; and the more so as this task is one 
 upon the performance of which the patient's immediate 
 future is to cast so critical a light. Probably, also, 
 there are analysts who will welcome this collection of 
 analytical facts and methods. 
 
 "While attempting to bring the majority of the tests 
 and processes within the scope of everyday clinical 
 diagnosis, or of the reports required for Life Assurance, 
 I have also described other methods which cannot be 
 applied except by those accustomed to analytical 
 work, and who are possessed of the appliances of a 
 well-appointed laboratory. 
 
 While desiring to give special prominence to the 
 methods of examining diabetic, albuminous, and gouty 
 urine, it appeared undesirable to omit all reference to 
 subjects of collateral interest, such as the proportions 
 of urea and total nitrogen in urine, the recent researches 
 on creatinine and on xanthine derivatives, and the 
 behaviour of urinary colouring matters. On the other 
 hand, the book is not planned as a complete guide to 
 Urinary Analysis. Thus, I have omitted all mention 
 of the methods of determining phosphates and most 
 of the other mineral constituents of urine ; firstly, 
 because they are not of great pathological interest, and 
 secondly, because I have nothing to say about them 
 which cannot be found in every physician's and 
 analyst's library. Should this production meet with 
 such a reception as to call for the issue of a second 
 edition, it may be desirable to supply this and other 
 omissions. 
 
 It is with great pleasure that I acknowledge the 
 valuable assistance rendered me by Dr James Edmunds, 
 
PREFACE. Vll 
 
 of Dover Street, Piccadilly, to whom I am greatly 
 indebted for the perusal of the whole of the proofs, 
 and for many valuable suggestions, both scientific and 
 literary. I am also obliged to Messrs A. W. Gerrard, 
 F. G. Hopkins, and G. Stillingfleet Johnson for the 
 perusal and correction of particular proof-sheets. 
 Much time and labour have been devoted in my 
 laboratory to the examination of many of the tests 
 and processes described, and my cordial thanks are 
 due to Mr G. Bernard Brook and Mr Arnold E. 
 Tankard for their zealous and painstaking assistance 
 in this arduous work. 
 
 The progress of further investigations which I have 
 in hand would be materially facilitated by increased 
 opportunities of examining abnormal specimens of 
 urine. I therefore take this opportunity of soliciting 
 the co-operation of clinical workers, who would greatly 
 oblige me by forwarding for examination specimens 
 of any urines which appear to justify such a course. 
 There is reason to believe that some of the less known 
 constituents of urine, such as glycuronic acid and its 
 compounds, creatinine, xanthine, the indoxyl and skat- 
 oxyl derivatives, and particular pigments, are greatly 
 augmented under certain pathological conditions at 
 present not fully understood, and the systematic 
 examination of urines of abnormal character would 
 probably materially extend our knowledge of this 
 difficult and obscure subject. In sending such samples, 
 I would request that the urine be poured into a clean, 
 strong eight-ounce bottle, which should then be at 
 once securely corked, carefully packed, and distinctly 
 labelled, with the date and hour of passing, and with 
 
Vlll PREFACE. 
 
 the name and address of the sender. The sample 
 should be forwarded at once by parcel-post, and full 
 information as to the patient's history should be sent 
 to me at the same time by letter-post. Of course the 
 complete examination of urine for abnormal constitu- 
 ents cannot be effected on so small a quantity as eight 
 ounces, but this amount will suffice to ascertain whether 
 a more extended analysis of urine from the same 
 source is desirable. 
 
 ALFRED H. ALLEN. 
 
 67, Storey Street, 
 Sheffield, June 1st, 1895. 
 
CONTENTS. 
 
 GENERAL COMPOSITION OF URINE. 
 
 PAGE 
 
 Constituents of Urine, 1 
 
 Analyses of Urine, 3 ; Quantity of Urine, 3 ; Influence of 
 Food on Urine, 5. 
 
 PRELIMINARY EXAMINATION OF URINE. 
 
 Physical and General Characters of Urine, ... 7 
 
 Volume, 7 ; Appearance, 7 ; Urinary Sediments, 8 ; Odour, 
 8 ; Taste, 9 ; Reaction of Urine, 9 ; Determination of 
 Acidity and Alkalinity of Urine, 10 ; Specific Gravity 
 and Total Solids of Urine, 11. 
 
 DIABETIC URINE. 
 
 Characters of Diabetic Urine, 14 
 
 Existence of Sugar in Normal Urine, 17. 
 
 Carbohydrates of Urine, 21 
 
 Constitution of Carbohydrates, 21 ; Dextrose or Grape-Sugar, 
 22 ; Laevulose or Fruit-Sugar, 26 ; Lactose or Milk- 
 Sugar, 29 ; Maltose, 31 ; Sucrose or Cane-Sugar, 31 ; 
 Dextrin, 31; Animal Gum, 31; Glycogen, 32; Inosite, 
 35 ; Glycuronic Acid, 37 ; Glycosuric Acid, 41. 
 
 Detection and Determination of Sugar in Urine, . . 42 
 Isolation of Sugar from the Urine, 43 ; Fermentation Test 
 for Sugar, 44 ; Polarimetric Determination of Diabetic 
 Sugar, 49 ; Detection and Determination of Diabetic 
 Sugar by its Reducing Action, 50 ; Behaviour of Urinary 
 Constituents with Precipitants and Oxidising Agents, 
 52 ; Reaction of Glucose with Copper Solutions, 56 ; 
 Trommels Test, 58 ; Fehling's Test, 58 ; Substances 
 reducing Fehling's Solution, 59; Modified Fehling's 
 Test, 62 ; Pavy's Ammoniacal Cupric Solution, 66 ; 
 
CONTENTS. 
 
 Glycerol-Cupric Solutions, 72 ; Gerrard's Cyano-Cupric 
 Process, 74 ; Reaction of Glucose with Bismuth Com- 
 pounds, 77 ; Eeaction of Glucose with Mercuric Com- 
 pounds, 78 ; Eeaction of Glucose with Coal-Tar Dyes, 
 78 ; Picric Acid Test, 78 ; Methylene-Blue Eeaction for 
 Glucose, 81 ; Safranine Test for Glucose, 83 ; Eeaction 
 of Glucose with Phenyl- Hydrazine, 85 ; Characters of 
 Osazones, 86 ; Eeaction of Glucose with Benzoyl 
 Chloride, 90. 
 
 ACETONUEIA, 90 
 
 Acetone, 91 ; Aceto-Acetic Acid, 97 ; Aceto-Acetic Ether, 
 97 ; Hydroxybutyric Acid, 99. 
 
 ALBUMINOUS UEINE. 
 
 Classification and Characters of Proteids, . . . 101 
 Eeactions of Urinary Proteids, 102. 
 
 Detection of Albumin in Urine, 104 
 
 Heat Test for Albumin, 104 ; Nitric Acid Test, 107 ; Eerro- 
 cyanide Test, 108; Picric Acid Test, 110; Esbach's 
 Test, 111 ; Trichloracetic Acid Test, 112 ; Salicyl-Sul- 
 phonic Acid Test, 112 ; Metaphosphoric Acid Test, 113 ; 
 Spiegler's Test for Albumin, 113; Tanret's Eeagent, 
 114. 
 
 Determination of Albumin in Urine, . . . . 116 
 
 Distinction and Separation of Urinary Proteids, . . 118 
 Paraglobulin, 118 ; Albumoses or Proteoses, 120 ; Peptones, 
 122 ; Mucin, 124. 
 
 THE NITEOGENISED CONSTITUENTS OE UEINE. 
 
 Determination of the Total Nitrogen of Urine, . . 127 
 Kjeldahl's Process, 127 ; Modified Kjeldahl's Method, 128. 
 
 Urea, 133 
 
 Urea Nitrate, 135 ; Urea Oxalate, 136 ; Determination of 
 Urea, 138 ; Use of the Nitrometer, 144 ; Influence of 
 Varying Conditions on the Excretion of Urea, 150. 
 
 Creatinine, 152 
 
 Creatine, 152 ; Creatinine Compounds, 155 ; Eeactions of 
 Creatinine, 156 ; Determination of Creatinine, 158. 
 
CONTENTS. XI 
 
 Uric Acid, . 160 
 
 Xanthine Derivatives, 162 ; Detection of Uric Acid, 165 ; 
 Determination of Uric Acid, 167. 
 
 Urates, 173 
 
 Quadri-urates, 173 ; Acid Sodium Urate, 177 ; Acid Lithium 
 Urate, 179 ; Neutral or Normal Urates, 181. 
 
 Hippuric Acid, 183 
 
 COLOURING MATTERS OF URINE. 
 
 Relation op Colour to Pathological Conditions, . . 189 
 Urobilin, 191 ; Bile-Pigments in Urine, 193 ; Blood Pig- 
 ments in Urine, 196 ; Hsematoporphyrin, 197 ; Urinary 
 Indigogens, 199 ; Potassium Indoxyl-Sulphate, 199. 
 
 APPENDIX. 
 
 Weights and Measures, 203 
 
 The Metric System, 203 ; Relations of Metric to English 
 Weights and Measures, 204. 
 
 Relations op Thermometric Degrees, 205 
 
 Tensions op Aqueous Vapour, 205 
 
 Symbols and Combining Weights op Elements, . . . 206 
 
 Normal and Standard Solutions, 206 
 
 Determination of Chlorine, 207. 
 
 INDEX, 209 
 
GENERAL COMPOSITION OF URINE. 
 
 Urine is an excretion of extremely complex composi- 
 tion, even in a state of health, while pathological 
 urine contains a variety of other bodies absent 
 from or existent only in traces in the normal excre- 
 tion. 
 
 W. D. Halliburton gives the following list of 
 constituents, the arrangement being based onHoppe- 
 S e y 1 e r ' s classification : — 
 
 1. Urea and related substances. Urea, uric acid, 
 allantoin, oxalic acid, xanthine, guanine, creatinine, 
 thiocyanic (sulphocyanic) acid. 
 
 2. Fatty and other non - nitrogenous substances. 
 Fatty acids of the series C n H 2n 2 ; oxalic, lactic, and 
 glycero-phosphoric acids ; minute quantities of certain 
 carbohydrates. 
 
 3. Aromatic substances. The ethereal sulphates of 
 phenol, cresol, pyrocatechin (catechol), indoxyl, and 
 skatoxyl ; hippuric acid ; aromatic oxy-acids. 
 
 4. Other organic substances. Pigments, ferments 
 (especially pepsin), mucus, humous substances ; and 
 in dogs, cynurenic and urocanic acids. 
 
 5. Inorganic compounds. Sodium and potassium 
 chlorides ; potassium sulphate ; sodium, calcium and 
 
 h A 
 
2 COMPOSITION OP URINE. 
 
 magnesium phosphates ; silicic acid ; calcium carbon- 
 ate ; ammonia salts. 
 
 6. Gases. Nitrogen and carbonic acid. 
 
 In addition to the above constituents of normal 
 urine, there may be present in certain pathological 
 conditions : — albumin and other proteids, haemoglobin, 
 methsemoglobin, bile-pigments, bile-acids, abnormal 
 urinary pigments, leucine and tyrosine, oxymandelic 
 acid, dextrose, milk sugar, glycuronic acid, fats, leci- 
 thin, cholesterin, cystin ; constituents derived from 
 food or drugs ; and organised bodies, such as blood- 
 corpuscles, urinary casts, and renal epithelium. 
 
 Several other urinary constituents of considerable 
 importance are omitted from Halliburton's list. 
 Among these may be named the acetone, aceto-acetic 
 ether, and hydroxy-butyric acid often present in 
 diabetic urine during the latter stages of the disease ; 
 hypoxanthine ; and substances resulting from the im- 
 perfect metabolism of food or tissue. 
 
 The specific gravity of urine is roughly a measure 
 of the contained solids. 
 
 The constituents of urine of chief importance are 
 the urea, uric acid, and phosphates, and it is to the 
 determination of these that the quantitative exami- 
 nation of healthy urine is generally limited. 
 
 Constituents of secondary importance are the creati- 
 nine, hippuric acid, chlorides, and sulphuric acid in 
 the two forms of metallic sulphates and ethereal 
 salts. 
 
 In healthy urine, sugar and albumin exist only in 
 traces, if at all ; but in typical diabetic urine a variety 
 of sugar is the leading constituent. Albumin some- 
 times co-exists with sugar in diabetic urine, but 
 its presence is more characteristic of certain other 
 diseases. 
 
 The urine passed in twenty-four hours by a man 
 
COMPOSITION OP URINE. 
 
 weighing 66 kilogrammes (= 144|- lbs.) is stated by 
 Parkes to measure 1500 c.c. (52 oz.), and to contain 
 about 72 - 5 grammes (or 2 J oz.) of solids, having the 
 following composition : — 
 
 
 Percentage 
 Composi- 
 tion of 
 Solids. 
 
 Absolute 
 Weight of 
 
 Solids in 
 Grammes. 
 
 Weight per 
 
 1000 of 
 
 Body 
 
 Weight. 
 
 Urea, CH 4 N 2 0, 
 
 45-75 
 
 33-18 
 
 0-5000 
 
 Creatinine, C 4 H r N 3 0, 
 
 1-25 
 
 0-91 
 
 00140 
 
 Uric acid, C 5 H 4 N 4 3 , 
 
 0-75 
 
 0-55 
 
 0-0084 
 
 Hippuric acid, C 9 H 9 N0 3 , . 
 
 055 
 
 0-40 
 
 0-0060 
 
 Pigment and other organic aub- ) 
 stances, J 
 
 13-79 
 
 10-00 
 
 0-1510 
 
 Sulphuric acid, S0 3 , . 
 
 2-77 
 
 2-01 
 
 0-0305 
 
 Phosphoric acid, P 2 5 , 
 
 4-36 
 
 3-16 
 
 0-0480 
 
 Calcium, ..... 
 
 0-35 
 
 0-26 
 
 0-0004 
 
 Magnesium, < 
 Potassium, .... 
 
 0-28 
 3-45 
 
 0-21 
 2-50 
 
 0-0003 
 00420 
 
 Sodium, ..... 
 
 15-29 
 
 11-09 
 
 0-1661 
 
 Chlorine,* .... 
 
 10-35 
 
 7-50 
 
 0-1260 
 
 Ammonia, .... 
 * = Sodium Chloride, 
 
 1-06 
 
 0-77 
 
 00130 
 
 100-00 
 
 72-54 
 
 1-1057, 
 
 17-04 
 
 12-36 
 
 0-1852 
 
 According to J. L. W. Thudichum (Pathology 
 of the Urine, 1887), the average volume of urine 
 excreted in twenty-four hours, by men weighing from 
 60 to 65 kilogrammes, is from 1400 to 1600 c.c. (48 
 to 56 fluid ounces), of a specific gravity averaging 
 1*020. The solids contained in this quantity of urine 
 range from 55 to 66 grammes, and are stated to be 
 composed as follow : — 
 
 Urea, .... 
 
 Creatine, 
 
 Creatinine, . 
 
 Xanthine and its derivatives, 
 
 Uric acid, . 
 
 Hippuric acid, 
 
 Cryptophanic acid, 
 
 }o- 
 
 75 
 
 30 to 40 grammes. 
 
 0-30 
 
 0-45 
 
 undetermined. 
 
 0-50 
 
 0-50 
 
 0*65 
 
COMPOSITION OF URINE. 
 
 Colouring matters, 
 
 . undetermined. 
 
 Biliary acids, . . .. 
 
 - 012 grammes 
 
 Oxalic; oxaluric, and nitrogenised 
 
 deri- 
 
 vative of sarco-lactic acid, . 
 
 . undetermined. 
 
 Acetic acid, .... 
 
 . 0-288 
 
 Formic acid, .... 
 
 . 0-05 
 
 Carbonic acid, 
 
 . undetermined. 
 
 Sulphuric acid, 
 
 . 1-5 to 2-5 „ 
 
 Sulphur in other forms, . 
 
 . 0-20 
 
 Phosphoric acid as alkaline phospha 
 
 tes, . 3'66 „ 
 
 Earthy phosphates, 
 
 . 1-28 
 
 Lime, ..... 
 
 . 0-17. 
 
 Magnesia, .... 
 
 . 0-19 
 
 Iron, . . . 
 
 . undetermined. 
 
 Potassium and sodium chlorides,* 
 
 . 10- to 13 
 
 Potash and soda, . . 
 
 undetermined. 
 
 Ammonia, .... 
 
 . 0-7 
 
 Methylamine, 
 
 . trace. 
 
 * Containing chlorine, , 
 
 6 to 8 „ 
 
 Yvon and Berlioz have published the results 
 of numerous analyses of normal urine {Lancet, ii., 
 1888, page 629). Their mean figures are as follow : — 
 
 Male. Female. 
 
 Per litre. Per diem. Per litre. Per diem. 
 Specific gravity, P0225 1-0215 
 
 Volume, . . 1360 c.c. 1100 c.c. 
 
 Urea, . . 21-5 grms. 26-5 grms. 19"0 grms. 20-5 grms. 
 
 Uric acid,. . 0-5 „ 0-6 „ 0-55 „ 0-57 „ 
 
 Phosphoric acid, 2'5 „ 3-2 „ 2-4 „ 2-6 „ 
 
 With the exception of the uric acid, which is almost 
 the same for the two sexes, the amounts under each 
 head are higher for males than for females. The pro- 
 portion of urea to uric acid is 40 : 1, and that of urea 
 to phosphoric acid 8:1. 
 
 W. Camerer (Zeit. Biol., xxiv. 306) has recorded 
 the amount of total nitrogen contained in normal 
 urine, and has compared it with that eliminated 
 in the form of urea. Thus the mixed urine from a 
 
INFLUENCE OF FOOD ON URINE. 
 
 number of persons measured on the average 1840 
 c.c., had a specific gravity of X'016, and contained ':■ — 
 
 Total nitrogen, 
 Nitrogen as urea, 
 Nitrogen in other forms, 
 
 Per diem. Per cent. 
 16-06 grammes, 0-873 
 14-15 „ 0-769 
 
 1-91 „ 0-114 
 
 The nitrogen passed in the form of urea is about 
 90 per cent, of the total. 
 
 The foregoing analyses probably fairly represent 
 the average composition of urine, but they take no 
 account of the variation in composition resulting from 
 change of food. The two following analyses by G. 
 Buhge furnish interesting information in this con- 
 nection. They were carried out on the urine of a 
 young man in good health, who was fed in succession 
 on animal and vegetable diet. The urine was collected 
 on the second day, after an exclusive diet of roast beef, 
 with a little salt and spring water. In the second case 
 the urine was also collected on the second day, after 
 an exclusive diet of wheat-bread, butter, a little salt, 
 and spring water. Bunge points out that these analy- 
 ses are probably unique among those published, in that 
 all the important constituents were determined on 
 the same sample of urine. The following were his 
 results : — 
 
 
 
 
 Meat Diet. Bread 
 
 Diet. 
 
 Total measure of 
 
 urine in 24 
 
 
 
 
 hours, 
 
 
 1672 c.c 
 
 1920 
 
 c.c. 
 
 Urea, 
 Creatinine, 
 
 
 
 67-2 gran 
 2-163 
 
 imes. 20-6 grammes 
 0-961 „ 
 
 Uric acid, 
 
 . 
 
 
 1-398 
 
 0-253 
 
 
 , 
 
 Sulphuric acid, 
 
 
 4-674 
 
 1-265 
 
 
 » 
 
 Phosphoric 
 Lime, 
 
 acid, 
 
 
 3-437 
 0-328 
 
 1-658 
 0-339 
 
 
 
 Magnesia, 
 Potash, . 
 
 
 
 0-294 
 3-308 
 
 0-139 
 1-314 
 
 
 
 Soda, 
 
 
 
 3-991 
 
 3-923 
 
 
 > 
 
 Chlorine, 
 
 
 
 3-817 
 
 4-996 
 
 
 » 
 
6 SULPHUR COMPOUNDS IN URINE. 
 
 The figures for sulphuric acid were obtained by- 
 boiling the urine with hydrochloric acid and barium 
 chloride, and hence include both that existing as 
 metallic sulphates and that present as ethereal salts. 1 
 
 It will be observed that the urea, creatinine, and 
 uric acid are all greatly increased by a meat diet. 
 The phosphoric and sulphuric acids, which are largely 
 derived from the oxidation of albuminoids, also show 
 a marked increase. 
 
 1 In the foregoing analyses the sulphuric acid and chlorine are sufficient to 
 convert all the inorganic bases into sulphates and chlorides. The ammonia 
 was not determined, but if present in normal proportion {i.e., from 0'4 to 0"9 
 gramme), it would suffice to convert the phosphoric acid into AmH 2 P0 4 . Fur- 
 ther, the saturating power of the sulphuric acid is over-estimated, since some 
 of it existed in the form of phenyl-sulphuric acid, C 6 H 6 .HS0 4 , or allied com- 
 pounds, exerting only a mono-basic function. The quantity of sulphur in the 
 form of ethereal salts in human urine averages one-tenth of that present as 
 , metallic sulphates. On acidulating the urine with acetic acid, and adding 
 barium chloride, the metallic sulphates are precipitated. If the liquid be 
 filtered, and the filtrate rendered strongly acid by hydrochloric acid and boiled, 
 the conjugated sulphuric acid salts are broken up, and the resultant sulphuric 
 acid can then be thrown down as barium sulphate.- If the liquid be again 
 filtered, evaporated to dryness, and the residue fused with nitre, an additional 
 quantity of sulphuric acid is formed, corresponding, in human urine, to 10- 
 20 per cent, of the total sulphur excreted, but rising in anomalous cases to a 
 larger proportion. This unoxidised sulphur exists in the urine partly in the 
 form of cystin, a body having the composition CaHyNSOj ; but a portion is 
 excreted in the form of thiocyanates. The ethereal sulphates are represented 
 by the potassium salts of phenyl-sulphuric acid, C 6 H 6 .HS0 4 , indoxyl-sulphuric 
 acid, C 8 H 6 N.HS0 4 (the so-called urinary indican), skatoxyl-sulphuric acid, 
 C 8 H B (CH 3 )N.HS0 4 , and similar derivatives of catechol (pyro-catechin) and 
 quinol (hydrequinone.) 
 
PRELIMINARY EXAMINATION OF URINE. 
 
 Volume. — The quantity of urine passed by a healthy 
 man in twenty-four hours is commonly stated, at 
 about 50 oz. (1450 c. c), but the observations 
 on which this statement is founded have been 
 chiefly made on the inhabitants of gaols, workhouses, 
 or barracks, where the inclination and opportunity for 
 drinking is limited. Many men in a state of perfect 
 health habitually pass a considerably larger volume 
 than 50 oz. daily. An excessive excretion indicates 
 polyuria, and is a very common, but not invariable, 
 accompaniment of glycosuria. 
 
 Whenever practicable, all observations on urine 
 should be made on portions of the mixed excretion 
 of twenty-four hours, otherwise very misleading con- 
 clusions may be formed. Thus the acidity, specific 
 gravity, colour, and other characters of urine vary 
 greatly with the period of the day and the time which 
 has elapsed since the last meal. In cases of diabetes 
 of a mild type, or which are progressing towards re- 
 covery, sugar is often nearly or entirely absent 
 from the morning urine, but returns for several hours 
 after partaking of a meal of which carbohydrates have 
 formed a portion. 
 
 Appearance. — The colour of normal urine is pale 
 yellow or amber. Typical diabetic urine is very 
 pale, but this peculiarity appears to be due to its 
 greater volume and consequent dilution of the urinary 
 
8 URINARY SEDIMENTS. 
 
 pigments, as compared with the normal excretion, 
 rather than to actual diminution of colouring matter. 
 
 Dark urines owe their colour to bile-pigments, blood, 
 tinctorial drugs {e.g., rhubarb), or to excess of the 
 pigments present in normal, urine. The subject is 
 discussed more fully in a subsequent section. 
 
 Urinary Sediments. — Normal human urine is 
 clear, or contains a fine flocculent precipitate of mucus. 
 f The urine of herbivorous animals is turbid when ex- 
 creted, from the separation of earthy phosphates, and 
 human urine is not unfrequently passed in the same 
 condition. In certain diseases very turbid urine is 
 voided, and may contain mucus, pus, tube-casts, epi- 
 thelium, chyle, blood, &c. 
 
 However clear the urine may appear when freshly 
 passed, a deposit almost always becomes visible on 
 allowing it to stand for a few hours, In normal 
 urine this deposit is nearly transparent, very light 
 and flocculent, and consists of mucus. A heavy pul- 
 verulent deposit, of a buff or fawn colour, generally 
 consists Of urates ; but earthy phosphates are not un- 
 frequently deposited from urine of an alkaline reaction. 
 A deposit of urates disappears on heating, while one 
 consisting of phosphates is permanent, and often be- 
 comes denser on boiling the urine. 
 
 On standing for several days, normal urine deposits 
 the whole, or at any rate the greater part, of its uric acid 
 in the form of acid urate of sodium. On still further 
 standing, the urine becomes alkalinefrom the decomposi- 
 tion of the urea with formation of ammonium carbonate. 
 
 Odour. — .The smell of urine is peculiar and slightly 
 aromatic. The odour of the normal excretion is said 
 to be due to phenol, and taurylic and damoluric acids. 
 The odour of diabetic urine often recalls that of hay, 
 but in the later stages of the disease the smell of 
 acetone is frequently perceptible. When cystin is 
 
CHARACTERS OF URINE. 9 
 
 present the smell of the urine is at first like that of 
 sweet-briar, but afterwards becomes offensive. Tur- 
 pentine produces an odour like that of violets, and 
 the essential oils of cubebs, copaiba, santal-wood, &c, 
 when taken internally, communicate characteristic 
 smells to the urine. On long keeping, or when 
 it has undergone fermentation in the bladder, urine 
 has an ammoniacal odour, while an excretion contain- 
 ing blood or pus is often putrid, and occasionally 
 evolves sulphuretted hydrogen. 
 
 Taste. — The taste of urine is said to be at once salt 
 and bitter. Diabetic urine has a distinctly sweet 
 taste, owing to the presence of sugar, the existence 
 of which was first recognised by this character. The 
 French chemists still lay stress on the "saveur" as 
 a test for urine. 
 
 Eeactiok of Urine. — Normal urine has commonly 
 a marked acid reaction to litmus, but during the so- 
 called " alkaline tide " which follows meals the urine 
 has not unfrequently a distinct alkaline reaction. 
 Diabetic urine is very generally acid, and the reaction 
 is not readily altered by food or alkaline medicines. 
 
 The acid reaction of urine has been attributed to 
 the presence of traces of hippuric or other acids in an 
 uncombined state, but is in all probability due to the 
 occurrence of sodium dihydrogen phosphate (acid 
 phosphate of sodium), NaH 2 P0 4 , produced by the re- 
 action of the uric and sulphuric acids resulting from the 
 metabolysis of the food on the basic sodium phosphate 
 of the blood. Hence it follows that the- acidity of 
 urine is increased by muscular exercise and the con- 
 sumption of highly albuminous food, as of course by 
 the internal administration of mineral acids. 
 
 Normal urine becomes alkaline after a vegetable 
 diet containing potassium salts of organic acids. Thus 
 the acid potassium salts of tartaric, citric, malic, and 
 
10 REACTION OF URINE. 
 
 other vegetable acids are largely present in acid fruits, 
 and on combustion are converted into potassium car- 
 bonate. Hence the urine passed after a fruit diet has 
 a strong alkaline reaction and effervesces on addition 
 of a mineral acid. Potatoes cause the urine to be 
 strongly alkaline, because they contain but a small 
 proportion of proteids, and hence yield but little 
 sulphuric acid on oxidation ; while on the other hand 
 they are rich in potassium malate, which is converted 
 into carbonate on ignition. The cereals and legumi- 
 nous seeds, on the contrary, yield a urine as acid as 
 that excreted under a meat diet, since they are rich 
 in both albuminoids and phosphates. 1 
 
 The alkalinity of urine may be due to the presence 
 of carbonates or basic phosphates of the alkali-metals, 
 or to the presence of carbonate of ammonium. This 
 last body is derived from the hydrolysis of the urea, 
 according to the following equation : — CH 4 N 2 + 
 2H 2 = (NH 4 ) 2 C0 3 . This change occurs spontane- 
 ously in urine when kept for some days, and is owing 
 to the action of a ferment called torula ureas. If any 
 such ferment find access to the bladder, as by the use 
 of a septic catheter, decomposition may occur before 
 the urine is voided, and the excretion may have an 
 alkaline reaction to litmus even when freshly passed. 
 The nature of this alkalinity may be readily ascer- 
 tained by moistening red litmus paper with the urine. 
 If the alkalinity be due to ammonia, the blue colour 
 will disappear as the paper dries, but otherwise will be 
 permanent. 
 
 The quantitative determination of the acidity of 
 urine may be made by titrating 100 c.c. of the sample 
 
 1 Reasoning from these facts, F. B u n g e regards cheese as a particularly 
 unsuitable food for persons inclined to gravel, since in its manufacture the 
 alkaline salts are largely lost in the whey, while the casein yields much 
 sulphuric and phosphoric acid on combustion. Salted meat and fish he con- 
 siders objectionable for similar reasons. 
 
REACTION OP URINE. 11 
 
 with decinormal caustic soda, using phenolphthalein 
 or alizarin as an indicator. The addition of the alkali 
 is continued till a deep red colour is obtained with the 
 former indicator or a violet with the latter. Each 1 c.c. 
 of standard alkali required represents 0*012 gramme of 
 sodium dihydrogen phosphate, NaH 2 P0 4 , to which salt 
 the acidity of normal urine is very probably due. 
 
 Similarly, according to Freund and Toepfer, 1 
 the alkalinity of urine can be determined by titrating 
 100 c.c. with decinormal sulphuric or hydrochloric acid, 
 the end-reaction being the point at which the red colour 
 due to phenolphthalein disappears, or the violet of 
 alizarin changes to yellow. Each 1 c.c. of decinormal 
 acid required is stated to represent, when the former 
 indicator is used, 0"0106 gramme of sodium car- 
 bonate, Na 2 C0 3 ; or 0'0164 of trisodium phosphate, 
 Na 3 P0 4 . When alizarin is the indicator used, each 
 1 c.c. of acid represents half these quantities. 
 
 Whatever variations in the reaction of urine may be 
 observed hourly under the influence of food, &c, the 
 mixed excretion of twenty-four hours,if of normal charac- 
 ter, will invariably be found to exhibit an acid reaction. 
 
 Specific Gravity and Total Solids of Urine. — 
 The specific gravity of normal human urine varies from 
 1015 to 1025, averaging about 1020 ; but after great 
 physical exertion and consequent profuse perspiration 
 it has been known to be as high as 1035 in the case 
 of healthy persons. On the other hand, after exces- 
 sive drinking it has fallen as low as 1002. Typical 
 diabetic urine is of very high specific gravity, usually 
 exceeding 1030 and occasionally rising as high as 1074. 
 
 The specific gravity of urine is most conveniently 
 ascertained by means of the hydrometer, an instru- 
 ment which, if accurately constructed, gives, with 
 reasonable care, indications quite delicate enough for 
 
 .*■ Zeit. physiol. Chem,, xix. 84, and Jour. Chem. Soc, lxvi, part ii. page 260. 
 
12 SPECIFIC GRAVITY OP URINE. 
 
 ordinary purposes. It must be remembered, however, 
 that the readings are very materially affected by 
 alterations of temperature. Hence the urine should 
 be brought as nearly as possible to a temperature of 
 60° F. ( = 15'5° C), and on no account should the 
 hydrometer reading be taken when the urine is 
 sensibly warm. 1 
 
 The specific gravity of urine is roughly an indica- 
 tion of the proportion of solid matter contained in it; 
 and hence of the total waste of the system. It would 
 be an accurate measure of this if the solids were of a 
 homogeneous nature, but as solutions of equal strength 
 of sodium chloride, urea, sugar, and other urinary con- 
 stituents have different specific gravities, the density 
 of the urine will vary with the nature of the contained 
 solids, as well as with their amount. 
 
 The following are the specific gravities at 60 F. 
 (=15"5 C.) of solutions of urinary constituents con- 
 taining 10 per cent, of the solids : — 
 
 Substance. Specific Gravity. Observer. 
 
 Sodium Chloride (common salt), 1073-35 Gerlach. 
 
 Urea, . . . . . 1028-2 A. H. Allen. 
 
 Dextrose, .... 1040-0 F. Salomon. 
 
 Albumin, .... 1026-0 
 
 Hence to deduce accurately the percentage of solids 
 from the gravity of urine it would be necessary to 
 ascertain the proportion of each of the leading con- 
 stituents ; but when that was effected, the specific 
 gravity, would no longer have any interest. 
 
 If the specific gravity of a sample of urine above 
 that of water ( = 1000) be multiplied by 2\33, accord- 
 
 1 F. W. Fletcher (North London Chemical Works, Holloway) has devised 
 a " thermo-hydrometer " which allows the temperature of the liquid in which 
 it is immersed being read with great facility. One side of the ivory scale 
 enclosed in the stem indicates the specific gravity of the liquid, while the 
 other shows the temperature ; the stem.of a delicate thermometer, the bulb of 
 which is situated below that of the hydrometer, funning up inside the hollow 
 hydrometer stem. An approximate correction for temperature may be made 
 by adding one degree to the observed specific gravity for each 8° F. above 60°. 
 
SOLID MATTERS OF URINE. 13 
 
 ing to Haeser and Christison ; by 2, according to 
 Trapp ; or by 2*2, according to Loebisch ; the products 
 will be the number of grammes or grains of solids 
 contained in 1000 c.c. or fluid grains of the sample. 
 By dividing the product by 1000, and multiplying by 
 the volume in centimetres or fluid grains passed in 
 twenty-four hours, the total weight of solids excreted 
 by the kidneys in that time will be found. 1 The varia- 
 tion in the factors given by the authorities quoted 
 shows that, for reasons above stated, the method is 
 only roughly approximate. 
 
 . Bouchardat has proposed a somewhat similar 
 formula for ascertaining approximately the proportion 
 of sugar contained in diabetic urine from the specific 
 gravity of the liquid. The gravity above 1000 is 
 multiplied by 2, and the product by the number of 
 litres of urine passed in twenty-four hours. From 
 the product 60 is deducted, this correction represent- 
 ing the influence of the solids other than sugar, when 
 the remainder will represent the weight of sugar in 
 grammes passed in twenty-four hours. 2 
 
 When an accurate determination of the solid matters 
 of urine is required, it is best effected by evaporating 
 an exactly weighed or measured quantity in a flat- 
 bottomed porcelain or platinum capsule on the water- 
 bath, exposing the apparently dry residue for some 
 hours in the water-oven, and weighing the residue 
 with precautions to prevent re-absorption of water. 
 5 c.c, or 100 fluid grains, is a suitable volume of 
 urine to use for the determination. 
 
 1 Thus if the urine passed in twenty-four hours measure 1450 c.c, and have 
 
 a specific gravity of 1032 '5, the total solid matters excreted by the kidneys 
 
 in that time will be, by Haeser and Ohristison's formula, 
 
 32-5 x 2-33x1450 ,„ , 
 
 jOPQ = 101-1 grammes. 
 
 s Thus, taking the same urine as before :— 32-5 x 2 = 65-0 ; 65 x l-45 = 94'25. 
 94-25-60 = 34-25, which figure represents the weight in grammes of sugar 
 excreted by the kidneys in twenty-four hours. 
 
DIABETIC URINE. 
 
 Glycosuria, or the excretion of urine containing 
 a kind of sugar known as glucose, is especially- 
 characteristic of the disease known as Diabetes 
 Mellitus. 1 It is commonly, but by no means invari- 
 
 1 " According to the writings of Celsus, Aretseus and Galen, the disease 
 termed 'diabetes' (Sic£, 'through,' fraivo, 'I go') seems to have been 
 recognised in a general way by the ancients. The progressive emaciation 
 characteristic of the malady was observed as being accompanied by inordinate 
 thirst, voracious appetite, and excessive discharge of urine. It was not, 
 however, until 1674 that the urine in certain cases was discovered to possess a 
 sweet taste, and the honour of this discovery, on which followed the establish- 
 ing of the distinction between diabetes insipidus and glycosuria diabetes, is due 
 to Willis, an English physician. A hundred years subsequently, Dobson, 
 of Liverpool, discovered that the blood as well as the mine contained sugar ; 
 and he inferred therefrom that this sugar was separated from the blood, and 
 not formed by the kidney. In 1778, Cowley separated the sugar from the 
 urine iu a free state. In 1815, Chevreul pointed out that the sugar existing 
 in the urine in cases of diabetes mellitus was different from cane sugar and 
 closely resembled that of the grape ; and in 1825Tiedmann andGmelin 
 ascertained that during its passage along the alimentary canal starchy matter 
 was transformed into sugar." — Urine and Urinary Analysis, by D, Campbell 
 Black. 
 
 Glycosuria may be temporarily induced (so long as the liver is charged with 
 glycogen) by puncturing the floor of the fourth ventricle of the brain, while 
 the lesion of a closely adjacent part of the same ventricle has been found to 
 produce polyuria. Hence it has been suggested that in cases of diabetes in 
 which both glycosuria and polyuria are present both these localities of the 
 fourth ventricle have suffered irritation or injury. 
 
 Complete removal of the pancreas from an animal produces a condition of 
 diabetes, which is relieved if the pancreas from another animal be grafted into 
 the abdomen of that from which the pancreas was extirpated. Similarly, 
 disease of the pancreas in man causes diabetes. 
 
 Diabetes kills the person suffering from it by starving the tissues and 
 emaciating the body. Sometimes death ensues from uremia due to exhaus- 
 tion of the kidneys and their consequent failure to excrete urinary materials 
 from the blood. 
 
 The physiology of diabetes is very imperfectly understood. The appearance 
 of sugar in the urine depends upon some failure at points where the sugar is 
 
DIABETIC URINE. 15 
 
 ably, associated with Polyuria, or the passing of an 
 excessive volume of urine. This may exist without 
 the presence of glycosuria, in which case the disease 
 is known as Diabetes Insipidus. 1 In typical cases 
 of diabetes mellitus the volume of urine excreted, 
 is frequently from 100 to 130 oz. per diem, and 
 sometimes reaches twice this volume. An excretion 
 of 400 oz. per diem has been observed. 
 
 Typical diabetic urine is very pale, probably owing 
 to the dilution of the urinary pigments, but in mild 
 cases, unaccompanied by marked polyuria, the urine 
 has often a very dark colour. The odour sometimes 
 resembles that of hay, but in advanced cases of 
 diabetes the excretion has often an odour of acetone 
 or alcohol. The taste of diabetic urine is distinctly 
 sweet from the presence of sugar. 
 
 The urine of diabetic persons has a tendency to 
 froth on agitation. Its reaction is usually distinctly 
 acid, even after meals or alkaline medicines have been 
 taken. 
 
 The specific gravity of typical diabetic urine is very 
 
 absorbed and assimilated, or at some other point (e.g., the muscular tissue) 
 where sugar properly assimilated in the blood fails to be oxidised and made 
 use of. Sugar injected directly into the blood is not oxidised in the muscle, 
 but runs off through a healthy kidney and is found in the urine. The 
 significance of sugar in the urine of those who eat redundantly of sugar and 
 other carbohydrates, who take little muscular exertion, and whose persons 
 are already laden with fat, is quite different from that which it has in the case 
 of persons who are emaciated. Similarly, sugar in the urine of an over- 
 charged system is very different in its significance from glucose in the urine of 
 the same person when all sugar has been stopped, the bulk of the starch has 
 been excluded from the diet, and a fair amount of daily walking or other gentle 
 muscular exercise is taken. 
 
 1 Thudichum mentions a case in which 5600 c.o. ( = 196 oz.) of urine 
 containing 0'8 per cent, of sugar was excreted in twenty-four hours, while at 
 a later period the volume fell to 4333 c.c. , and contained only traces of sugar. 
 In many cases of diabetes with a minimum excretion of sugar, no material 
 increase in the volume of urine is observed, though the patients have a strong 
 desire to micturate. In these cases it is assumed that the sugar exerts an 
 irritating action on the bladder, and causes its frequent evacuation. In many 
 cases polyuria appears to be the result of an irritation of the nerves and con- 
 sequent congestion of the kidneys. 
 
16 DIABETIC URINE. 
 
 high, the usual range being from 1030 to 1040 ; but 
 a density of 1065 was observed by Seegen in a 
 urine containing 10 per cent, of sugar. 1 Bernard 
 states the maximum density at 1074. 
 
 Albumin is sometimes present in the urine in 
 chronic cases of diabetes, but tube-casts are of rare 
 occurrence. 
 
 Unless previously concentrated, diabetic urine does 
 not usually deposit uric acid when acidified and left 
 at rest. Whether this behaviour is due to the dilute 
 condition of the excretion, or to the presence of less 
 than the usual amount of uric acid, is not certain. 
 
 As a rule, diabetic persons excrete more urea than 
 persons in health, owing to the greater amount of 
 proteids they consume. There is no relation between 
 the urea and the sugar excreted. The latter varies 
 with the amount of starchy and saccharine food taken, 
 except in cases where sugar is produced under a 
 strictly albuminous diet. 
 
 Dieting experiments on diabetic patients show that 
 more proteids are used than by healthy persons, since 
 the carbohydrates are not available as a proteid-saving 
 food. More fat undergoes combustion in the system 
 of diabetics than in that of normal persons. Muscular 
 work is stated to increase the excretion of urea, but 
 not to affect sensibly the elimination of sugar. 
 
 The quantity of sugar excreted by diabetic persons 
 varies from mere traces to as much as 600 grammes 
 (20 oz.) in the twenty -four hours. The percentage of 
 sugar present is, of course, dependent on the volume 
 of the urine. In severe cases it not unfrequently 
 reaches 8 or 10 per cent., and, according to A. H. 
 H a s s a 1 1, as much as 1 5 per cent, is present in some 
 
 1 As a 10 per cent, solution of pure sugar in water has a specific gravity of 
 1040, the difference between this figure and the observed density may be 
 regarded as due to the normal constituents of urine. Thus, eliminating the 
 effect of the sugar, the urine would have had a specific gravity of 1025. 
 
SUGAR IN NORMAL URINE. 17 
 
 cases. The morning urine contains least sugar, while 
 that passed three or four hours after a meal is the richest 
 in sugar. Under strictly animal diet, the sugar will 
 often fall to 1 per cent., while a meal of starchy or 
 saccharine food will raise it to five or even to ten 
 times that amount. Hence the pathological signifi- 
 cance attaching to the excretion of 50 grammes of 
 sugar daily when under strictly animal diet is much 
 greater than the elimination of a larger amount when 
 saccharine and amylaceous foods are being freely 
 taken. 
 
 As the proportion of sugar in diabetic urine varies 
 largely according to the time of day and the nature 
 of the food taken, it is highly important that any 
 analytical examination should be conducted on the 
 united excretion of the previous twenty-four hours. 
 
 The question of the occurrence of traces of sugar in 
 
 normal human urine has been the occasion of much 
 
 controversy, and the last word on the subject still 
 
 remains to be said. B r ii c k e ( Wien. Ahad. Sitzung- 
 
 ber, xxix. 346) appears to have been the first to state 
 
 that all normal urine contained sugar, and this view was 
 
 supported byBence Jones {Jour. Chem. Soc, xiv. 
 
 22), Kiihne, Tuchen, and many other observers ; 
 
 but opposed by Friedlander, Wiederhold, 
 
 Maly, and Kiilz. The question was re-examined 
 
 in 1871, by Seegen, who pointed out many fallacies 
 
 in the methods of those who had found sugar, and 
 
 concluded that it was either absent from normal 
 
 human urine, or present in such small proportion that 
 
 the then existing methods were insufficient for its 
 
 positive recognition in the presence of co-occurring 
 
 substances which simulate many of its reactions. On 
 
 the other hand, F. W. Pavy, in 1878, concluded that 
 
 sugar was a normal constituent of urine, and that no 
 
 sharp line of demarcation could be drawn between the 
 
18 SUGAR IN NORMAL URINE. 
 
 excretion in health and in diabetes, except quantita- 
 tively ( Guy's Hospital Reports, xxi. 413). Molisch, 
 from the examination of a large number of samples of 
 healthy human urine by the alpha-naphthol and thymol 
 tests, came to the conclusion that traces of sugar are 
 met with frequently in human urine ; but the value of 
 his tests, and hence the accuracy of his conclusions, 
 have been disputed by Leuken and also by S e e g e n 
 {Jour. Soc. Chem. Industry, vi. 149, 150). E. 
 Luther, again (Chem. Centr., 1891, ii. 90, and 
 Jour. Chem. Soc, lx. 1559), as the result of the 
 application of the furfuraldehyde and alpha-naphthol 
 tests to a large number of samples, concluded that 
 glucose is present in all human urine, the amount 
 found in the excretion of adults averaging O'l per 
 cent., while the total carbohydrates amount to 0"2 per 
 cent. According to E. Eoos (Zeit. physiol. Chem., 
 xv. 513), the normal urine of the dog, horse, and 
 rabbit always contains more or less carbohydrates, as 
 indicated by the furfuraldehyde reaction and confirmed 
 \ by the benzoic chloride test. Human urine is stated 
 by Eoos always to give an affirmative reaction with 
 phenyl-hydrazine, and the same is true of dogs' urine, 
 while the excretion from rabbits gives especially well- 
 formed crystals. The urine of all these animals was 
 found to be slightly lsevo-rotatory. Wedenski 
 (Zeit. physiol. Chem., xiii. 122), by agitating a large 
 volume of urine with benzoic ehloride, obtained a 
 separation of the insoluble benzoyl compounds of 
 carbohydrates. On separating the precipitate and 
 treating it with soda, a portion dissolved and appeared 
 to consist of animal gum, while the undissolved portion 
 gave the reactions of dextrose. 
 
 G. Stilling fleet Johnson denies the presence 
 of traces of sugar in normal urine, on the ground that 
 strictly negative reactions were obtained on testing 
 
SUGAR IN NORMAL URINE. 19 
 
 the filtrate from the precipitate produced on treating 
 the urine with mercuric acetate. But while mercuric 
 acetate effectually and conveniently removes from solu- 
 tion the creatinine and other interfering substances, it 
 also exerts an oxidising action on glucose itself, so that 
 where mere traces are present they might be expected 
 to suffer complete destruction. Direct experiments, 
 made by the author to test the point, showed that the 
 oxidation of the glucose was but trifling in the highly 
 dilute solutions employed. The writer, by what was 
 apparently substantially the same method as that 
 by which G. S. Johnson obtained negative results, 
 but confirmed by the results of the phenyl-hydrazine 
 test, 1 satisfied himself that minute quantities of sugar 
 are present in some specimens of urine from persons 
 apparently in perfect health. 
 
 Similarly, relying on the failure of normal urine to 
 yield a red-brown coloration with an alkaline solution 
 of picric acid, after the creatinine, &c, have been 
 removed with mercuric acetate, Sir G. Johnson 
 maintains strongly the absence of sugar from the 
 excretion ; and meets the objection that such urine 
 often gives a crystalline product with phenyl- 
 hydrazine by the suggestion that, " for some reason, 
 the test must be difficult to apply and uncertain in 
 its results." 1 (See articles and correspondence in 
 the Lancet during July and August, 1894.) It is 
 contended that the mere presence of traces of sugar 
 in urine thereby proves the excretion to be abnormal, 
 though sugar admittedly appears temporarily on very 
 slight provocation. 2 But such an argument begs the 
 
 1 Glycuronic acid and its compounds give with phenyl-hydrazine a crystal- 
 line compound closely resembling phenyl-glucosazone, but having a different 
 melting-point, but with proper precautions no confusion between the two 
 bodies is possible. (See pages 41 and 88.) 
 
 2 In the discussion of a paper by the author, "On the Examination of Urine 
 for Small Quantities of Sugar," read before the Society of Public Analysts, 
 Mr G. Stillingfleet Johnson is reported to have said : — " The question was, 
 
20 SUGAR IN NORMAL URINE. 
 
 very question at issue, which is whether normal urine 
 does not sometimes contain traces of sugar. 
 
 The latest experimental contribution to the con- 
 troversy is by F. W. P a v y {Physiology of the Carbo- 
 hydrates, 1894), who, by operating on large quantities 
 of urine, precipitating the sugar in combination with 
 oxide of lead, and recovering it from this compound, 
 obtained a liquid which exhibited the chief chemical 
 reactions of glucose. No attempt appears to have 
 been made to obtain the glucose in a solid form, and 
 the method employed for effecting its fermentation 
 by yeast is open to criticism. 1 
 
 W. D. Halliburton( Chemical Physiology and 
 Pathology, 1891) considers the balance of evidence 
 clearly in favour of the existence of a small quantity 
 of sugar in normal urine. 2 
 
 What is a normal urine? This was simply reduced to the further question, 
 Who is a healthy man ? Everybody knew that a man might rise in the 
 morning a healthy man, and that he might go to bed at night anything hut 
 a healthy man. He was quite sure that slight errors of diet — such, for 
 instance, as taking a late dinner, or dining out — were sufficient to produce a 
 temporary glycosuria, which was of no importance whatever. If the test were 
 applied carefully to the urine of healthy individuals, it would be found in the 
 long run to give practically negative results. He had no doubt that the 
 reason why the idea that normal human urines were saccharine in character 
 had existed so long was that the reducing action of creatinine had been mis- 
 taken for the reducing action of sugar." — Analyst, xix. 185. 
 
 1 See a correspondence in the Lancet, January, February, and March, 1895. 
 
 2 In a letter published in the Lancet for February 9th, 1895, Halliburton 
 writes : — " I think that a careful study of the researches of Seegen and of 
 Baisch, in which more stringent chemical methods were employed than in those 
 of Dr Pavy, will convince the impartial observer that a small quantity of 
 glucose is obtainable. Whether this is artificially produced by the method of 
 analysis, as suggested by Sir George Johnson, is a subject that demands 
 renewed research. Dr Pavy gives the percentage of sugar in normal urine 
 about five times greater than has been found by these observers, and I think 
 his high figure is most easily explicable by Sir George Johnson's hypothesis — 
 namely, a neglect to take into consideration the reducing action of creatinine." 
 This attempted explanation does not appear valid, since Pavy relies largely on 
 the separation of the glucose in the form of an insoluble lead compound. It is 
 nevertheless remarkable that not the slightest reference to creatinine or its proved 
 reducing power on copper solutions, &c, is to be found throughout Dr Pavy's 
 writings, and in a letter published in the Lancet for March 2nd, 1895, Pavy de- 
 clines a direct challenge of Dr Halliburton to explain his views as to creatinine. 
 
CARBOHYDRATES OE URINE. 21 
 
 THE CARBOHYDRATES OF URINE. 
 
 The "sugars" belong to the class of organic com- 
 pounds known to chemists as Carbohydrates. 
 This name is a survival of the period when they were 
 regarded as hydrates of carbon, or compounds of 
 carbon with the elements of water, if not with water 
 itself. Thus the carbohydrates contain carbon, hydro- 
 gen, and oxygen, the two latter elements being in the 
 proportion in which they exist in water. Hence the 
 carbohydrates, as a class, may be represented by the 
 generic empirical formula : — C x H 2n O n . Great progress 
 has been made of late years in the knowledge of the 
 constitution of sugars and other carbohydrates, the 
 researches of Emil Fischer being of pre-eminent 
 importance. Fischer has shown that the class of sugars 
 known as glucoses, of which grape-sugar is the type, 
 are really the aldehydes or ketones of the 
 hexatomic alcohol mannite or mannitol, having the 
 formula : — C 6 H 8 (OH) 6 . Just as aldehyde is obtained 
 by oxidising ordinary alcohol, C 2 H 6 0, so from 
 mannitol, C 6 H u 6 , the corresponding compound, 
 C 6 H 12 6 , is obtained. The carbohydrates of this 
 composition are called Monosaccharides, the best 
 known members of the class being dextrose 
 (sometimes called dextro-glucose, or simply glucose), 
 lsevulose, and galactose. By the loss of the 
 elements of water, two molecules of these glucoses 
 may coalesce to form Disaccharids, thus : — 2C 6 H 12 6 
 — H 2 = C 12 H 22 O u . The disaccharides thus formed, 
 when boiled with dilute acids, readily take up the 
 elements of water again, becoming split into two 
 molecules of monosaccharid. These two molecules 
 may be either identical or dissimilar in nature. 
 Thus, maltose splits into two molecules of dextrose ; 
 cane-sugar yields one of dextrose and one of lsevulose : 
 while milk-sugar is hydrolysed to dextrose and galac- 
 
22 HYDEOLYSIS OF CARBOHYDRATES. 
 
 tose. Trisaccharids, resulting from the coalescence 
 of three molecules of glucose, are known, the typical 
 member of this class being raffinose, C 18 H 32 16 . 
 
 Starch and dextrin are still more complex 
 molecules, but, like the more simply constituted 
 saccharides, tend to hydrolyse into their constituent 
 disaccharide and monosaccharide molecules under the 
 influence of dilute acids or other hydrolysing agents. 
 The following represents the chief features of existing 
 knowledge on the subject : — 
 
 Saccharid. 
 
 Hydrolysis Products. 
 
 a- and /J-Amylan. 
 Cellulose. 
 
 Dextrose. 
 Dextrose. 
 
 Dextrin. 
 
 Dextrose. 
 
 Glycogen. 
 Inulin. 
 
 Dextrose. 
 Lsevulose. 
 
 Lichenin. 
 
 Dextrose. 
 
 Tunicin. 
 
 Dextrose. 
 
 Starch. 
 
 Dextrose. 
 
 Maltose. 
 
 Dextrose. 
 
 Lactose (milk-sugar). 
 
 Cane-Sugar. 
 
 Raffinose. 
 
 Dextrose and Galactose. 
 Dextrose and Laevulose. 
 Dextrose, Laevulose, and Galactose. 
 
 Dextrose. Dextro-glucose. Glucose. Grape- 
 sugar. C 6 H 12 6 ; CH 2 (OH).(CHOH) 4 .COH ; or, 
 CH 2 (OH).CH(OH).CH(OH).CH(OH).CH(OH).COH. 1 
 Dextrose is a constant, or at least a very frequent, 
 product of the hydrolysis of the polysaccharides by 
 boiling with dilute acids. It is also produced by 
 the hydrolysis of the natural glucosides amygdalin, 
 populin, salicin, hesperidin, lupulin, phloridzin, and 
 ruberythric acid. iEsculin, arbutin, and coniferin 
 also yield dextro-rotatory glucoses on hydrolysis, 
 but it is not certain that they are identical with 
 
 1 This constitutional formula shows the presence o£ the COH group, and 
 consequently classes dextrose among the aldehydes. On the other hand, 
 laevulose contains a carboxyl group, CO, and has the constitution of a 
 ketone :-CH 2 (OH).CH(OH).CH(OH).CH(OH).CO.CH li (OH). 
 
CHARACTERS OP DEXTROSE. 23 
 
 ordinary dextrose. On the other hand, lsevulose is 
 obtained when inulin is boiled with dilute acids. 
 Dextrose occurs in the free state in grapes, many- 
 other fruits, the seeds and sap of plants, &c. It also 
 occurs in the blood, liver, and other parts of the 
 body, and is the characteristic sugar of diabetic urine. 1 
 Dextrose often crystallises from honey, in which it 
 co-exists with lsevulose. As already stated, a mixture 
 of dextrose and lsevulose in equal proportions results 
 from the action of dilute acids on cane-sugar, and it is 
 also formed by the hydrolysis of milk-sugar and maltose. 
 
 Dextrose usually crystallises from its aqueous solu- 
 tion in granular hemispherical warty masses or tabular 
 crystals, containing C 6 H 12 6 + H 2 0, but hot concen- 
 trated solutions often deposit anhydrous dextrose in 
 prisms. Dextrose loses its water of crystallisation 
 when gently heated. The anhydrous substance melts 
 at 146°, and at about 170° C. loses water and is con- 
 verted into dextrosan, C 6 H 1() 05, and at higher 
 temperatures (200° C. ) yields caramel. 
 
 Dextrose is less soluble than cane-sugar in cold 
 water, requiring 1^ times its own weight, but it 
 dissolves in all proportions in boiling water, forming 
 a syrup having a sweetening power inferior to a solu- 
 tion of cane-sugar or lsevulose of the same strength. 
 
 Solutions of dextrose exert a powerful dextro- 
 rotatory action on polarised light, and one of the 
 most accurate methods of estimating dextrose, in the 
 absence of other optically active substances, is based 
 on this fact. The specific rotation of dextrose solu- 
 tions for a concentration of 10 per cent, is +52 '7° 
 for the sodium ray, and +57*0° for the transition-tint. 
 
 When treated with yeast and exposed to a moderate 
 temperature, solutions of dextrose readily undergo the 
 
 1 It is not absolutely certain that the dextro-rotatory glucose of diabetic 
 urine is strictly identical with the sugar of grapes. 
 
24 moore's test for sugar. 
 
 alcoholic fermentation, the weight of alcohol generated 
 being approximately half that of the glucose fermented. 
 The best conditions for obtaining quantitative results 
 are described on page 46, et seq. 
 
 Dextrose is not affected when heated for a moderate 
 time with dilute acids. Prolonged treatment is said 
 to result in the formation of products having the pro- 
 bable formula C 6 H 14 7 . When quite pure, dextrose 
 is not readily charred by concentrated sulphuric acid, 
 but combines with it to form a compound decomposed 
 by water. By treatment with nitric acid, dextrose is 
 oxidised to saccharic acid, C 6 H ]0 O 8 . 
 
 When a caustic alkali is added to a solution of 
 dextrose, the liquid acquires a colour ranging from 
 yellow to dark brown, according to the amount of 
 glucose present. The change of colour occurs slowly 
 in the cold, but almost instantaneously on heating. 
 The reaction results in the formation of acetal, acetone, 
 and formic, acetic, and lactic acids ; besides the little- 
 known bodies termed glucinic, japonic, and saccharumic 
 acids. On acidulating the brownish liquid with nitric 
 acid, the colour is lessened or destroyed, and the solu- 
 tion acquires the characteristic odour of caramel (burnt 
 sugar). 
 
 Moore applied the foregoing facts to the detection 
 of sugar in diabetic urine. The sample to be tested 
 is first freed from albumen, if present, by rendering it 
 faintly acid with acetic acid and boiling. The filtered 
 liquid, or, in the absence of albumin, the original 
 urine, is mixed with an equal measure of normal caustic 
 soda or potash (or Liquor potassse, B.P.), and filtered, 
 without heating, from the precipitated earthy phos- 
 phates. The filtered liquid is then boiled in a test- 
 tube, in such a manner that only the upper part is 
 heated, when in presence of much glucose a yellow or 
 brown colour will be developed in the heated part of 
 
REACTIONS OF DEXTROSE. 25 
 
 the solution, and contrasts well with the unchanged 
 lower portion. The test is simple, and occasionally- 
 very useful, but does not indicate with certainty less 
 than 2 per cent, of glucose. Cane-sugar, uric acid, 
 and creatinine give no similar reaction. Urine con- 
 taining the colouring matters of rhubarb and senna 
 becomes reddish-brown with alkali before heating ; 
 while samples containing catechol (pyrocatechin) 
 acquire a brown colour on exposure to air. 
 
 A solution of dextrose dissolves the alkaline earths, 
 forming yellow solutions precipitated by alcohol. By 
 boiling with excess of lime dextrose is rapidly acted 
 on and destroyed. 
 
 Dextrose, when pure, is not precipitated by neutral 
 or basic lead acetate, but on subsequent addition of 
 ammonia it yields a white insoluble compound of 
 dextrose and lead hydroxide, which may be washed 
 without decomposition if carbonic acid be excluded. 
 This lead compound affords a means of isolating 
 dextrose from urine and freeing it from various co- 
 occurring substances. (See page 43.) 
 
 When heated with water and silver oxide, dextrose 
 yields glycollic, oxalic, and carbonic acids, but not 
 acetic acid. 
 
 Dextrose is a powerful reducing agent, especially in 
 presence of free alkali and at a high temperature. 
 Thus at the boiling-point, in alkaline solution it 
 reduces silver, mercury and bismuth to .the metallic 
 state, cupric oxide to cuprous, ferricyanides to ferro- 
 cyanides, blue indigo to white indigo, &c, &c. Further 
 details of these and similar reactions, and their applica- 
 tion to the detection of urinary sugar, are given on 
 page 50, et seq. 
 
 By the action of nascent hydrogen in neutral or 
 alkaline solution, but not in presence of acid, dextrose 
 is reduced to m a n n i t o 1, C 6 H 14 6 . 
 
26 LSEVULOSE. 
 
 With phenyl-hydrazine dextrose reacts to form a 
 well defined osazone, crystallising in tufts of 
 needles. The properties and method of preparing this 
 important body are fully described on page 86, et seq. 
 
 On agitating a solution of dextrose with benzoyl 
 chloride, a compound of the two separates in white 
 crystals. This reaction has been employed to demon- 
 strate the presence of glucose in urine. 
 
 LSEVULOSE. LuEVO-GLPCOSE. FRUIT SUGAR. C 6 H 12 6 ; 
 
 or CH 2 (OH).CH(OH).CH(OH).CH(OH).CO.CH 2 (OH). 
 Lsevulose occurs with dextrose in honey and in many 
 fruits, and is a product of the hydrolysis of cane-sugar 
 and raffinose. The hydrolysis of the starch-like body 
 i n u 1 i n yields lsevulose without dextrose. The conver- 
 sion of dextrose into lsevulose has been effected by 
 E. Fischer by acting on the former with phenyl- 
 hydrazine, reducing the resultant glucosazone to iso- 
 glucosamine by zinc and acetic acid, and treating the 
 last body with nitrous acid. 
 
 Lsevulose is separated from dextrose by converting 
 them into the lime compounds, when the former sugar 
 forms an insoluble product, which may be separated 
 from the soluble lime dextrosate, washed, and decom- 
 posed by oxalic acid. 
 
 Lsevulose is generally described as a syrup, but on 
 treating this with absolute alcohol the lsevulose sepa- 
 rates after some time as a mass of fine shining needles, 
 which melt at 95° and undergo partial decomposition 
 at 100° C. 
 
 With phenyl-hydrazine in excess, lsevulose reacts 
 to form an osazone said, to be identical with that 
 yielded by dextrose under similar treatment. 
 
 In its other chemical reactions lsevulose presents 
 the closest resemblance to dextrose, but differs from 
 it in the characters of its lime compound as already 
 mentioned, and in being readily affected by treatment 
 
DISTINCTION OF DEXTROSE FROM LSEVULOSE. 27 
 
 with dilute acids, or even by boiling its aqueous 
 solution. Formic, propionic, and other acids are 
 formed by boiling lsevulose with, dilute hydrochloric 
 or sulphuric acid. 
 
 Lsevulose also differs from dextrose in the product 
 of the action of bromine. When a solution of dextrose 
 is heated with bromine-water, and the liquid then 
 treated with silver oxide (care being taken to avoid 
 an excess of the latter), gluconic acid, HC 6 H n 7 , 
 is formed, and may be obtained as a syrup on evapora- 
 tion. If slaked lime be added in excess to its luke- 
 warm solution, and the liquid filtered and heated to 
 boiling, the acid is almost completely precipitated as 
 a basic calcium gluconate. When lsevulose is similarly 
 treated with bromine-water and oxide of silver, it yields 
 glycollic acid, HC 2 H 3 3 , the calcium salt of 
 which crystallises in silky needles, which require 
 about 90 parts of cold water or 18 of boiling water 
 for solution. 
 
 The most characteristic distinction between dextrose 
 and lsevulose is their action on polarised light. Thus, 
 while dextrose, as its name denotes, exerts a dextro- 
 rotatory action, lsevulose is still more strongly lsevo- 
 rotatory ; and while the optical activity of dextrose is 
 unaffected by temperature the activity of lsevulose 
 rapidly diminishes as the temperature increases. Thus 
 at about 87° C. its specific rotation for the D ray is 
 — 527°, being exactly the same as that of dextrose, but 
 in the opposite direction. At 20° C, Ho nig and 
 J e s s e r give the specific rotation as — 90*72° for a 10 
 per cent, solution. Jungfleisch and Grimbert 
 (Compt. rend., cvii. 390) give the following formula 
 for the specific rotation of solutions of lsevulose for 
 temperatures (t) between 0° and 40° C. and con- 
 centrations (jp) less than 40 per cent. : — 
 
 [ct] D = -101-38 -0-56Z + 0108 (p-10). 
 
28 URINARY L./EVULOSE. 
 
 The rotation decreases by 0-56 degrees for every rise 
 of 1° C. in temperature. In a later paper (Compt. 
 rend., cviii. 144) they find that the rotation of 
 hevulose prepared from inulin is - 90-02°, while that 
 obtained from invert-sugar exhibits a rotation of 
 - 95-74°; both at a temperature of 20° C. This they 
 attribute to the influence of the mineral acid used 
 for inverting the cane-sugar, the same difference 
 not being observed when acetic acid was substituted, 
 but becoming at once apparent on adding hydrochloric 
 acid. Ost (Berichte, xxiv. 1636) has to some extent 
 confirmed the foregoing observations, and gives the 
 following formula for the specific rotation of pure 
 lsevulose at 20° C. p is the weight in grammes of 
 lsevulose in 100 grammes of the solution: — 
 
 [a] D 20 °= - (91-90° + 0'1 lip). 
 
 Lsevulose is stated by some observers to occur at 
 times in the urine of diabetic persons. Its presence 
 has been commonly inferred from discordance between 
 the estimations of glucose by the polarimetric and 
 oxidation methods. But as lsevulose does not appear 
 ever to have been actually isolated from urine, the 
 evidence of its presence is at best inconclusive. On 
 a priori grounds, the existence of lsevulose in urine 
 under ordinary conditions is improbable, as it is much 
 more oxidisable than dextrose. This fact is so well- 
 established that the use of lsevulose is actually per- 
 mitted to diabetic patients. 1 According to J. B. 
 Haycraft, patients suffering from chronic diabetes 
 can oxidise 50 grammes ( = If oz.) of lsevulose 
 per diem. In some acute cases, a portion of the 
 lsevulose is burnt off, a second converted into and 
 
 1 Lsevulose is now an article of commerce, and can be obtained at a price of 
 3s. 6d. per lb. from Messrs A. & M. Zimmermann, 6 and 7 Cross Lane, 
 London, E.C. 
 
MILK SUGAR. 29 
 
 excreted as dextrose, while a third passes into the 
 urine unchanged. The same result may be expected 
 from the ingestion of cane-sugar. In rabbits, lsevulose 
 undergoes conversion into glycogen, which accumulates 
 in the liver. 
 
 H. Leo (Chem. Centr., 1887, page 193) isolated 
 from diabetic urine a reducing substance which, after 
 being purified from the co-occurring dextrose by 
 repeated treatment with a solution of barium hy- 
 droxide in methyl alcohol, was obtained as a bright 
 yellow syrup. It was Isevo-rotatory (a D = —36 '07°), 
 was not fermentable with yeast even after boiling 
 with hydrochloric acid, and had a cupric oxide reduc- 
 ing power much less than that of dextrose. The body 
 was a carbohydrate of the formula C 6 H 12 6 . 
 
 Milk-sugar or Lactose, CJEL^OhjILO, has been 
 found in the urine of nursing women by Biot, 
 Kaltenbach, Sinetz, and others. It was isolated by 
 Hofm'&i. ster (Zeit. physiol. 
 Chem., i. 101) by precipitating the 
 liquid with neutral lead acetate, 
 treating the filtrate with ammonia, (v/^ <^ 
 and precipitating the refiltered /^ ^> ^ 
 liquid once again with lead acetate ^ n^^\ 
 and ammonia. These two last pre- xfx^^ 
 cipitates were decomposed by sul- \1. 
 
 phuretted hydrogen, the filtrate Kg. i.-Milk-Sugae. 
 shaken with oxide of silver, and the filtered liquid 
 freed from silver by sulphuretted hydrogen. Barium 
 carbonate was next added, and the liquid evaporated. 
 On treating the residue with alcohol, the milk-sugar 
 was dissolved, and obtained in a crystalline form by 
 evaporating the solution in vacuo over sulphuric acid. 
 (See fig. 1.) Kaltenbach obtained mucic acid 
 and galactose from the body thus isolated, thereby 
 conclusively identifying it with milk-sugar. 
 
30 GALACTOSE. 
 
 Milk-sugar yields with the phenyl-hydrazine test 
 a crystalline phenyl-lactosazone, somewhat 
 resembling the analogous compound given by dextrose ; 
 but the crystals are much broader than those of 
 phenyl-glucosazone, melt at 200° C. instead of at 
 204°, and dissolve more readily in alcohol. 
 
 Milk-sugar reduces alkaline solutions of copper, and 
 has a specific rotation of +55*8° for the sodium ray. 
 
 When boiled with dilute mineral acids (citric acid 
 has no action) milk-sugar is split up into two glucoses, 
 dextrose and galactose. The latter has been identified 
 with the sugar obtained by Thudichum by the action 
 of dilute sulphuric acid on certain constituents of the 
 brain, and called by him cerebrose. 
 
 Eesearches by F. W. P a v y {Physiology of the Car- 
 bohydrates, 1894) also appear to show that a reducing 
 sugar analogous to, if not identical with, galactose is 
 produced by the direct treatment of proteid matters 
 with dilute sulphuric acid. After this treatment the 
 product is nearly neutralised with baryta, and the 
 neutralisation completed with barium carbonate. The 
 filtered liquid is concentrated, and heated on the 
 water-bath for at least an hour with acetic acid and 
 phenyl-hydrazine (or phenyl-hydrazine hydrochloride 
 and sodium acetate). Crystals of an osazone occa- 
 sionally make their appearance in the hot liquid, but 
 more frequently are deposited during and after cool- 
 ing. From the crystalline osazone thus obtained the 
 sugar itself is susceptible of recovery by the process 
 described by Fischer. The "sugar" thus obtained 
 gives a clear and definite reaction with Fehling's solu- 
 tion, attended with distinct deposition of red cuprous 
 oxide ; and it yields a crystalline osazone melting at 
 189° to 190° C. 1 It is, however, optically inactive, 
 and unfermentable with yeast. 
 
 1 Galactosazone melts at 193° to 195° C. 
 
DEXTRIN. ANIMAL GUM. 31 
 
 Maltose, C 12 H 22 O n , is said to have been found in 
 diabetic urine by Le Nobel. It is dextro-rotatory 
 (a D = +138 '9°), reduces alkaline solutions of copper, 
 &c, and yields with phenyl- hydrazine a compound 
 which crystallises in yellow tables melting at 82° C. 
 
 Sucrose or Cane-sugar, C 12 H 22 0h, is alleged to 
 be liable to occur in the urine of persons who con- 
 sume large quantities of sugar. It is dextro-rotatory 
 (a D = +66 "5°), does not reduce alkaline cupric solu- 
 tions, and forms no compound with phenyl-hydrazine. 
 Dextrin, C 6 H 10 O fi . In some cases of diabetes in 
 which the sugar gradually disappeared, Reichard 
 (Pharm. Zeitschrift fur Russland, xiv. 45) found 
 Fehling's solution to become gradually green, then 
 yellow, and sometimes finally dark brown. He 
 suspected this to be due to the presence of dextrin, 
 w 7 hich he succeeded in isolating by the following 
 process. The urine was evaporated to a syrup, 
 absolute alcohol and caustic potash added, the pre- 
 cipitate allowed to settle, and the liquid decanted. 
 The precipitate was washed with absolute alcohol, and 
 then dissolved in acetic acid. On repeated addition 
 of absolute alcohol to this solution the dextrin was 
 deposited as a white substance, which after drying 
 was tasteless, gave the dextrin reaction with Fehling's 
 test, a brown coloration with iodine solution, and 
 was readily converted into glucose by boiling with 
 dilute sulphuric acid. 
 
 Animal gum is a carbohydrate obtained by 
 Landwehr by the action of hydrochloric acid on 
 mucin, and said to occur in small quantities in urine. 
 It forms an opalescent solution in water, the resultant 
 liquid giving a sticky precipitate with ferric chloride 
 and cupric sulphate solutions. It does not reduce 
 alkaline cupric solutions until after boiling with dilute 
 sulphuric acid. Landwehr's body yields oxalic acid 
 
32 CHARACTERS OF GLYCOGEN. 
 
 on oxidation with nitric acid, and lsevulic acid by- 
 treatment with hydrochloric acid. It does not ferment 
 with yeast, is precipitated by alcohol, and gives no 
 coloration with iodine. 
 
 Glycogen, (C 6 H 10 O 5 ) n , has been termed " animal 
 starch," and presents the closest analogy to soluble 
 starch. It was first found in the liver, but has 
 more recently been met with in many other parts of 
 the body. 
 
 Pure glycogen is a white, amorphous powder, 
 readily soluble in water to form a solution which is 
 usually, but not invariably, opalescent, and becomes 
 more limpid on adding acetic acid or an alkali. 
 Glycogen is precipitated from its aqueous solution, by 
 alcohol whenever the alcohol amounts to 60 per cent, 
 of the liquid. If the solution be quite free from salts, 
 the separation is sometimes very difficult, but takes 
 place instantly on adding a minute quantity of com- 
 mon salt. The precipitation of glycogen in liquids 
 containing 60 per cent, of alcohol distinguishes it 
 from the different varieties of dextrin, none of which 
 are precipitated by alcohol of less than 85 per cent, 
 strength. Glycogen exactly simulates dextrin in its 
 behaviour with a solution of iodine, which produces a 
 port-wine colour, disappearing on heating, and return- 
 ing as the liquid cools. 
 
 Glycogen is strongly dextro-rotatory, the value of 
 [a] D varying from +203° to +23.4°, according to the 
 concentration of the solution. 
 
 Glycogen does not reduce Fehling's solution. It is 
 precipitated by baryta-water as BaO(C 6 H 10 O 6 ) 3 , and 
 by basic lead acetate as PbO(C 6 H 10 O 6 ) 2 . 
 
 When boiled with dilute nitric acid, glycogen yields 
 oxalic acid. ^ Boiled with dilute sulphuric or hydro- 
 chloric acid, it is converted into dextrose. It does not 
 ferment with yeast, but diastase and saliva convert it 
 
PREPARATION OF GLYCOGEN. 33 
 
 into maltose and achroo-dextrin, a little dextrose being 
 also formed. On the other hand, in the hydrolysis of 
 glycogen in the liver, dextrose and not maltose is the 
 chief product. 
 
 Glycogen is best prepared by rapidly cutting up the 
 liver of an animal killed immediately previously, and 
 throwing the fragments into five times their weight of 
 boiling water. After boiling for a short time, the 
 fragments of liver are mixed with sand and reduced 
 to powder in a mortar, and then returned to the 
 water, which is again boiled. The liquid is strained, 
 and faintly acidified with acetic acid while still hot. 
 The filtrate from the coagulated proteids is rapidly 
 cooled, and the remaining proteids precipitated by the 
 alternate addition of hydrochloric acid and potassio- 
 mercuric iodide. The filtered liquid is mixed with 
 such a volume of strong spirit as to make it contain 
 60 per cent, of absolute alcohol, when the precipitated 
 glycogen is filtered off, washed first with 60 per cent, 
 spirit, and then with absolute alcohol and ether. 1 
 
 The researches of Claude Bernard first estab- 
 lished the fact of the formation of a substance in the 
 liver which, from the circumstance of its ready con- 
 version into glucose, he termed " glycogen." Bernard 
 
 1 Brautigara and Edelmann (Chem. Gentr., 1894, i. 485; Jour. 
 Chem. Soc, Ixvi. ii. 336) have proposed to distinguish horse-flesh from the 
 flesh of other animals by a reaction based on its high content of glycogen. 
 Ten per cent, of horse-flesh or liver can be detected by this means, the propor- 
 tion present ranging from - 37 to 1*07 per cent., while the flesh of other 
 animals used for food contains little or none — ox-flesh coming next with "20 
 per cent. The finely-divided flesh is boiled with four times its weight of 
 water, and the resulting broth treated with dilute nitric acid to precipitate 
 albumin, and filtered. Saturated hydriodic acid is now added, so the two 
 liquids may remain as distinct layers, when a red or violet zone will be seen 
 at the junction of the two strata if glycogen be present. The reaction is said 
 to be quite characteristic of horse-flesh. In the event of the extraction of 
 the glycogen with water proving inadequate, a solution of caustic potash 
 containing KOH equal to 3 per cent, of the weight of the flesh must be sub- 
 stituted. Humbert substitutes a saturated solution of iodine in boiling 
 water for the hydriodic acid {Jour. Pharm. et Chim., 1895, 195 ; Analyst, 
 xx. 95). 
 
 C 
 
34 FOBMATION OF GLYCOGEN. 
 
 affirmed, as the result of his experiments, that glyco- 
 gen is normally, and as a physiological action of the 
 liver regularly, transformed into glucose; that in health, 
 this glucose, after being carried into the circulation by 
 the hepatic veins, is destroyed by oxidation ; but that 
 in certain abnormal conditions this natural trans- 
 formation is arrested. These views of Bernard have 
 been strongly combated by P a v y and others, accord- 
 ing to whom sugar is not found in the liver when 
 examined instantly after death, and is not present in 
 any quantity in the blood of the right side of the 
 heart, as it would be if sugar were constantly being 
 formed in the liver and conveyed away from that 
 organ by the hepatic vein. 1 
 
 Researches by Voit, Otto, Abbott, Lusk, 
 and others render it probable that the formation of 
 glycogen in the liver may arise in two ways : as a 
 temporary store of carbohydrate food, and as the 
 result of proteid metabolism. Doses of dextrose, 
 lsevulose, cane-sugar, and maltose increased the hepatic 
 glycogen, while lactose and galactose did not, or only 
 very slightly, but both these sugars increased the 
 proportion of sugar eliminated in diabetes. In the 
 alimentary canal, cane-sugar and maltose undergo in- 
 version. Lsevulose, lactose, and galactose are absorbed 
 as such, and after large doses pass unchanged into the 
 urine. The liver appears unable to form glycogen 
 from sugars which, like lactose and galactose, do not 
 pass into the condition of either dextrose or lsevulose. 
 Dextrose and lsevulose appear to be the only kinds of 
 sugar which, when present in the blood, lead to the 
 storage of glycogen in the liver, and the liver-cells 
 
 1 The blood in the right side of the heart consists of the venous blood re- 
 turned from the whole body, plios that returned from the liver by the hepatic 
 vein. The amount of sugar in the blood of the hepatic vein is the datum 
 requisite for completing the argument. 
 
INOSITE. 35 
 
 either convert lsevulose into glycogen direct, or after 
 previously changing it to dextrose. 
 
 If carbohydrates be introduced into the stomach or 
 blood of rabbits, the liver of which has been rendered 
 quite free from glycogen by six days' starvation, a. 
 large amount of glycogen will be found in the liver 
 after a few hours. 
 
 It is probable that the glycogen stored in the liver 
 and the muscles is not derived exclusively from the 
 carbohydrates of the food. It appears that the 
 albuminous and gelatinous constituents of the food 
 are also concerned in its formation.. Thus animals 
 which have been fed wholly on lean meat for a con- 
 siderable period exhibit large stores of glycogen in 
 their liver and muscles. In the severer forms of 
 diabetes, under a protracted and exclusive flesh diet, 
 the , secretion of sugar does not cease, and even in- 
 creases in proportion to the amount of albumin 
 consumed. 
 
 Von Mering found that when phloridzin, a 
 glucoside contained in the root-bark of cherry and 
 apple trees, was administered to a dog, sugar appeared 
 in the urine after a few hours. This glycosuria ceased 
 in a few days, and the liver and muscles are then 
 wholly free from glycogen. On again administering 
 phloridzin, a still larger amount of sugar was excreted. 
 Unless the sugar was produced from proteids, it must 
 be assumed to have been formed from fat, for which 
 assumption there is no foundation. 
 
 Inosite, C 6 H 12 6 , sometimes improperly called 
 "muscle-sugar," has been met with in healthy urine 
 after the use of diuretics or a large excess of water. 
 Dahnhardt isolated O'l gramme of inosite from 8 
 litres of ox urine. 
 
 Inosite was found byG-alloisin the urine of five 
 diabetic persons out of a total of thirty examined. 
 
36 CHARACTERS OF INOSITE. 
 
 Five of these samples contained sugar in various pro- 
 portions in addition to inosite. Inosite has also been 
 found in the urine in cases of albuminuria, syphilis, 
 phthisis, and typhus fever. 
 
 For the detection of inosite, several litres of the 
 urine should be treated with excess of neutral lead 
 acetate, filtered, and the filtrate warmed and treated 
 with basic lead acetate till precipitation is complete. 
 The liquid is filtered after standing forty-eight hours, 
 and the washed precipitate suspended in water and 
 decomposed with sulphuretted hydrogen. The filtrate 
 deposits uric acid on standing, when it is again 
 filtered, evaporated to a syrup at 100°, and treated 
 with absolute alcohol. The precipitate is dissolved 
 in hot water, and three or four volumes of rectified 
 spirit added. Ether is then gradually added until a 
 permanent turbidity is produced, when the inosite 
 crystallises on standing. 
 
 Pure inosite crystallises in rhombic prisms sometimes 
 grouped in rosettes, or, from its solution in hot rectified 
 spirit, in shining scales, or in tables which, under the 
 microscope, somewhat resemble cholesterin (fig. 2). The 
 < ^ crystals easily effloresce, 
 
 and sometimes turn red. 
 When impure, inosite is 
 often obtained in mam- 
 millated forms. It dis- 
 solves in 16 parts of cold 
 water, and is soluble in 
 ^\^ rectified spirit, especially 
 ^^, when hot, but is insoluble 
 
 (ft in absolute alcohol or 
 
 ether. Inosite melts at 
 . Fig ' 2 -_- lN0SI ™- 210° C, has a sweet taste, 
 
 is optically inactive, and does not ferment with yeast, 
 but by treatment with putrid albumen it is said to 
 
REACTIONS OP INOSITE. 37 
 
 yield sarcolactic acid. Inosite does not reduce Fehling's 
 solution. It is not precipitated by neutral lead acetate, 
 but is precipitated by the basic acetate, especially 
 on heating, as the compound C 6 H ]2 6 ,2PbO. It 
 yields no osazone with phenyl-hydrazine. 1 
 
 According to G alio is, if an aqueous solution of 
 inosite be treated in porcelain with a drop of solution 
 of mercuric nitrate a yellowish precipitate will form, 
 which, if spread out on the edge of the dish quickly 
 and heated carefully, becomes dark red. On cooling, 
 the colour disappears, but is reproduced on again 
 heating. The reaction is not produced by glycogen, 
 starch, mannite, milk-sugar, urea, uric acid, glycocine, 
 taurin, or cystin. Albumin assumes a rose colour, 
 and sugar becomes black under similar treatment. 
 
 According to Scherer (Ann. Chem. und Pharm., 
 1852, page 375), if a solution of inosite be evaporated 
 nearly to dryness at 100° with a few drops of nitric 
 acid, and the nearly dry product moistened with 
 ammonia and calcium chloride solution, a bright pink 
 or rose-red coloration is obtained on completing the 
 evaporation. 
 
 Glycuronic Acid. C 6 H 10 O 7 ; or COH(CH.OH) 4 .COOH. 
 Glycuronic acid doubtless has its origin in the 
 dextrose of the body, to which compound it is 
 closely related. 2 It was first obtained in the conju- 
 gated form ofcampho-glycuronic acid in the 
 
 1 Although having the empirical formula of a glocuse, inosite is not a true 
 carbohydrate. It really belongs to the aromatic series, and may be represented 
 
 2 The relation between glycuronic acid and bodies of the sugar group is 
 shown by the following constitutional formula; : — 
 
 Dextrose, .... CH 2 (0H).(CH.0H) 4 .C0.H 
 
 Gluconic acid, . 
 Saccharic acid, 
 Glycuronic acid, 
 Gulonic acid, 
 
 CH,(OH). (CH. OH) 4 . CO. OH 
 CO(OH). (CH. OH) 4 . CO. OH 
 CO(OH).(CH.OH) 4 .CO.H 
 CO(OH). (CH. 0H) 4 . CH 2 . OH 
 
 Gulose, CO(H).(CH.OH) 4 .CH 2 .OH 
 
38 PURREE OR INDIAN YELLOW. 
 
 urine of dogs to which camphor had been administered, 
 and subsequently as uro-chloralic acid after 
 the administration of chloral. It is remarkable for its 
 tendency to form ethereal or glucosidal compounds 
 when appropriate substances are introduced into the 
 body. Traces, of such compounds probably occur nor- 
 mally in urine, especially i n d o x y 1- and skatoxyl- 
 glycuronic acids; in addition to the combination 
 with urea, having probably the constitution of u r o- 
 glycuronicacid, which appears to be the ordinary 
 form in which glycuronic acid exists in urine. 
 
 Baeyer {Annalen, civ. 257) has shown that 
 euxanthic acid, which exists in combination with 
 magnesia in the " purrde " or " Indian yellow " of 
 commerce, 1 is decomposed on boiling with hydro- 
 
 1 Piuri or Purrie, now used as a pigment under the name of " Indian 
 yellow," is obtained in Bengal from the urine of cows which are fed exclusively 
 on the leaves of the mango tree and water. The urine is heated, and the pre- 
 cipitate separated and dried. Analyses of very pure specimens of purree by 
 C. G r a e b e (Annalen, ccliv. 265) showed : euxanthic acid, 51 ; silica and 
 alumina, l - 5 ; magnesia, 4'2 ; lime, 3 - 4 ; and water and volatile substances, 
 39 per cent. The analyses of Stenhouse and Erdmann show much less lime. 
 Urea, uric acid, and hippuric acid have also been found in purree. The poorer 
 qualities contain considerable quantities of euxanthone, partly free and 
 partly in combination . For the isolation of the euxanthic acid and euxanthone, 
 and the assay of purree, the colouring matter should be triturated with dilute ' 
 hydrochloric acid until the whole has assumed the bright yellow colour of 
 euxanthic acid. The residue is then well washed with cold water to remove 
 the salts, and the euxanthic acid extracted from the residue by ammonium 
 carbonate solution. It is precipitated from the nitrate by hydrochloric acid, 
 and purified by crystallisation from alcohol. The euxanthone, left undissolved 
 by the ammonium carbonate, is treated with caustic soda, the solution preci- 
 pitated with an acid, and the precipitated euxanthone shaken out with ether 
 or filtered off and dried at 100°. 
 
 Euxanthic acid has the constitution : — ■ 
 
 0H.C 6 H 3 | c ° }c 6 H 3 .0.CH(0H).(CH.0H) 4 .C00H. 
 
 It forms pale yellow needles, which melt at 156-158°. It has a sweet taste . 
 and bitter after-taste, is but slightly soluble in cold water, very sparingly in 
 ether, but readily in boiling alcohol. Alkalies colour the solution deep yellow. 
 Euxanthic acid does not reduce Fehling's solution, nor form a compound with 
 phenyl-hydrazine. 
 
 Euxanthone is a neutral substance, crystallising in pale yellow needles 
 soluble in alkalies but not in dilute acids. It forms no compound with 
 phenyl-hydrazine. 
 
GLYCURONIC ACID. 39 
 
 chloric acid or dilute sulphuric acid, with, formation of 
 euxanthone and an acid which has been shown by 
 Spiegel {Ber., xv. 1965) to be identical with gly- 
 curonic acid, C 19 H 18 O n = C 13 H 8 4 + C 6 H 10 O 7 . In 
 fact purree is the best material for the preparation 
 of glycuronic acid, which can be obtained on the small 
 scale by the following process :-■ — The artists' water- 
 colour known as " Indian yellow " is ground up with 
 sand, and then treated with dilute hydrochloric acid, 
 which dissolves out calcium and magnesium salts, &c. 
 The residue is washed with water and treated with a 
 solution of ammonium carbonate, which dissolves the 
 euxanthic acid, leaving euxanthone and sand undis- 
 solved. From the filtered liquid the euxanthic acid 
 is precipitated by dilute hydrochloric acid, washed 
 with cold water, and then heated with water in a 
 closed soda-water bottle to 125° C. for three or four 
 hours. The requisite temperature can be conveniently 
 obtained by immersing the bottle in a bath of molten 
 paraffin wax (candles). From the cooled product the 
 euxanthone is dissolved by agitation with ether, and 
 the glycuronic anhydride crystallised from the concen- 
 trated aqueous liquid. 
 
 Glycuronic acid is a syrupy liquid, miscible with water 
 or alcohol. When the aqueous solution is boiled, eva- 
 porated, or even allowed to stand at the ordinary 
 temperature, the acid loses the elements of water and 
 yields the anhydride or lactone. 
 
 Glycuronic Anhydride, C 6 H 8 6 , forms monoclinic 
 tables or needles, having a sweet taste, and melting 
 at about 160° when heat is gradually applied, or at 
 170-180° when heated rapidly. The anhydride is in- 
 soluble in alcohol, but dissolves readily in water to form 
 a dextro-rotatory solution. [<x D ] = 19'25°. The solu- 
 tion prevents the precipitation of cupric solutions by 
 alkalies, and powerfully reduces hot Fehling's solution, 
 
40 GLYCURONIC ACID. 
 
 the cupric oxide reducing power being 98*8, compared 
 with glucose as 100. On distillation with hydro- 
 chloric acid, glycuronic anhydride (as also uro-chloralic 
 acid) yields furfuraldehyde, a trace of which substance 
 is also obtainable on similarly treating normal urine. 
 
 Glycuronic acid itself is dextro-rotatory ([a] D = +35°), 
 but many of its compounds are lsevo-rotatory. 1 It 
 reduces Fehling's solution on heating, and precipitates 
 the metals from hot alkaline solutions of silver, mercury, 
 and bismuth. 
 
 On oxidation, glycuronic acid yields camphoric and 
 formic acids. By treatment with bromine it yields 
 saccharic acid, C 6 H 10 O 8 , a reaction which in- 
 dicates the presence of an aldehyde group and the 
 close relation between glycuronic acid and dextrose. 
 Saccharic acid can again be reduced to glycuronic acid 
 by treatment with sodium amalgam, further treatment 
 yielding gulonic acid, C 6 H 12 7 , a body which 
 does not reduce Fehling's solution. (Fischer and 
 P i 1 o t y, Ber., xxiv. 521 ; Jour. Chem. Soc, lx. 667.) 
 
 "When boiled with caustic alkali, glycuronic acid 
 yields oxalic acid as an invariable product. Catechol 
 and protocatechuic acid are also formed if concentrated 
 alkali be employed for the treatment. 
 
 Glycuronic acid is distinguished from glucose by not 
 undergoing the alcoholic fermentation when treated 
 with yeast. On the other hand, when fermented in 
 presence of cheese and chalk it yields lactic and acetic 
 acids. 
 
 Glycuronic acid forms a potassium salt which 
 crystallises in needles. The sodium salt is similar. 
 The zinc, cadmium, copper, silver, and calcium salts 
 are uncrystallisable. The barium salt is amorphous 
 
 1 After taking chloral hydrate the urine contains trichlorethyl-glycuronic 
 acid ("uro-chloralic acid"), a lsevo-rotatory body which is decomposed into 
 trichlorethyl alcohol and dextro-rotatory glycuronic acid. 
 
GLYCOSURIC ACID. 41 
 
 and soluble in water. It is the compound employed 
 for the isolation of glycuronic acid from urine. 
 
 With phenyl-hydrazine, glycuronic acid forms a 
 yellow crystalline compound melting at 114° to 
 115° C, but under modified conditions an amorphous, 
 brownish-yellow body, melting at 150° C, is produced. 
 According to J, A. H i r s c h 1, normal urine yields this 
 compound (see page 88). 
 
 For the actual isolation of glycuronic acid from 
 urine a large quantity of the excretion is required. 
 The method is described in an interesting report by 
 H. H. Ashdown (Brit. Med. Jour., 1890, i. 169, 
 and Pharm. Jour., [3], xx. 607). 
 
 Glycuronic acid occurs in the urine to a very not- 
 able extent after the administration of morphine, 
 chloroform, chloral, butyl-chloral, nitrobenzene, cam- 
 phor, curare, and certain other drugs. It was un- 
 doubtedly mistaken for glucose by the older observers. 
 In one case recorded by Ashdown large amounts of 
 glycuronic acid occurred in the urine of a healthy 
 young man, whose excretion was not abnormal either 
 in volume or density. 
 
 Glycosuric Acid is a body extracted by J. 
 Marshall from pathological urine (Arc h. Pharm., 
 [3], xxv. 593, and Jour. Chem. Soc, lii. 1047). It 
 is isolated by a process based on the insolubility of the 
 lead salt in alcohol of 45 per cent. Glycosuric acid 
 crystallises in opaque tetragonal prisms, which melt at 
 140°, are readily soluble in water, alcohol, and ether, 
 less readily in chloroform, and insoluble in benzene, 
 toluene, and petroleum spirit. Glycosuric acid con- 
 tains no nitrogen, is readily and completely absorbed 
 by animal charcoal, and appears to be a phenolic de- 
 rivative. It reduces Fehling's solution more powerfully 
 than glucose, and also reduces silver nitrate ; but not 
 bismuth compounds or alkaline solutions of picric acid. 
 
42 URINARY SUGAR. 
 
 It gives a brown coloration when boiled with aqueous 
 soda. A dilute and neutral solution of ferric chloride 
 gives an evanescent blue colour. On evaporating the 
 ethereal solution of glycosuric acid at 60° the liquid 
 takes a wine-red colour, which is imparted to the 
 crystals which separate, but these form a colourless 
 solution in water. 
 
 A. Geyger (Pharm. Zeit, 1892, page 1488) ex- 
 tracted glycosuric acid from a diabetic urine by 
 acidulating it with sulphuric acid and agitating with 
 ether. The ether left on evaporation a crystalline sub- 
 stance, melting at 143° C, which proved to be the acid 
 in question. He suggests that diabetic urine should 
 always be examined in this manner. 
 
 DETECTION AND DETERMINATION OF 
 SUGAR IN URINE. 
 
 A great variety of methods have been proposed for 
 the recognition and estimation of sugar in urine. 
 Many derive the only value they possess from being 
 simple in application, and therefore not beyond the 
 manipulative skill and appliances of the general medi- 
 cal practitioner. Not a few are utterly worthless and 
 misleading in their indications, while others fail to 
 show the presence of proportions of sugar (e.g. , below 
 ■§■ per cent.) which are not without grave pathological 
 significance, and of the greatest importance as a warn- 
 ing to the patient. 
 
 With the exception of a few colour-reactions of little 
 value, the processes for detecting and determining 
 sugar in urine may be arranged in the following 
 classes : — 
 
 1. Isolation of the dextrose as such. 
 
 2. Methods based on the fermentation of the 
 glucose. 
 
ISOLATION OF URINARY SUGAR. 43 
 
 3. Methods based on the optical activity of dextrose 
 
 4. Methods based on the reducing action of glucose. 
 These include the various modifications of the copper, 
 mercury, and bismuth tests, and the colour-changes 
 produced by glucose in hot alkaline solutions of 
 various coal-tar colours. 
 
 5. Methods dependent on the conversion of the 
 sugar into definite crystalline compounds, such as 
 those yielded with benzoyl chloride and phenyl- 
 hydrazine. 
 
 Isolation of Sugar from the Urine. 
 
 When typical diabetic urine, containing from 5 to 
 1 per cent, of glucose, is in question, it is not difficult 
 to isolate the dextrose in a pure state. On evaporat- 
 ing such urine on the water-bath to the consistence of 
 a syrup, and then leaving it at rest for some days, 
 dextrose will frequently separate in tabular crystals 
 or irregular warty masses. More frequently it is 
 deposited in crystals of a compound of dextrose with 
 sodium chloride containing 2C 6 H 12 6 ,NaCl+H 2 0, 
 which is less soluble in alcohol but more soluble in 
 water than glucose itself. Not unfrequently the 
 syrup may remain for months without any crystals 
 appearing. Sometimes treatment of the syrup with 
 ether, which is subsequently allowed to evaporate 
 spontaneously, facilitates the formation of crystals. 
 The syrup, with any crystals contained in it, is 
 treated with cold absolute alcohol, which dissolves 
 out urea and extractive matter, but leaves most of the 
 glucose. On boiling the residue with rectified spirit 
 the sugar is dissolved, sulphates, phosphates, urates, 
 &c, remaining insoluble. The liquid is filtered hot 
 and evaporated to a small bulk, when it will deposit 
 crystals of glucose on cooling ; these may be purified 
 by recrystallisation from boiling rectified spirit. 
 
44 ISOLATION OF URINARY SUGAR. 
 
 For the isolation of the minute quantity of sugar 
 existing in non-diabetic urine, F. W. Pavy employs 
 the following adaptation of a process devised by 
 Briicke : — A large quantity of the urine is treated 
 with half its measure of a 10 per cent, solution of 
 neutral lead acetate. The precipitate, which contains 
 urates, sulphates, phosphates, colouring matter, &c, 
 is filtered off, and the filtrate treated with basic lead 
 acetate, any further precipitate being removed as 
 before. The filtered liquid is then treated with 
 ammonia and more lead acetate, unless a distinct 
 excess has been already used. This causes the pre- 
 cipitation of the glucose as a definite compound with 
 lead oxide. The precipitate is filtered off and washed 
 with cold water till the greater part of the ammonia 
 is removed, when it is suspended in water and de- 
 composed by a stream of sulphuretted hydrogen. 
 Sulphuric or oxalic acid may be substituted for the 
 last reagent, but the product is more strongly coloured. 
 In any case, the strongly acid liquid should be 
 treated cautiously with sodium carbonate solution till 
 neutral, when colouring matter will separate and may 
 be filtered off. Further decolorisation mav be effected 
 
 ml 
 
 by treating the liquid, rendered slightly acid with 
 acetic acid, with a little animal charcoal (previously 
 carefully purified from phosphates, &c, by boiling 
 with hydrochloric acid and washing till free from acid 
 reaction to litmus). 
 
 From this clarified liquid, dextrose can be obtained 
 in crystals recognisable under the microscope, but for 
 its identification by chemical tests the solution itself 
 is quite suitable. Working on the product from 
 healthy persons, Pavy obtained all the chief reactions 
 of sugar, including the production of carbonic acid 
 gas by fermentation with yeast and the formation 
 of a crystalline compound with phenyl-hydrazine. 
 
FERMENTATION TEST FOR GLUCOSE. 45 
 
 Pavy's results go far to decide in the affirmative the 
 long-vexed question of the existence of traces of 
 sugar in normal human urine (compare, however, 
 page 20). 
 
 Fermentation Test for Sugar. 
 
 The recognition of sugar in urine, by its conversion 
 into alcohol and carbon dioxide when treated with 
 yeast, has the advantage of simplicity and almost 
 absolute certainty that the effect produced is really 
 due to sugar. On the other hand, the experiment 
 extends over a comparatively long time, and the 
 method is deficient in delicacy and accuracy. 
 
 In its simplest form, the fermentation test is ap- 
 plied by introducing a fragment of pressed yeast the 
 size of a nut into a 4-oz. bottle, filling the bottle 
 completely with the urine to be tested, and then 
 inverting the vessel over a small capsule or saucer of 
 the urine, in such a manner as not to allow any air to 
 enter. The whole is then left in a warm place — from 
 25° to 30° C. ( = 77° to 86° F.)— for twelve or fifteen 
 hours, when, if sugar be present in quantity, the liquid 
 will become lighter in colour, and diminish in gravity, 
 while at the same time a notable quantity of carbon 
 dioxide will collect in the upper part of the bottle. 
 The test fails, or answers but imperfectly, when the 
 proportion of sugar is less than 0*5 per cent. By 
 keeping the bottle in an erect position, and allowing 
 the fermentation to continue for twenty-four hours, a 
 good approximation to the content of sugar may be 
 obtained by noting the decrease in the density of the 
 liquid. As pointed out by Sir W. Eoberts, every 
 degree of gravity lost by the fermentation corresponds 
 closely with 1 grain of sugar per fluid ounce of the 
 urine. Another portion of the same urine, untreated 
 with yeast, should be kept side by side with the 
 
46 FERMENTATION TEST FOR GLUCOSE. 
 
 fermentation test, and its density observed under 
 similar conditions to the latter. Thus, if the untreated 
 urine be found to have a gravity of 1042, and the 
 portion treated with yeast have on completion of the 
 fermentation a density of 1015, the urine will have 
 contained 27 grains of sugar per fluid ounce (1042 
 -1015 = 27). Attempts have been made to obtain 
 more accurate results by this process, but the method 
 has too many sources of error to make such refine- 
 ments of practical value. 
 
 Instead of observing the diminution in density, the 
 proportion of sugar may be deduced from the weight 
 or measure of carbon dioxide (carbonic acid) gas evolved 
 Many ways of effecting this have been devised, but 
 one of the simplest and most effective is to introduce 
 a known measure of the urine to be tested into a 
 bottle furnished with a perforated cork, and attached 
 by elastic tubing to the three-way tap of a nitrometer 
 filled with brine (see " Urea"). Yeast is added to the 
 urine, and the arrangement left for twenty-four hours 
 in a warm place, as previously described. The gas 
 which has collected in the nitrometer is then measured 
 with the usual precautions. One c.c. of C0 2 , measured 
 over brine at the ordinary pressure and temperature 
 (about 30 inches of barometric pressure, and at 15° 
 to 20° C, = 59° to 69° F.), represents 0'002 gramme of 
 glucose in the urine taken. 
 
 Instead of using a separate vessel for the urine, 5 c.c. 
 may be introduced, through the tap, into a nitrometer 
 filled with mercury, 5 c.c. of water containing as much 
 yeast as possible in suspension run in through the 
 tap, and the apparatus kept in a warm place until 
 the fermentation is complete. 
 
 Where a nitrometer and the other appliances of a 
 laboratory are not available, a very fair determination 
 of the sugar in diabetic urine may be made by the 
 
FERMENTATION TEST FOR GLUCOSE. 
 
 47 
 
 arrangement shown in fig. 3, constructed from a feed- 
 ing-bottle and ordinary wash-bottle. The glass tube is 
 removed from the inside of the feeding-bottle, and the 
 india-rubber tube fixed air-tight into the stopper by 
 means of sealing-wax. The nipple is removed from 
 the other end of the india-rubber tube, which is then 
 slipped on to the mouth-tube of an ordinary wash-bottle. 
 Three fluid drachms ( = | oz.) of the urine to be tested, 
 or an equivalent measure of the diluted sample, 1 should 
 then be placed in the feeding-bottle, about an ounce 
 of water and a piece of pressed yeast added, and the 
 stopper with its attached tubes firmly adjusted. The 
 wash-bottle is filled with water, or preferably with 
 brine, and its cork (carrying the tubes) firmly fixed 
 in. This will cause the exit tube to fill with water. 
 A suitable measuring vessel, such as a graduated 8-oz. 
 medicine-bottle, is placed below the outlet tube so 
 
 Fig. 3. — Apparatus for estimating diabetic sugar by fermentation. 
 
 as to receive the brine displaced by the gas gener- 
 ated by the fermentation. The whole arrangement is 
 kept at a temperature of from 25° to 30° C. ( = 77° to 
 86° F.) for twenty-four hours, when the volume of 
 
 1 1 oz. of the sample may bo diluted with' 3 oz. of water, and 1J oz. 
 of the diluted liquid ( = § oz. of the original sample) taken for the experi- 
 ment. 
 
48 FERMENTATION TEST FOR GLUCOSE. 
 
 liquid collected in the measuring vessel is observed as 
 accurately as possible. 
 
 In any experiment where it is sought to determine 
 sugar by fermentation, it is of course essential that 
 the yeast employed be free from starch. Much of the 
 pressed yeast met with in commerce contains a con- 
 siderable proportion of starch, which is said to be 
 added as a preservative, but is certainly not essential. 
 The presence of starch may be readily detected by 
 mixing up a little of the yeast with tincture of iodine, 
 which will produce a blue or violet colour if starch be 
 present. 
 
 If f ounce ( = 3 fluid drachms) was the measure of 
 urine used for the experiment, every ounce of fluid 
 displaced will represent 1 per cent, of sugar in the 
 sample; while every 100 grains measure will corre- 
 spond to 1 grain per fluid ounce of the urine. 1 
 
 Fresh brewers' yeast may be substituted for pressed 
 yeast if it be first well washed on a filter with cold water. 
 
 In all the foregoing modifications of the fermenta- 
 tion test it is important that the urine should be 
 moderately but distinctly acid. The safest plan is to 
 render the liquid slightly alkaline to litmus paper, to 
 ensure the neutralisation of any possible mineral acid, 
 and then to acidify the liquid with tartaric acid to the 
 extent of about 0"5 gramme per 100 c.c. (2 grains per 
 fluid ounce). 
 
 1 The main reaction occurring in the fermentation of glucose is expressed 
 by the equation C 6 H 12 6 =2C 2 H 6 + 2C0 2 . Hence 180 parts of sugar yield 92 
 of alcohol and 88 of carbon dioxide. (This statement is not strictly correct, 
 • but sufficiently so for the purpose in question. ) 88 grammes measure 44,760 c.c. 
 at the standard pressure and temperature, or about 48,000 c.c. at 20° C. ( = 68° 
 F. ). Hence this measure of gas will be yielded by 80 grammes of glucose, 800 
 by 3 grammes, or 800 grains measure by 3 grains. If the urine contain 1 grain 
 of glucose per fluid ounce, and f oz. is employed, the liberated gas will 
 measure &§£x§ = 100 grains measure. But if the content of sugar be 1 per 
 cent., or 4 "375 grains per fluid ounce, the gas will measure 437 J grains, or 
 exactly 1 oz. If 15 c.c. of urine be taken, then every 10 c.c. of liquid dis- 
 placed by the gas represents J per cent, of sugar in the sample. 
 
OPTICAL GLUCOSE DETERMINATION. 49 
 
 Polarimetric Determination of Diabetic Sugar. 
 
 The sugar contained in diabetic urine is commonly 
 assumed to be identical with the dextro-glucose pre- 
 sent in grapes and produced, together with hevulose, 
 by the inversion of cane-sugar. It is not quite certain, 
 however, that this assumption is correct, nor that the 
 dextrose is not sometimes associated with lsevulose or 
 some other kind of sugar. The subject requires further 
 investigation by competent observers, who will require 
 to determine the total sugar by some more certain 
 process than those commonly used for the purpose, 
 comparing the results so obtained with those yielded 
 by the optical method. In ordinary cases, however, 
 the polarimeter can be trusted to give excellent com- 
 parative, if not rigidly accurate, results, provided the 
 proportion of sugar is not too small. For the detection 
 or determination of the traces of sugar supposed to be 
 present in healthy urine, or even for the examination 
 of slightly diabetic urine, the optical method is useless, 
 since urine ordinarily contains traces of various sub- 
 stances other than sugar which are more or less 
 optically active. 1 
 
 The proportion of glucose in urine can be deduced 
 from the following formula, in which c is the concen- 
 tration, or grammes of sugar per 100 c.c. of urine 
 
 1 M. Carles {Jour, de Pharrn., 1890, page 108) states that all normal 
 urines have a lsevo-rotation varying from 0"25 to - 80° of the saccharometer, the 
 precise activity varying with the proportion of extractive matter, intensity of 
 colour and odour, amount of urea, and specific gravity. Carles inclines to the 
 opinion that the optical activity of normal urine is due, at least in part, to the 
 creatinine. He describes a urine which contained 33 grammes of urea and 2 - 27 
 of phosphoric acid per litre, and had a specific gravity of 1'025, which ex- 
 hibited a left-handed rotation of 1 "93°. Hence it might have contained 4'35 
 grammes of sugar per litre, and yet have been without dextro-rotation. 
 
 According to Haas, dextrose is the chief, and almost the only constituent 
 of urine producing dextro-rotation, while lsevo-rotation may be due to glyeu- 
 ronic acid, its phenyl and indoxyl derivatives, leucine, tyrosine, albumin 1 , 
 hemialbumoses, peptones, eystin, cholesterin, aspartic acid, pseudo-butyric 
 acid, and lsevulose. In addition may be mentioned the disturbing influence of 
 quinine and other alkaloids. 
 
 D 
 
50 OPTICAL GLUCOSE DETERMINATION. 
 
 a the angular rotation observed in the polarimeter ; 
 and I the length of the tube in decimetres, 
 
 (XxlOO 
 °~ 1x52-7°' 
 
 This equation applies only to instruments in which 
 monochromatic sodium light is employed. With sac- 
 charometers intended for observations of the transition- 
 tint, 57"0° must be substituted for 527°. 
 
 In cases where the optical method may be suitably 
 employed, its application is very simple. 1 Diabetic 
 urine is usually so pale in colour that its rotation may 
 be observed without previous clarification. If the 
 colouring matter of the sample should occasion any 
 difficulty in the observation, simple agitation of 100 
 c.c. of the cold urine with about 3 grammes of neutral 
 acetate of lead, and filtration through paper, will 
 generally produce an almost water- white filtrate. 
 
 Detection and Determination of Diabetic 
 Sugar by its Reducing Action. 
 
 The methods of examining urine for glucose most 
 generally employed are those based on its reducing 
 action, which is powerfully exerted in boiling alkaline 
 solutions. Thus the sugar of diabetic urine, which is 
 chiefly if not entirely dextrose, in hot alkaline solutions 
 effects the following reductions. The oxygen shown 
 in the reactions is not evolved as such, but converts the 
 glucose into gluconic acid and other oxidation-products. 
 
 (a) Cupric oxide, retained in solution by fixed 
 organic matter, ammonia, or cyanides, is reduced to 
 cuprous oxide : — 2CuO = Cu 2 + 0. 
 
 1 The method of using the polarimeter is familiar to those accustomed to 
 employ the instrument, and is described in great detail in various works 
 readily accessible. A Laurent instrument on the half-shadow principle is to 
 be preferred to other forms. A polarimeter of this kind, obtainable at a 
 moderate price, is described in The Analyst, vol. xi. page 141. Another 
 useful instrument is made by Messrs Philip Harris & Co., Birmingham. 
 
REDUCING ACTION OF GLUCOSE. 51 
 
 (b) Bismuihic oxide, in the solid state, or in alkaline 
 citrate solution, is reduced to metallic bismuth: — 
 Bi 2 3 = Bi 2 +0 3 . 
 
 (c) Mercuric oxide, in the form of potassio-mercuric 
 cyanide or iodide, and in presence of caustic alkali, is 
 reduced to metallic mercury : — HgO = Hg+0. 
 
 (d) OrthonitropJienyl-propiolic acid is reduced to 
 indigo-blue :— 2C 9 H 5 NO, = C 16 H 10 N 2 O 2 + 2C0 2 + 2 . 
 Indigo-blue is, by further treatment, reduced to white 
 indigo :— C 16 H 1 oN 2 2 +H 2 = C 16 H 12 N 2 2 +0. 
 
 (e) Ferricyanides, in presence of fixed caustic 
 alkali, are reduced to ferrocyanides : — 2K 3 (C 6 N 6 Fe) + 
 2KHO = 2K 4 (C 6 N 6 Fe) + H 2 + 0. 
 
 (/) Picric acid is reduced to picramic acid : — 
 C 6 H 2 (N0 2 ) 3 .OH+3H 2 = C 6 H 2 (NH 2 ) 3 .OH 9 . 
 
 (g) Methylene-blue is converted into the correspond- 
 ing leuco-compound : — C 16 H 19 N 3 SO = C 16 H 19 N 3 SO. 
 
 (h) Safranine is similarly reduced to the corre- 
 sponding leuco-compound. 
 
 Some of the foregoing decompositions take place at 
 the ordinary temperature, or considerably below the 
 boiling-point, if time be allowed for the reaction. 
 
 The reaction which occurs when a solution of glucose 
 is heated with an oxidising agent in presence of caustic 
 alkali is complex, and has been but imperfectly studied. 
 The principal products are said to be formic, oxalic, 
 glycollic and carbonic acids, but the products of the 
 action of the alkali itself on the sugar have evidently 
 to be taken into account (compare page 24). The 
 practical necessity of employing caustic alkali in con- 
 junction with the oxidising agent is a grave defect of 
 all quantitative tests based on the reducing action of 
 glucose. 
 
 The recognition of glucose in urine, by its reducing 
 action on the foregoing and similar yielders of oxygen, 
 is readily effected when the proportion of reducing 
 
52 REDUCING CONSTITUENTS OF URINE- 
 
 sugar is considerable ; but, when very small quantities 
 are in question, the indications of the reagents are not 
 to be depended on. This is not so much due to any 
 deficiency in the delicacy of the reactions, as a conse- 
 quence of the presence in urine of other bodies which 
 resemble glucose in their power of exercising more 
 or less reducing action on oxidising agents. Under 
 abnormal conditions urine may contain many such 
 reducing bodies, but even normal urine invariably 
 contains traces of some of such bodies, and under 
 special conditions of diet, or under the influence of 
 medicines, these and others may be increased to a 
 very material extent. 
 
 Of the constituents of normal urine which resemble 
 sugar in their reducing action, the most important are 
 uric acid and creatinine ; but hippuric. acid, creatine, 
 and glycuronic acid and its compounds exercise a 
 similar action, though their quantity is usually so 
 insignificant that they may safely be neglected. Very 
 little is known of the specific action of these urinary 
 bodies on the before-mentioned oxidising reagents, 
 and of their quantitative behaviour we know still less. 
 The alkaline solutions of copper are almost the only 
 reagents of which the oxidising power on urinary con- 
 stituents other than glucose is even approximately 
 known, and the published statements stand in much 
 need of verification and revision. It may, however, 
 be taken as an established fact that creatinine, uric 
 acid, and glycuronic acid and its compounds reduce 
 both Fehling's and Pavy's copper solutions. Creatinine 
 also reduces an alkaline solution of picric acid, with 
 production of a blood-red coloration, but uric acid does 
 not reduce picric acid even on boiling. Safranine in 
 alkaline solution is reduced to a leuco-compound by 
 glucose, but not by creatine, creatinine, uric acid, or 
 hippuric acid. 
 
REACTIONS OP URINARY CONSTITUENTS. 
 
 53 
 
 The following table shows the behaviour of various pre- 
 cipitants and alkaline oxidising agents when heated to 
 boiling with the chief urinary constituents. 
 
 Reagent. 
 
 Albumin. 
 
 Glucose. 
 
 Uric Acid. 
 
 Creatinine. 
 
 Xanthine. 
 
 Remarks. 
 
 Neutral lead - 
 
 Ppted. 
 
 Not 
 
 Ppted. 
 
 Not 
 
 Ppted. 
 
 
 acetate. 
 
 
 affected. 
 
 
 affected. 
 
 
 
 Basic lead ace- 
 
 Ppted. 
 
 Do. 
 
 Ppted. 
 
 Do. 
 
 Ppted. 
 
 ... 
 
 tate. 
 
 
 
 
 
 
 
 Cupric acetate 
 
 Ppted. 
 
 Imper- 
 
 Ppted. 
 
 Partially 
 
 Ppted. 
 
 Glucose is 
 
 (acidulated). 
 
 
 fectly 
 reduced. 
 
 
 ppted. 
 
 
 gradually 
 and im- 
 perfectly 
 oxidised. 
 
 Mercuric ace- 
 
 Ppted. 
 
 White 
 
 Ppted. 
 
 Ppted. 
 
 Ppted. 
 
 Creatinine 
 
 tate 
 
 
 ppte. of 
 
 
 
 
 ppted. 
 
 (HgCl 2 + 2NaA). 
 
 
 Hg-A, 
 gradually 
 formed. 
 
 
 
 
 very 
 
 slowly in 
 
 the cold. 
 
 All others, 
 except 
 glucose, 
 
 promptly. 
 
 Fehling's alka- 
 
 Red- 
 
 Immediate 
 
 Reduc- 
 
 Partial 
 
 Reduc- 
 
 ... 
 
 line cupric 
 
 violet 
 
 separation 
 
 tion and 
 
 pptn. and 
 
 tion and 
 
 
 tartrate solu- 
 
 colora- 
 
 of red 
 
 pptn. of 
 
 reduction. 
 
 pptn. as 
 
 
 tion. 
 
 tion. 
 
 Cu 2 0. 
 
 cuprous 
 urate. 
 
 
 cuprous 
 
 Xanthin- 
 
 ate. 
 
 
 Pavy's ammo- 
 
 No 
 
 Colour 
 
 Colour 
 
 Colour 
 
 ... 
 
 
 niacal cupric 
 
 change. 
 
 discharged. 
 
 dis- 
 
 discharged. 
 
 
 
 solution. 
 
 
 
 charged. 
 
 
 
 
 Gerrard's cyano- 
 
 Do. 
 
 Do. 
 
 D 
 
 
 ... 
 
 
 cupric solu- 
 
 
 
 
 
 
 
 tion. 
 
 
 
 
 
 
 
 Nylander's bis- 
 
 Do. 
 
 Pptn. of 
 
 * Pptn.! 
 
 No 
 
 
 
 muth citrate 
 
 
 grey or 
 
 " of Bi. 
 
 change. 
 
 
 
 solution. 
 
 
 black Bi. 
 
 
 
 
 
 Knapp's mer- 
 
 
 Pptn. of 
 
 
 
 
 
 cuiic cyanide 
 
 
 grey Hg. 
 
 
 
 
 
 solution. 
 
 
 
 
 
 
 
 Sacchse's (Ness- 
 
 
 Do. 
 
 
 
 
 
 ler's) mercuric 
 
 
 
 
 
 
 
 iodide solu- 
 
 
 
 
 
 
 
 tion. 
 
 
 
 
 
 
 
 Braun's alkaline 
 
 
 Brown- 
 
 No 
 
 Blood-red 
 
 
 
 picric acid 
 
 
 red colour. 
 
 change. 
 
 coloration. 
 
 
 
 solution. 
 
 
 
 
 
 
 
 Wender's alka- 
 
 
 Colour 
 
 Do. 
 
 Colour 
 
 
 
 line methyl- 
 
 
 discharged. 
 
 
 discharged. 
 
 
 
 ene-blue so- 
 
 
 
 
 
 
 
 lution. 
 
 
 
 
 
 
 
 Crismer's alka- 
 
 
 Do. 
 
 Do. 
 
 No 
 
 
 
 line safraniue 
 
 
 
 
 change. 
 
 
 
 solution. 
 
 
 
 
 
 
 
54 REMOVAL OF INTERFERING SUBSTANCES. 
 
 When very small quantities of sugar are to be 
 sought for, the removal of the interfering constitu- 
 ents of urine is absolutely necessary, and the neglect 
 of this preliminary treatment has given rise to many 
 erroneous observations on the presence of sugar. 
 
 Urine may be conveniently clarified, and freed from 
 albumin (if present), uric acid, phosphates, sulphates, 
 and colouring matters, by precipitation with neutral 
 lead acetate. One-fourth of its measure of a 10 per 
 cent, solution of lead acetate in water is sufficient to 
 throw down all the precipitable matters from almost 
 any urine. The precipitate is bulky, but filters readily, 
 especially if the urine be first heated to boiling and the 
 hot reagent added to the boiling liquid. This mode 
 of operating avoids the risk of loss from bumping 
 which is apt to occur when the liquid containing the 
 dense lead precipitate is heated. 1 Addition of basic 
 lead acetate to the filtrate from the precipitate pro- 
 duced by the neutral salt removes certain bodies which 
 escaped precipitation previously, but there is no 
 material advantage in its use. In either case, the 
 filtrate from the lead treatment is colourless, or nearly 
 so, and is well fitted for optical examination or for 
 testing by the phenyl-hydrazine reaction. 2 
 
 Mercuric chloride is a very perfect precipitant for 
 urinary constituents. When sodium acetate is also 
 
 1 Three per cent, of lead acetate in fine powder may be substituted for the 
 solution prescribed in the text. The powder should be added to the boiling 
 hot urine, which should then be well agitated and filtered. This plan avoids 
 dilution and the consequent correction for the volume of reagent added. 
 
 2 According to some observers, uric acid is incompletely precipitated by 
 treating the urine with neutral lead acetate only, addition of basic acetate 
 being requisite for its complete removal from solution. This is opposed to the 
 author's experience, who has specially examined the filtrate from the pre- 
 cipitate by neutral lead acetate by the murexide test with negative results. It 
 has been fully established that no sugar is carried down from solution by 
 either neutral or basic lead acetate, but if ammonia be subsequently added 
 glucose is completely removed as a compound with lead hydroxide, which can 
 be washed without decomposition. This reaction is employed by Briicke 
 and by P a v y for the isolation of sugar from urine. 
 
REMOVAL OF INTERFERING SUBSTANCES. 55 
 
 added, as recommended by G. Stillingfleet Johnson, 
 mercuric acetate is potentially present, and this is 
 even a more perfect precipitant than the chloride. 
 Johnson recommends that the urine should be treated 
 with 5 per cent, of its volume of a cold saturated 
 solution of sodium acetate, and 25 per cent, of a 
 cold saturated mercuric chloride solution. 1 This treat- 
 ment occasions an immediate voluminous precipi- 
 tate containing albumin (if present), uric acid, xan- 
 thine, phosphates, colouring matters, &c. Creatinine is 
 not precipitated immediately in the cold, but separates 
 completely in the course of a few days, even at the 
 ordinary temperature. If the liquid be boiled for a 
 few minutes the creatinine is completely precipitated, 
 and this behaviour affords by far the best means of 
 separating it from urine. The urea is partially, but 
 not completely, precipitated on boiling. Glucose is 
 gradually oxidised at a boiling heat, but the reaction 
 does not materially affect the detection of traces in 
 the filtrate. The separation of the excess of mercury 
 from the filtrate from the mercurial precipitate presents 
 some difficulty. Sulphuretted hydrogen is not available 
 for the purpose, as a fixed reducing body is stated by 
 Johnson to be formed by its action, which body inter- 
 feres with the subsequent detection of sugar. G. S. 
 Johnson recommends the careful addition of ammonia, 
 with subsequent filtration from the resultant precipi- 
 tate. It is essential that the precipitation of the 
 mercury should be perfect, as any remaining in the 
 liquid will be reduced to metal by the glucose when 
 the liquid is subsequently treated with caustic alkali 
 and boiled, as it necessarily is whether Pavy's am- 
 moniacal cupric solution or picric acid be employed 
 for determining the glucose. The author has found 
 
 1 The proportion of sodium acetate used by Johnson is considerably in 
 excess of that requisite to react with the mercuric chloride. 
 
56 REMOVAL OF INTERFERING SUBSTANCES. 
 
 ammonia uncertain in its action, and prefers to boil 
 the liquid for five minutes or so with zinc-dust. 1 
 This precipitates the mercury perfectly, and the zinc, 
 which passes into solution in its place, does not inter- 
 fere with the subsequent application of the phenyl- 
 hydrazine or picric acid tests, or the determination 
 of the glucose by Pavy's solution. When the latter 
 method is to be used, it is convenient to treat the 
 filtrate from the precipitate produced by zinc with an 
 equal measure of strong ammonia, which will ensure 
 the zinc remaining in solution, and will furnish a con- 
 stant supply of ammonia during the titration. 
 
 As pointed out by the author, in cases where 
 the mere detection of glucose is sufficient, there is 
 no occasion to remove the mercury from the solution. 
 An excess of potassium iodide may be added, which 
 will prevent precipitation on subsequently adding 
 caustic alkali, and if the liquid be then boiled a grey 
 precipitate of mercury will be produced if sugar be 
 present. Unfortunately, this reaction cannot readily 
 be made quantitative. 
 
 Reaction of Glucose with Copper Solutions. 
 
 The reduction of cupric compounds to cuprous by re- 
 action in alkaline solution with glucose is one of the 
 best known and deservedly popular methods of test-, 
 ing for sugar in urine. There are, however, several 
 serious sources of error connected with the reaction, 
 and ignorance or neglect of these has not unfrequently 
 led to mistaken diagnosis and consequent improper 
 medical treatment. 
 
 Solutions of the ordinary blue and green salts of 
 copper on treatment with an alkali yield a pale blue 
 precipitate of cupric hydroxide or hydrated 
 
 1 Dr James Edmunds recommends that the cold liquid should be poured 
 through a column of zinc-dust contained in a Mohr's burette (one-third filled) 
 and retained in position by a plug of glass- or cotton-wool. 
 
REDUCTION OF CUPRIC OXIDE. 57 
 
 oxide of dopper, having the composition Cu0 2 H 2 , or 
 CuO,H 2 0. When the liquid containing this precipi- 
 tate is boiled, the hydroxide loses its water of consti- 
 tution and is converted into black cupric oxide, 
 CuO. The cuprous salts, corresponding to cuprous 
 oxide, Cu 2 0, are mostly insoluble in water, and are 
 readily converted into the blue cupric salts by oxida- 
 tion. On addition of a caustic alkali to a cuprous 
 salt, either in the solid state or in solution, a bright 
 yellow cuproushydroxide, CuOH, or Cu 2 0,H 2 0, 
 is precipitated, and this, on boiling the liquid contain- 
 ing it, is dehydrated and converted into red or brownish- 
 red cuprous oxide, Cu 2 0. 
 
 The precipitation of cupric oxide from cupric salts 
 by alkalies is prevented by the presence of tartaric 
 acid, citric acid, glycerin, the sugars, and various 
 other kinds of organic matter ; but these substances 
 exert no similar interfering action on the precipitation 
 of cuprous oxide from cuprous solutions. Various 
 oxidisable bodies convert cupric salts to cuprous, by 
 assimilating one-half of the oxygen (2CuO = Cu 2 + 0), 
 and this occurs with special facility at the boiling 
 point and in the presence of free caustic alkali. It 
 follows, therefore, that if caustic potash or soda be 
 added to a solution of cupric sulphate to which 
 sufficient of an interfering agent has been added, no 
 precipitate will be formed, the copper remaining in 
 solution and communicating a deep blue colour to the 
 liquid. If a body capable of abstracting oxygen from 
 cupric oxide be now added, reduction to the cuprous 
 state will ensue, and the yellow cuprous hydroxide or 
 red cuprous oxide (according to the temperature of the 
 liquid and other conditions of the experiment) will 
 separate. "With phenyl-hydrazine, reduction and con- 
 sequent precipitation occurs immediately, and at the 
 ordinary temperature. With glucose, it occurs slowly 
 
58 TROMMERS TEST. 
 
 in cold solutions, but immediately on boiling ; while 
 cane-sugar does not reduce the copper solution even 
 at a boiling heat, although it shares with glucose the 
 property of preventing the precipitation of cupric 
 solutions by alkalies. 
 
 Upon the foregoing facts the various modifications 
 of the copper test for glucose have been based. 
 
 Trommer's Test. — If urine or other liquid contain- 
 ing glucose, be treated with sufficient of a solution of 
 copper sulphate to render the liquid greenish, and a 
 solution of caustic potash or soda be then added, a 
 deep blue liquid will be formed if sugar in notable 
 amount be present. On allowing the liquid to stand 
 for some hours in the cold, a yellow precipitate of 
 cuprous hydroxide or red precipitate of cuprous 
 oxide will be produced, and the same precipitate 
 will be obtained immediately on boiling the liquid. 
 Trommer's modification of the copper test is merely 
 of qualitative value, and should be resorted to only 
 in cases where the conditions make the application 
 of Fehling's form of the test impossible. 
 
 Fehling's Test. — In Fehling's form of the copper 
 test for glucose, reliance is not placed on the sugar 
 itself for the prevention of the precipitation of the 
 cupric solution by an alkali, but sufficient tartrate of 
 alkali-metal is added to form a double tartrate of 
 copper and sodium, which is not decomposed by 
 alkalies. One molecule of tartaric acid is able to 
 prevent the precipitation of one molecule of copper 
 by a caustic alkali, owing to the formation of the salt 
 (CHO) 2 Cu(C0 2 K) 2 , but it is better to employ twice 
 the amount of tartaric acid requisite for the production 
 of this body. Fehling's solution is therefore essentially 
 a solution of the double tartrate of copper (cupric) and 
 sodium or potassium, with a considerable quantity of 
 caustic alkali. It is best prepared as follows: — (a) 
 
FEHLING S SOLUTION. 59 
 
 34*64 grammes weight of pure crystallised sulphate of 
 copper (free from iron and moisture) is dissolved in 
 warm distilled water, the solution filtered if necessary, 
 and when cold diluted to 500 c.c. with distilled water ; 
 (b) 180 grammes of crystallised Eochelle salt (potas- 
 sium sodium tartrate) is dissolved in about 300 c.c. of 
 hot water, the liquid filtered if necessary, and 70 
 grammes of good caustic soda or 100 grammes of 
 caustic potash added to the nitrate, which is then 
 cooled and made up to 500 c.c. 
 
 Fehling's Solution is prepared by carefully adding 
 an accurately measured volume of the above copper 
 sulphate solution to an equal measure of the alkaline 
 tartrate mixture. The precipitate first formed dis- 
 solves on agitation with formation of a clear azure- 
 blue solution. 
 
 Fehling's solution is affected by air and light, under- 
 going a change which causes a precipitation of cuprous 
 oxide on boiling. The alteration is often attributed 
 to the formation of the isomeric modification of tar- 
 taric acid known as racemic acid, but the evidence on 
 which this statement is made appears very insufficient. 
 It is, at any rate, an undoubted fact that Fehling's 
 solution spoils by keeping, and hence the indication 
 obtained with a particular sample of the reagent 
 cannot be trusted unless the solution be proved to 
 give no precipitate of cuprous oxide when diluted 
 with an equal measure of water and boiled for a few 
 minutes. The best plan is to store the cupric 
 sulphate and alkaline tartrate solutions separately, 
 and to mix them only when required for use ; then 
 proving by actual trial that the mixture suffers no 
 reduction when diluted and boiled as just described. 
 
 Fehling's solution is reduced by the following bodies 
 when their neutralised solutions are heated to boiling 
 with it : — 
 
6Q REDUCTION OF FEHLING's SOLUTION. 
 
 Carbohydrates, &c. — 'Dextrose, laevulose, maltose, 
 isomaltose, lactose, galactose, xylose, arabinose. 
 
 Alcohols, aldehydes, phenols, &c. — Acetaldehyde, 
 valeraldehyde, chloral, chloroform, resorcinol. 
 
 Acids. — Gallotannic, glycosuric, glycuronic,indoxyl- 
 sulphuric, hippuric, salicylic, trichloracetic, uric. 
 
 Bases. — Creatine, creatinine, xanthine, phenyl- 
 hydrazine. (The last substance reduces Fehling's 
 solution in the cold, with evolution of nitrogen.) 
 
 Fehling's solution is not reduced when boiled with 
 neutral or alkaline solutions of the following bodies : — 
 
 Carbohydrates, &c. — Cane-sugar, raffinose, mannitol, 
 dulcitol, dextrin, arabin, cellulose. 
 
 Alcohols, aldehydes, phenols, &c. — Alcohol, glycerin, 
 benzoic aldehyde, salicylic aldehyde, phenol. 
 
 Acids. — Acetic, citric, benzoic, formic, gluconic, 
 lactic, lactonic, mucic, oxalic, protocatechuic, sulphur- 
 ous, sulphamido-benzoic (" saccharine "), tartaric. 
 
 Bases. — Urea. 
 
 Among the products of the action of Fehling's 
 solution on glucose are said to be the following 
 bodies: — (1) Acetic and formic acids: (2) Certain 
 non-volatile acids, especially tartronic ; an acid form- 
 ing uncrystallisable salts ; and an acid which is decom- 
 posed with formation of humus-like products on 
 heating its alkaline solution : (3) A gummy substance 
 (compare page 24). 
 
 Fehling's copper solution, when properly applied, 
 affords one of the simplest and most popular means of 
 detecting glucose in urine. The usual method of 
 employing it is to remove albumin, if present, by heat- 
 ing to boiling the slightly acid or acidulated urine, and 
 filtering from any precipitate (see page 106). The 
 filtrate is then rendered distinctly alkaline to litmus 
 paper by a few grains of sodium carbonate, heated to 
 boiling, and filtered from the precipitate of earthy 
 
fehling's test. 61 
 
 phosphates, &c. Ten c.c. of recently prepared Fehling's 
 solution 1 is heated to boiling in a test-tube, some plat- 
 inum wire or a few fragments of clay tobacco-pipe or 
 pumice being added to prevent bumping. When the 
 liquid boils, 1 c.c. of urine, prepared as above described, 
 is added, and the liquid boiled again for a few seconds. 
 If sugar be present in abundance, as in a decidedly 
 diabetic urine, a yellowish or brick-red opacity and 
 deposit will be produced. If a negative reaction be 
 obtained, traces of sugar are tested for by adding 7 to 
 8 c.c. of the prepared urine to the hot liquid, heating 
 again to ebullition, and then setting the tube aside for 
 some time. If no turbidity is produced as the liquid 
 cools, the urine is regarded as either quite free from 
 sugar or containing less than 0'2 per cent. If the pro- 
 portion of sugar be moderate — that is, under - 5 per 
 cent. — the precipitation of the yellow or red cuprous 
 oxide does not take place immediately, but occurs 
 as the liquid cools, the appearance being somewhat 
 peculiar. The liquid first loses its transparency, and 
 passes from a clear bluish-green to an opaque light 
 greenish colour. This green milky appearance is said 
 to be very characteristic of dextrose, but it would be 
 more correct to say that its appearance indicated the 
 presence of some substance interfering with the normal 
 reaction of sugar. The number of these interfering 
 bodies is very considerable, including, as it does, uric 
 acid, hippuric acid, glycuronic and glycosuric acids, 
 xanthine, hypoxan thine, creatinine, indoxyl-sulphates, 
 mucus, peptones, and the milk-sugar sometimes present 
 in the urine of nursing women. Some of these sub- 
 
 1 It is absolutely necessary that the Fehling's solution employed for the 
 test described in the text should remain clear when diluted with its own 
 volume of distilled water and boiled for a few minutes. The cuprie sulphate 
 and alkaline tartrate solutions should be kept separate, and the Fehling's 
 solution made when required by mixing them in equal volumes. Compare 
 page 59. 
 
62 MODIFIED FEHLINGS TEST. 
 
 stances exert a reducing action on the copper solution, 
 while others, such as xanthine, uric acid, peptones and 
 pigments, modify the reaction by causing the forma- 
 tion of a greenish precipitate instead of the separation 
 of yellow or red cuprous oxide as normally obtained. 
 
 Modified Fehling's Test. — The presence of such 
 interfering bodies render the indications of Fehling's 
 solution uncertain and difficult to interpret when only 
 small quantities of sugar are in question. A great 
 improvement in the precision and delicacy of the test, 
 as applied to urine, without in any way adding to the 
 time required or to the complexity of the manipula- 
 tion, can be effected by operating in a manner devised 
 by the author. This consists in a preliminary 
 treatment of the urine with cupric sulphate or 
 acetate, in neutral or slightly acid solution, whereby 
 many of the constituents of urine are precipitated 
 without in any way affecting any form of sugar. 
 
 Cupric sulphate yields little or no precipitate with 
 normal urine in the cold, but on standing or boiling a 
 whitish or pale green precipitate is thrown down, which 
 has a tendency to darken if the heating be continued. 
 If cupric acetate be substituted for the sulphate, or if 
 sodium acetate be added to the cupric sulphate solution, 
 this precipitation is much more complete, uric acid, 
 xanthine, hypoxanthine, colouring matter, and albumin 
 (if present) being entirely thrown down, and the phos- 
 phates and creatinine partially. The filtered liquid 
 cannot be used for the phenyl-hydrazine test, and the 
 presence of copper unfits it for titration by Pavy's 
 solution, but it is admirably adapted for the detec- 
 tion of small quantities of glucose by FehliDg's test. 
 The procedure is as follows : — 
 
 { From 7 to 8 c.c. ( = 3 oz.) of the sample of urine 
 \to be tested is heated to boiling in a test-tube, and, 
 without separating any precipitate of albumin which 
 
MODIFIED FEHLING'S TEST. 63 
 
 may be produced, 5 c.c. (about 80 minims) of the 
 solution of cuprie sulphate used for preparing Fehling's 
 test is added. This produces a precipitate containing 
 uric acid, xanthine, hypoxanthine, phosphates, &c. 
 To render the precipitation complete, however, it is 
 desirable to add to the liquid, when partially cooled, 
 from 1 to 2 c.c. (20 to 30 minims) of a saturated 
 solution of sodium acetate having a feebly acid reac- 
 tion to litmus. 1 The liquid is next filtered. To the 
 filtrate, which will have a bluish-green colour, 5 c.c. 
 (80 minims) of the alkaline tartrate mixture used for 
 preparing Fehling's solution is next added, and the 
 liquid boiled for fifteen to twenty seconds. In the 
 presence of more than 0'25 per cent, of sugar, separa- 
 tion of cuprous oxide occurs before the boiling-point 
 is reached ; but with smaller quantities precipitation 
 takes place during the cooling of the solution, which 
 becomes greenish, opaque, and suddenly deposits 
 cuprous oxide as a fine orange-yellow precipitate. 2 
 
 The foregoing description is intended to apply more 
 especially to the examination of urine containing a 
 comparatively small proportion of sugar, as it is in such 
 cases that the interference of the co-occurring sub- 
 stances is most marked and objectionable. But the 
 method is equally applicable to the examination of 
 strongly diabetic urine. In such cases, however, the 
 
 1 Sufficient acetic acid should be added to the sodium acetate solution to 
 render it feebly acid to litmus paper. A saturated solution of sodium 
 acetate keeps well, but weaker solutions are apt to become mouldy, and then 
 possess the power of reducing Fehling's solution. Hence it is essential in 
 all cases of importance to make a blank test by mixing equal measures of 
 cuprie sulphate solution, alkaline tartrate solution, and water, adding a little 
 sodium acetate solution, and heating the mixture to boiling. 
 
 2 The sudden separation of the precipitate during cooling is a phenomenon 
 difficult of explanation. Operating as prescribed in the text on urine contain- 
 ing but a small quantity of sugar, the precipitate is of a bright orange-yellow 
 colour ; but when typical diabetic urine is under treatment, or a solution of 
 pure sugar is employed, the cuprous oxide is thrown down as a bright brick- 
 red or brown precipitate. The modified colour of the precipitate yielded by 
 slightly saccharine urine appears to be due to the extractive matters present. 
 
64 MODIFIED FEHLINGS TEST. 
 
 measure of urine taken should be reduced from 7 or 8 
 to 1 or 2 c.c. ( = 15 to 30 minims), or even less, water 
 being added to bring the volume up to 7 or 8 c.c. 
 
 It is evident that in this modification of the ordi- 
 nary Fehling's test, advantage is taken of the very 
 general precipitating power of cupric acetate to 
 remove from the urine the great majority of those 
 substances which interfere with the detection of dia- 
 betic sugar ; and which by themselves either reduce the 
 alkaline copper solution, retaining the cuprous oxide 
 in solution, or produce a flocculent precipitate which 
 masks the true reaction of sugar. Operating as above 
 described, no greenish turbidity refusing to settle is 
 produced, and hence the separation of any cuprous 
 oxide is very readily observed. 
 
 In employing the foregoing modification of the 
 copper test, it must not be forgotten that a solution 
 of cupric acetate, even when acidulated with acetic 
 acid, yields a precipitate of cuprous oxide when boiled 
 with a solution of glucose. This reaction, first ob- 
 served by Barfoed, and confirmed by M filler 
 and the author, is liable to vitiate the test for 
 small quantities of sugar, unless the prescribed method 
 of working is strictly followed. Hence it is clearly 
 important that the sodium acetate should not be 
 added to the boiling hot solution, or the liquid boiled 
 after its addition, or there will be danger of partial 
 reduction by the sugar before adding the alkaline 
 tartrate. But if the sodium acetate solution have 
 a distinctly acid reaction, and its addition to the 
 liquid be not made till the latter has somewhat cooled, 
 the author has fully satisfied himself that there is no 
 possibility of error from this cause. 
 
 A further practical advantage attending the employ- 
 ment of the foregoing modification of Fehling's test, 
 is the complete precipitation of any albuminous 
 
MODIFIED FEHLINGS TEST. 65 
 
 matters by the cupric sulphate solution without the 
 necessity for an additional nitration ; and the removal 
 of these bodies at a prior stage prevents the formation 
 of ammonia, which unavoidably ensues to a greater or 
 less extent on boiling their solutions with caustic 
 alkali. The formation of ammonia in this manner 
 interferes with the detection of minute quantities of 
 sugar by Fehliug's test, since the cuprous oxide pro- 
 duced is held in permanent (colourless) solution. 
 
 The presence of any considerable quantity of 
 ammonia, whether due to fermentation of the urea 
 or other cause, absolutely invalidates the indications 
 of Fehling's test, except, of course, when using Pavy's 
 modification of it (page 66), in which a strongly 
 ammoniacal solution of the copper reagent is inten- 
 tionally employed. 
 
 On adding Fehling's solution to a solution of crea- 
 tinine, a green liquid is produced, and on boiling this 
 a yellow coloration is observed, without, however, any 
 separation of cuprous oxide. In the case of urines 
 rich in creatinine, this behaviour causes interference 
 with the detection of glucose, the combination of the 
 yellow and blue colours resulting in a green, and in 
 addition the creatinine compound is said to have the 
 power of preventing the precipitation of cuprous oxide 
 by glucose. (See also the effect of dextrin observed 
 by Eeichard and described on page 31.) 
 
 In cases where the glucose present is more than 
 equivalent to the cupric oxide in solution, as may 
 happen when a urine containing a large proportion of 
 sugar is being treated, the cuprous oxide precipitated 
 often has a dark brown colour, while the supernatant 
 liquid also has a brownish tint. This behaviour is 
 due to the caramelisation of the excess of glucose by 
 the caustic alkali, a portion of the colouring matter 
 produced being carried down by the precipitate. 
 
66 AMM0NI0-CUPRIC PROCESS. 
 
 Volumetric Determination of Glucose by Fehling's 
 Solution. — The last described modification of Fehling's 
 test cannot be applied quantitatively, since an un- 
 known amount of copper passes into the precipitate, 
 while, owing to the insurmountable tendency, in many 
 cases, of the cuprous oxide to pass through a filter, 
 the gravimetric 1 and those modifications of the volu- 
 metric process in which filtration is necessary are not 
 suitable for the determination of sugar in urine. In 
 the absence of ammonia, either existing in the original 
 urine or resulting from the decomposition of urea, 
 fairly good results may be obtained by operating as 
 follows : — 10 c.c. of recently prepared Fehling's solu- 
 tion is diluted with 40 c.c. of recently boiled water, 
 and the liquid heated to boiling in a porcelain basin. 
 The urine, previously diluted with from 2 to 20 
 measures of recently-boiled water, is then gradually 
 added from a burette, the liquid being kept gently 
 boiling until the blue colour has nearly disappeared. 
 The addition of urine is then continued more cautiously, 
 removing the source of heat and allowing the cuprous 
 oxide to subside after each addition, when, by inclin- 
 ing the basin, the slightest remaining blue tint can be 
 perceived. "When the decolorisation is complete, the 
 volume of urine which has been used is noted. This 
 measure contains 0'050 gramme of glucose. 
 
 Titration op Glucose by Pavy's Ammoniacal 
 Cupric Solution. — For the examination of typical 
 diabetic urine, containing a considerable proportion 
 of sugar and in a perfectly fresh state, the foregoing 
 process is fairly suitable, but it becomes troublesome 
 
 1 It is a fact Dot generally recognised, but strongly urged on the author by 
 Dr J. Edmunds, that the cuprous oxide separated in Fehling's test undergoes 
 reoxidation with extreme facility, and to this behaviour is probably attribute 
 able the discordant results obtained by many observers who have tried to 
 utilise the weight of the separated cuprous oxide as a means of estimating the 
 glucose present. 
 
PAVYS AMMONIAC AL CUPRIO SOLUTION, 67 
 
 or inapplicable where the amount of sugar is small 
 and the extractives correspondingly high. In such 
 cases, and indeed for general urine testing, it may be 
 advantageously replaced by a process devised by E. W, 
 Pavy (Chem. News, xl. 77), who employs an am- 
 moniacal cupric solution prepared by mixing 120 c.c. 
 of ordinary Fehling's solution with 300 c.c. of strong 
 ammonia (sp. gr. 0-880), and with 100 c.c. of a 10 per 
 cent, solution of caustic soda, or the same volume of a 
 14 per cent, solution of caustic potash. 1 The mixture 
 is then made up with distilled water to 1 litre. 
 
 The oxidising value of Pavy's solution, prepared as 
 above, is one-tenth that of Fehling's solution ; i.e., 50 
 milligrammes (= 0'050 gramme) of glucose reduce 
 10 c.c. of Fehling's or 100 c.c. of Pavy's solution. 2 
 
 According to C. W. P u r d y, 3 the ratio of reduction 
 of Fehling's and Pavy's solutions by glucose varies 
 according as they are prepared with caustic potash 
 or caustic soda. The existence of such a difference 
 has been disproved by direct experiments made in the 
 a u t h o r's laboratory. G. B. B r o o k found the oxidis- 
 ing action of Pavy's solution to be absolutely the same 
 whether prepared with 56 of KHO, 40 of NaHO, or 
 56 of NaHO. In fact, the writer's experience is 
 that the amount of caustic alkali and ammonia may be 
 further varied very considerably without the oxidising 
 value of Pavy's solution being appreciably affected. 
 
 When Pavy's solution is heated with a liquid con- 
 taining glucose the cuprous oxide produced does not 
 
 1 If Fehling's solution be not at hand, Pavy's solution may be made up 
 directly as follows : — 4*157 grammes of pure crystallised sulphate of copper is 
 dissolved in about 100 c.c. hot water and the solution cooled ; 21 "6 grammes 
 of crystallised Eochelle salt and 18'4 grammes of caustic soda (or 25 - 8 grammes 
 caustic potash) are dissolved in hot water, cooled, and added to the copper 
 sulphate solution. To the mixture is added 300- c.c. strong ammonia 
 (sp. gr. "880), and the whole made up to 1 litre. 
 
 2 One molecule of glucose reduces to cuprous oxide 6 CuO existing in Fehling's 
 solution, but only 5 CuO in Pavy's solution prepared as described in the text. 
 
 3 Practical Uramalysis and Urinary Diagnosis, 1894. 
 
68 
 
 pavy's volumetric process. 
 
 separate as a precipitate, but remains in colourless 
 solution. The progress of the reaction is therefore 
 indicated by the gradual disappearance of the blue 
 colour of the liquid, and when the reaction is com- 
 plete — that is, when all the copper is reduced to the 
 cuprous state — the liquid becomes perfectly colour- 
 less. But ammoniacal cuprous solutions are extremely 
 oxidisable, and hence the blue colour indicative of 
 
 Fig. i. — Apparatus for Pavy's Test. 
 
 the existence of a cupric compound returns rapidly on 
 exposing the reduced liquid to the air. 1 It is necessary, 
 therefore, to operate in such a manner as will ensure 
 the complete exclusion of air during the process. This 
 is best done by attaching the nose of the Mohr's 
 
 1 J. Edmunds has pointed out, in a private communication to the author, 
 that the cuprous oxide precipitated in Fehling's form of the test is also highly 
 oxidisable, and he attributes the variable results obtained in the quantitative 
 application of the test to insufficient recognition of this source of error. 
 
TITRATION WITH PAVY S SOLUTION. 69 
 
 burette containing the urine to a tube passing through 
 the india-rubber stopper of a flask containing the 
 copper solution, as shown in figure 4. A second 
 tube conveys away the steam and ammoniacal gas, 
 while, by means of a third tube, a slow current 
 of coal-gas may be kept constantly passing through- 
 out the operation. 1 The coal-gas may be dispensed 
 with by allowing the exit-tube to pass into an empty 
 Woulffe's bottle, which is connected with a flask con- 
 taining cold water, as suggested by A. W. Stokes 
 (Analyst, xii. 47). If the exit-tube be provided with 
 a valve, the vapours may be passed direct into water 
 without the intervention of a Woulffe's bottle. (See 
 J. St ein er, Cliem. News, xl. 139.) The author has 
 suggested the employment of a layer of paraffin oil to 
 prevent contact with the air, and the same proposal 
 has been subsequently made by L. P e s k e. 
 
 It is a good plan to whitewash the iron plate which 
 supports the flask, as by that means a marked contrast 
 is obtained to the blue colour of the copper solution. 
 Stokes supports the half of an opal gas-globe behind 
 the flask (Analyst, xii. 47). H. M. Smith uses a 
 flask the lower third of which is made of white 
 opalescent glass 2 (Chem. News, lxxi. 165). 
 
 In carrying out Pavy's process, the author finds 25 
 c.c. of the copper solution a convenient quantity to em- 
 ploy. This measure of the ammoniacal cupric solution 
 should be placed in the flask and a few fragments of 
 pumice or tobacco-pipe added to prevent bumping. 
 The tubes and burette are then adjusted, a slow cur- 
 rent of coal-gas allowed to pass, and the contents of 
 the flask brought to ebullition. The urine, preferably 
 
 1 When coal-gas is employed, a brick-red film is formed on the surface of 
 the liquid towards the end of the titration. This is- not cuprous oxide, as 
 might be supposed, but cuprous acetylide, C 2 Cu 2 ,H 2 0, formed from the 
 acetylene invariably present in coal-gas. 
 
 2 Obtainable from C. E. Miiller & Co., 148 High Holborn, London, W.C. 
 
70 PAVYS VOLUMETRIC PROCESS. 
 
 freed from earthy phosphates by adding ammonia and 
 filtering, is then gradually run in from the burette, the 
 boiling being' continued." The process is at an end 
 when' the blue colour of the liquid is wholly destroyed. 
 The end-reaction is very sharply marked, but the 
 reduction occurs more slowly than with the ordinary 
 Fehling's solution, and hence the process must not be 
 hurried or too low a result will be obtained. In the 
 case of strongly diabetic urine it is desirable to dilute 
 it to a definite volume with distilled water. On the 
 other hand, when the proportion of sugar is small 
 {e.g., 0-2 per cent.), the volume of Pavy's solution may 
 be conveniently reduced to 10 c.c. When 25 c.c. is 
 the volume employed, the measure of urine required 
 to decolorise it, multiplied by four, contains 0'050 
 gramme of glucose. 
 
 As Pavy's solution, like Fehling's original reagent, 
 is reduced by uric acid, xanthine, creatinine, &c, as 
 well as by glucose itself, it follows that this method also 
 is apt to give inaccurate indications when only small 
 quantities of urinary sugar are in question, or when 
 an abnormally large proportion of one or more other 
 reducing substances happen to be present. A series 
 of experiments made in the author's laboratory by G. 
 B. Brook showed that the interfering substances can 
 be most readily and perfectly removed by a plan pro- 
 posed by Mai y and improved by G-. S til ling- 
 fleet Johnson, which has been described at length 
 on page 55. In practice, 60 c.c. of the urine to be 
 tested should be treated with 1 5 c.c. of a cold saturated 
 solution of mercuric chloride and 3 to 5 c.c. of a cold 
 saturated solution of sodium acetate (see footnote 
 on page 55). The liquid is boiled for five minutes, 
 and filtered hot. The precipitate contains all the uric 
 acid, xanthine, creatinine, phosphates, and albumin 
 of the urine, and a portion of the, urea. The preci- 
 
DETERMINATION OP TRACES OP GLUCOSE. 71 
 
 pitate is washed twice with water, the filtrate acidulated 
 with a few drops of acetic acid, boiled for ten minutes 
 with zinc-dust, 1 and again filtered. The precipitate is 
 washed, and the filtrate diluted to 120 c.c. with strong 
 ammonia. This liquid has half the concentration of 
 the original urine, and is added to not more than 
 50 c.c. of boiling Pavy's solution from a burette in 
 the usual manner. The large excess of ammonia has 
 no prejudicial influence on the result, while it keeps 
 the zinc in permanent solution and ensures a constant 
 but gradual supply of ammonia during the progress 
 of the titration. The requisite washing of the mer- 
 curic and zinc precipitates can be avoided if 50 c.c. of 
 the urine be boiled with powdered mercuric chloride and 
 sodium acetate, the liquid filtered, the filtrate treated 
 with zinc-dust and again filtered, and a known volume 
 of the last filtrate mixed with an equal volume of 
 strong ammonia. 
 
 Experiments made in the author's laboratory in the 
 foregoing manner, by G. B. Brook, upon a limited 
 number of samples of urine from apparently healthy 
 persons, showed the purified liquid to have a reducing 
 action upon Pavy's. solution corresponding to the 
 presence of from 0*05 to 0*13 gramme of glucose per 
 100 c.c. of the original urine, and yielded crystals 
 of phenyl-glucosazone by the phenyl-hydrazine test 
 (see page 85). Previously to the removal of the 
 interfering substances by the mercury reagent, the 
 same urines had a cupric oxide reducing power cor- 
 responding to from 0'1 to 0"3 gramme per 100 c.c, 
 equal to # 44 to 1*3 grain of glucose per ounce. The 
 difference between the amounts of apparent sugar 
 shown before and after clarification is an indication of 
 the proportion of interfering substances present. 
 
 G. Stillingfleet Johnson finds normal urine to have 
 
 1 Or by Edmunds' plan, described in footnote on page 56. 
 
72 GLYCEROL-CUPRIC SOLUTIONS. 
 
 absolutely no reducing action after clarification by the 
 mercury reagent. This implies that an infinitely 
 large quantity of the filtrate resulting from this 
 treatment could be added to a limited volume of 
 Pavy's solution without the blue colour of the latter 
 being destroyed. But as the reduction of the mer- 
 curic solution with separation of calomel, and the 
 simultaneous oxidation of the glucose, commences after 
 a few minutes' ebullition, it is conceivable that when 
 mere traces of sugar are present they might be 
 entirely destroyed at this stage of the process, and 
 that the amount found in the filtrate would be merely 
 that which had survived the treatment. 1 
 
 Eecognising the objections attaching to any process 
 in which glucose is subjected to the action of a hot 
 solution containing fixed caustic alkali, A 1 1 e i n and 
 Gaud {Jour. Pharm. Chem., [5], xxx. 305; and 
 Jour. Chem. Soc, lxviii. ii. 92) have recently pro- 
 posed to employ a strongly ammoniacal solution of 
 cupric sulphate for determining sugar. The process 
 has been tried in the author's laboratory, but the 
 reduction was found to occur so slowly that the 
 method cannot be recommended. 
 
 Glycerol-Cupric Solutions. — In 1870, J. Lowe 
 suggested the use of glycerin as a substitute for 
 alkaline tartrate in preparing an alkaline cupric solu- 
 tion. He gave a recipe for preparing the solution 
 from cupric sulphate, glycerin, and caustic soda, but 
 preferred to employ freshly precipitated cupric hydr- 
 oxide instead of the sulphate (Zeits. anal. Chem., x. 
 452). More recently, a glycerol-cupric solution has 
 been advocated by W. S. Haines (Chicago), who 
 prepares it by dissolving 30 grains of crystallised 
 
 1 Experiments made in the author's laboratory, to test this point, showed 
 that the oxidation by mercuric acetate of glucose in very dilute solutions (0 - 05 
 to 0'10 per cent. ) was very imperfect, and that 80 to 90 per cent, of the glucose 
 survived boiling with the mercuric reagent for ten minutes. 
 
GLYCEROL- CUPRIC SOLUTIONS. 73 
 
 copper sulphate in 240 grain-measures of distilled 
 water, adding \ troy-ounce ( = 240 grain-measures) of 
 glycerin, mixing thoroughly, and adding 5 troy-ounces 
 (5 "4 English fluid ounces) of liquor potassw. In using 
 this solution, it is recommended to boil about 1 drachm 
 in a test-tube and then add two or three drops of the 
 urine to be tested and again boil. In presence of 
 much sugar a copious yellow or yellowish-red precipi- 
 tate will be thrown down. If a negative result be 
 obtained, a further addition of urine should be made, 
 and the boiling repeated, but it is absolutely necessary 
 that the total volume of urine employed should not 
 exceed six or eight drops for 1 drachm of the glycerol- 
 cupric solution, otherwise a precipitate may be pro- 
 duced even in the absence of sugar. 
 
 It is desirable previously to remove the earthy 
 phosphates from the urine to be tested by adding a 
 few drops of sodium carbonate and filtering. Even 
 with this precaution, the author does not find the 
 glycerin solution so delicate a test for sugar as is 
 the ordinary tartrate solution of Fehling. But the 
 glycerin mixture has the advantage of being more 
 readily extemporised, and is said to keep indefinitely. 
 Hence it deserves recognition as a convenient clinical 
 test for diabetic sugar. 
 
 A glycerol-cupric solution of practically the com- 
 position of that prepared by Haines' formula may 
 be obtained by mixing 2 measures of the cupric sul- 
 phate solution employed for preparing Fehling's test 
 with 1 measure of pure glycerin, and then adding 
 10 measures of liquor potassse, B.P. 
 
 C. W. Purdy {Practical Uranalysis, page 110) 
 has proposed to employ Haines' glycerol-cupric solu- 
 tion with the addition 'of ammonia in place of Pavy's 
 solution, but experiments on the process conducted in 
 the author's laboratory showed that the method was 
 
74 gerrakd's process. 
 
 open to grave objections, and he is unable to recom- 
 mend it. 
 
 Gerrard's Cyano-cupric Process of Glucose- 
 titration.— A volumetric method of sugar titration, 
 which will probably supersede all the other forms of 
 copper determination of diabetic glucose, has been de- 
 vised by A. W. G e r r a r d. The process, as originally 
 published (Year-Booh Pharm., 1892, page 400, and 
 Pharm. Jour., [3], xxiii. 208), did not yield good 
 results, but the writer has obtained further parti- 
 culars from the inventor of the process, and has person- 
 ally investigated the sources of error and the pre- 
 cise conditions necessary to ensure success. Gerrard's 
 process is based on the following facts : — - 
 
 When a solution of potassium cyanide is added to 
 one of a cupric salt, a double cyanide of potassium 
 and copper is formed, according to the following 
 equation :— CuS0 4 + 4KCy = CuCy 2 ,2KCy + K 2 S0 4 . 
 The double cyanide of potassium and copper thus 
 formed is not decomposed by alkali nor precipitated 
 by sulphuretted hydrogen. Its solution is colourless 
 or faintly yellow. If Fehling's solution be employed 
 instead of one of cupric sulphate, the same colourless 
 double cyanide is formed, and the solution gives no ' 
 precipitate of cuprous oxide when boiled with glucose 
 solution. If Fehling's solution be present in quantity 
 more than sufficient to react with the potassium 
 cyanide used, this extra portion will suffer reduction 
 by glucose ; but instead of the cuprous oxide being 
 precipitated it will remain in solution, and the pro- 
 gress and end of the reduction will be indicated by the 
 gradual lessening and ultimate entire disappearance of 
 the blue colour. Hence in outward appearance the 
 method resembles that of Pavy (see page 67), but it 
 has the advantage of causing no evolution of ammonia. 
 Further, the re-oxidation of the reduced solution pro- 
 
CYANO-CUPRIC PROCESS. 
 
 75 
 
 ceeds slowly, so that, where the highest degree of accu- 
 racy is not sought, the titration, if conducted expe- 
 ditiously, may be performed in an open basin, instead 
 of in an apparatus jealously protected from ingress of 
 air. In its improved form, as modified by Gerrard 
 and the author, the procedure is as follows : — 
 
 Ten c.c. of freshly-prepared Fehling's solution, or 5 
 c.c. of each of the constituent solutions, both accurately 
 measured, should be placed in a porcelain dish, 40 c.c. 
 of water then added, and the liquid heated to boiling. 
 A solution of potassium cyanide, of about 5 per cent, 
 strength, is gradually added from a burette or pipette, 1 
 until the deep blue colour of the liquid is nearly 
 destroyed. The addition of the cyanide is continued, 
 very cautiously, drop by drop, the liquid being kept 
 constantly boiling and well stirred, until the blue 
 colour just disappears, or only a very slight tinge of 
 blue remains. Excess of cyanide solution must be 
 carefully avoided, so that it is safer to discontinue its 
 addition when the blue colour is still faintly per- 
 ceptible. 2 Another 10 c.c. of Fehling's solution, or 
 its equivalent, should now be added to the contents 
 of the basin, and the urine or other sugar-holding 
 
 1 Fletcher's graduated stopper-pipette (fig. 5) is 
 very convenient for this and similar work. It has a 
 small slit in the upper india-rubber ball which acts 
 as a valve, and by judicious pressure the liquid in 
 the pipette can be delivered drop by drop or rapidly 
 as may be desired The "Automatic Stopper" is 
 manufactured by Messrs Fletcher, Fletcher, & Co., 
 Holloway, London, N. 
 
 2 There is no occasion to go through this process 
 every time. The double solution of potassio-cupric 
 cyanide keeps unchanged for some weeks, so that 
 100 c.c. of Fehling's solution may be diluted with 
 about 300 c.c. of water, and the liquid decolorised 
 while boiling by a 5 per cent, solution of potassium 
 cyanide. The liquid is then diluted to exactly 500 c.c, 
 and preserved in a well-closed bottle. Fifty c.c. of 
 this solution, corresponding to 10 c.c. of Fehling, are 
 mixed with 10 c.c. of untreated Fehling's solution, the 
 liquid heated to boiling, and the titration conducted as described in the text. 
 
 Fig. 6.— 
 Fletchek's Pipette. 
 
76 CYANO-CUPRIC PROCESS. 
 
 liquid dropped in rapidly from a burette, with con- 
 stant stirring, the liquid being kept in ebullition. 
 The end-reaction is indicated by the disappearance of 
 the blue colour, a point which can be very sharply ob- 
 served when pure solutions of sugar are being treated, 
 but which is somewhat less distinct when urine is 
 under examination. 1 The volume of saccharine liquid 
 required to decolorise the solution contains 0050 
 gramme of glucose. Hence it follows that 10 c.c. of 
 urine containing 0*5 per cent, of sugar will be re- 
 quired, and proportionately less of stronger solutions. 
 Therefore urine suspected to contain more than 0'5 per 
 cent, of sugar should be diluted with water to a definite 
 measure before being used. For ordinary diabetic 
 urine, 10 measures should be diluted to 100. 
 
 Of course, if preferred, grains or minims may be 
 substituted for the metric weights and measures pre- 
 scribed above. In such case, 100 grain-measures of 
 Fehling's solution or its equivalent may be reduced 
 by cyanide, and a second 100 grain-measures added. 
 A. W. Gerrard has patented a special form of burette 
 for use with the process. 2 This is so graduated as to 
 allow the percentage of sugar contained in the urine to 
 be read off at once without calculation. 
 
 For simplicity, accuracy, and general convenience, 
 Gerrard's cyano-cupric process is preferred by the 
 w r i t e r to any other method of titration based on the 
 reducing power of glucose. No doubt the other 
 reducing constituents of urine (e.g., creatinine, uric 
 acid, &c.) exercise the same disturbing influence on 
 the results as in other methods, but they do not 
 visibly interfere with the indications. 
 
 1 Only the second measure of Fehling's solution suffers reduction. Experi- 
 ments in the writer's laboratory, by Arnold R. Tankard, have proved that the 
 reducing power of invert-sugar solution on the cyano-cupric reagent is secu- 
 rately as stated in the text. 
 
 2 Obtainable from Messrs Gibbs, Cuxson, & Co., Wednesbury. 
 
BISMUTH TEST FOR GLUCOSE. 77 
 
 Reaction of Glucose with Bismuth Compounds. 
 
 A test for glucose which has the merits of simplicity 
 and convenience, is the bismuth test due to B 6 1 tg e r. 
 The urine to be examined is mixed with an equal 
 measure of caustic potash or soda solution, and a small 
 quantity (as much as can be taken up on the point of 
 a pen-knife) of basic nitrate of bismuth added. On 
 boiling the liquid, metallic bismuth will separate as 
 a grey deposit or black metallic mirror if sugar be 
 present in notable amount. Nylander has improved 
 this test by substituting a solution of sodio-tartrate of 
 bismuth for the solid oxy -nitrate employed by Bottger. 
 2"5 grammes of the basic nitrate of bismuth (free from 
 silver) and 4 grammes of Bochelle salt are dissolved 
 in 100 c.c. of an 8 per cent, solution of caustic soda. 
 On boiling 10 c.c. of this reagent, filtered if necessary, 
 with 1 c.c. of urine for a short time, the liquid will turn 
 black if notable traces of glucose be present. Albumin, 
 blood, and other sulphuretted bodies are liable to cause 
 confusion by precipitating black bismuthous sulphide, 
 and hence must be removed before the test is applied. 1 
 
 A. Jo lies (Chem. Centr., 1890, ii. 609) states 
 that with Bottger's test 0'08 per cent, of sugar can be 
 detected in urine, and this limit is reduced to 0*01 
 per cent, if the urine be previously boiled with a 
 solution of sodium chloride. At least 50 c.c. of the 
 urine should be employed to 5 c.c. of Nylander's re- 
 agent, and the liquid should not be boiled more than 
 two minutes. The presence of such substances as 
 rhubarb, turpentine, kairine, quinine, sulphur, arsenic, 
 mercury, iodides, and salicylic acid should be avoided. 
 A brown colour is produced in presence of considerable 
 
 1 Experiments in the author's laboratory showed that 1 c.c. of a - l per 
 cent, solution of glucose, when boiled with 10 c.c. of Nylander's reagent, gave 
 a grey coloration, and a light grey mirror appeared on the tube. With 1 c.c. 
 of a l'Oper cent, glucose solution, the liquid became greenish-black, and a 
 metallic mirror was formed on the tube. 
 
 J 
 
78 REDUCTION OF MERCURIC COMPOUNDS. 
 
 quantities of uric acid, bat the depth of tint from this 
 cause does not increase beyond a certain point, whereas 
 that due to sugar continually darkens. 
 
 Reaction of Glucose with Mercuric Compounds. 
 
 Several methods of determining glucose have been 
 based on its reaction with mercuric compounds. Thus 
 an alkaline solution of potassio-mercuric cyanide has 
 been recommended by K n a p p ; an alkaline solution 
 of potassium mercuric iodide ("Nessler's reagent") by 
 S a c h s s e ; and a solution of mercuric acetate by 
 H a g e r. The mercury of the first two solutions is, on 
 boiling, promptly reduced to the metallic state, and 
 that of Hager's reagent more gradually to the mercur- 
 ous condition. Hence in presence of chloride, as when 
 the mercuric acetate is extemporised by mixing solu- 
 tions of mercuric chloride and sodium acetate, a pre- 
 cipitate of mercurous chloride is formed on boiling the 
 liquid for a short time (compare page 72). 
 
 The objections to the mercurial solutions are much 
 the same as apply to those of copper, and they present 
 no advantages to warrant an addition to the already 
 numerous methods of determining diabetic sugar. 
 
 Reaction of Glucose with Organic Colouring 
 Matters. 
 
 Picric Acid Test for Sugar. — Among the many 
 coal-tar dyes and other organic compounds which 
 suffer reduction in alkaline solution by glucose is the 
 substance commercially known as " picric acid." This 
 body has the constitution of a trinitro-phenol, 
 and is obtained by the action of excess of strong 
 nitric acid on carbolic acid : — 
 
 C 6 H 5 .OH + 3HNO s = C 6 H 2 (N0 2 ) 3 .OH+3H 2 0. 
 Picric acid, which occurs in pale yellow crystals, 
 sparingly soluble in cold water, but more readily in 
 
PICRIC ACID TEST. 79 
 
 alcohol, ether, and benzene, readily suffers reduction 
 in hot alkaline solution to picramic acid, a body 
 having the constitution of triamido-phenol : — 
 
 C 6 H 2 (N0 2 ) 3 .OH+9H 2 = C 8 H 2 (NH 2 ) 8 .OH+6H a O. 
 
 Picric Acid. Picramic Acid. 
 
 In practice, it is convenient to employ a cold satu- 
 rated aqueous solution of picric acid, mixed with an 
 equal measure of normal caustic soda or potash solution 
 (or Liquor potassse, B.P.). 1 The liquid is then boiled, 
 and the urine or other liquid to be tested for glucose 
 added, the boiling being repeated for a minute or so. 
 If glucose be present, the solution will darken from the 
 yellow-orange colour characteristic of a hot alkaline 
 solution of picric acid to an intense brownish-red 
 colour, so deep as to be opaque in presence of more 
 than traces of sugar. 1 
 
 This reaction, originally proposed as a test for sugar 
 by C. D. Braun (1865), has been strongly advocated 
 of late years by Sir G. J o h n s o n, who has described 
 a colorimetric application of it by which estimations 
 of diabetic sugar may be obtained. 2 Picric acid is 
 
 1 The picric acid can conveniently be mixed with the alkali before it is 
 required for use. If caustic potash be employed, an immediate deposition 
 of beautiful crystals of the sparingly soluble potassium picrate is produced, a 
 mere fraction of the picric acid remaining in solution. If caustic soda be 
 substituted, a similar effect is produced in cold weather, but the separation 
 of the sodium picrate is by no means so prompt or complete as that of the 
 potassium salt. Indeed, a permanent and serviceable reagent can be prepared 
 by mixing two measures of cold picric acid with one of normal soda solution, 
 disregarding any separation of crystals which may occur. 
 
 In either case it must be remembered that alkaline solutions of picric acid 
 are much deeper in colour than those of free picric acid, and that on heating 
 the colour becomes intensified. Impure picric acid gives a still deeper colour. 
 
 2 Sir George Johnson compares the colour yielded by the sample of urine in 
 question with a permanent standard solution prepared as follows : — 
 
 $.— Liq. Ferri Perchlor. Fort. (B.P., sp. gr. 1-42), . . 3j. 
 Acid. Acet. Glacialis (B.P., sp. gr. 1-05.8), . . . 5iv. 
 Liq. Ammonise (B.P., sp. gr. - 959), .... 3 T J- 
 
 Aquam Destill., ad 3iv. 
 
 Mix the iron solution with the acid, then add the ammonia, and dilute 
 with water up to 4 fluid ounces. This solution is of the same depth of colour as 
 that produced by a solution of glucose containing 1 grain per fluid ounce, when 
 
80 PICRIC ACID TEST. 
 
 not affected by uric acid, but unfortunately creatinine 
 gives with it a reaction very similar to that yielded 
 by glucose. As a consequence, normal urine yields 
 a blood-red colour when treated in the cold with 
 an alkaline solution of picric acid, and the colour 
 becomes more intense on boiling. This behaviour 
 renders the positive detection of co-occurring traces of 
 sugar very difficult. In fact, for use with untreated 
 
 boiled with picric acid and alkali. It should he preserved in a perfectly- 
 tight stoppered bottle, or preferably in the tube actually employed for com- 
 paring it with the glucose solution. 
 
 Sir G. Johnson employs for the comparison a tube about 12 inches long 
 and 1 inch in diameter, graduated into 100 c.c, accurately stoppered and' 
 lipped. The standard solution is contained in a similar but shorter tube of 
 exactly the same diameter, and accurately stoppered. These, under the 
 name of "Johnson's Picro-saecharometer," can be obtained from C. E. 
 Muller & Co., 148 High Holborn, London, W.C. 
 
 In the opinion of the author, Johnson's tubes are much wider than is 
 desirable, far better results being obtainable by the use of the narrow 
 graduated tubes employed for estimating carbon in steel by Eggertz' colour- 
 test, and obtainable under that description of any apparatus-maker. Five c.c. 
 measure of the sample of urine is treated with 5 c.c. of a saturated solution of 
 picric acid and 2 - 5 c.c. of normal caustic soda or potash. The liquid is 
 boiled for about a minute, cooled thoroughly, and made up to 20 c.c. with 
 distilled water. Five c.c. of this liquid is poured into the graduated tube, 
 which holds 25 or 30 c.c, and is graduated to 0'1 c.c. Five c.c. of the standard 
 solution of ferric acetate is placed in the twin tube, and the colours of the 
 two liquids compared by holding the tubes side by side up to a good light, 
 and looking through them transversely. The comparison is facilitated by 
 placing a piece of moistened filter-paper between the tubes and the light. 
 
 If the solution of the treated sample be lighter than the standard it may be 
 concluded that the urine contains less than 1 grain of sugar per ounce. 
 (" All normal urines reduce picric acid to an extent equivalent to J to 1 grain 
 of glucose per fluid ounce. This reduction is due to creatinine, and should be 
 allowed for when the proportion of glucose present is small." — Sir O. John- 
 son.) If, on the other hand, the solution of the sample be darker than the 
 standard, the former must be diluted with distilled water until they are 
 exactly of the same tint. If the sample require dilution to more than 30 c.c. 
 in order to match the tint of the standard, the process must be repeated on 
 the suitably diluted sample of urine. 
 
 The volume to which the treated urine requires to be made up, divided by 
 
 five, gives the number of grains of glucose contained in each fluid ounce of the 
 
 urine. Thus, if a sample of urine be diluted to four times its measure with 
 
 water, and 5 c.c. of this, after treatment with picric acid, require to be 
 
 diluted to 23 c.c. in order to match the standard, then : — 
 
 4 x 23 
 
 — -z — = 18 '4 grains of sugar per fluid ounce. 
 
 This result, divided by 4-375, gives 4-26 per cent, of sugar in the urine. 
 
METHYLENE-BLUE TEST. 81 
 
 urine, the test is not, in the opinion of the author, so 
 useful or trustworthy as that with safranine, where the 
 reaction consists in the discharge of a deep red colour 
 by sugar, instead of in its production. Further, the 
 safranine test is not affected by the constant presence 
 of creatinine. If the creatinine be previously removed 
 by mercuric acetate, as recommended by G. S. John- 
 son, and the mercury subsequently entirely removed 
 from the filtrate, as described on page 70, the picric 
 acid test will indicate very minute quantities of 
 glucose, and very fair cplorimetric estimations may be 
 made in the liquid thus clarified. 
 
 Methylene-Blue Reaction foe Glucose. — The 
 coal-tar colouring matter known in commerce as 
 methylene-blue is the hydrochloride or double zinc 
 salt of a base having the formula C 16 H 18 N 3 S.OH, and 
 the constitution of a tetramethyl-thionine 
 hydroxide. By the action of reducing agents, 
 including a boiling alkaline solution of glucose, this 
 body is reduced to tetramethyl-leucothion- 
 i n e, C 16 H 19 N 3 S, a colourless substance which under- 
 goes oxidation with extreme facility, forming methyl- 
 ene-blue and other products. N. Wender (Pharm. 
 Post., xxvi. 93, and Analyst, xviii. 131) has pro- 
 posed to utilise this reaction for the recognition 
 and rough estimation of diabetic sugar. The test 
 has been fully tried in the author's laboratory, and 
 the following mode of employing it is substantially 
 that prescribed by Wender : — 
 
 A solution of methylene-blue is prepared by dissolv- 
 ing 1 part of the commercial colouring matter in 3000 
 parts of distilled water ( = 0'333 gramme per litre). 
 Six c.c. ( = 90 minims) of this solution is first measured 
 into a test-tube, and 2 c.c. ( = 30 minims) of a normal 
 solution of caustic potash (or Liquor potassie, B.P.) 
 added. The sample of urine to be tested is then 
 
 p 
 
82 METHYLENE-BLUE TEST. 
 
 diluted to ten times its bulk with water, and 2 e.c. 
 ( = 30 minims) of the diluted urine added to the alka- 
 line solution of methylene-blue. The contents of the 
 test-tube are then boiled for a minute or two, avoiding 
 agitation or contact with air as much as possible, when 
 complete discharge of the blue colour will ensue if the 
 sample of urine contained 0'5 per cent, or more of 
 sugar. By repeating the test with 4 c.c. or still larger 
 volumes of the diluted urine, smaller proportions of 
 glucose may be approximately estimated. A good 
 plan is to immerse in a bath of boiling water four or 
 more test-tubes, each containing 6 c.c. of the methylene- 
 blue solution and 2 of caustic alkali, to which gradu- 
 ally increasing measures of urine have been added, 
 when a close indication of the amount of sugar can be 
 obtained in the time requisite for completing a single 
 test. Thus, if the blue colour is not discharged by 4 
 c.c. of the diluted urine, but is destroyed in the tube to 
 which 6 c.c. has been added, the proportion of sugar 
 present is less that 0"25, but greater than 0'17 per 
 cent. 
 
 The outward similarity of the reaction of glucose 
 with Pavy's solution to its behaviour with alkaline 
 methylene-blue, suggested to the author the possi- 
 bility of employing the latter reagent in precisely 
 the same manner as the ammoniacal copper solution. 
 A series of experiments in this direction, made in the 
 author's laboratory by G. B. Brook, have shown, how- 
 ever, that methylene-blue presents several practical 
 disadvantages. The discharge of the colour is not so 
 prompt nor the end-reaction so distinct as in the case 
 of Pavy's solution. The blue colour returns with the 
 utmost facility, a reduced solution of methylene-blue 
 being in fact one of the most delicate existing tests 
 for free oxygen. The employment of a layer of 
 paraffin oil is not feasible, since the free base of the 
 
SAFRANINE TEST FOR GLUCOSE. 83 
 
 colouring matter is liberated by the alkali and dis- 
 solves in the oil with red colour, and, of course, is 
 prevented from free reaction with the glucose, while 
 on the other hand it is exposed to the air. 1 
 
 As a clinical test for diabetic sugar, the methylene- 
 blue reaction is very serviceable. The discharge of 
 the colour is a phenomenon far more readily observed, 
 and requires less judgment, than the distinction be- 
 tween different shades of red and brownish-red re- 
 quired in applying the picric acid test. 2 
 
 Safranine Test for Glucose. Various other coal- 
 tar dyes are reduced and decolorised by heating with 
 glucose in alkaline solution. One of these, safra- 
 n i n e, recommended by M. Crismer [Pharm. Zeit. , 
 xxxiii. 65 ; Pharm,. Jour., [3], xix. 348), is especially 
 suitable for the purpose. Safranine occurs in com- 
 merce 3 as a brownish-red powder, readily soluble in 
 water to form a blood-red solution not liable to change 
 by keeping. No immediate precipitate is produced on 
 adding caustic alkali solution, but on standing more 
 or less separation occurs. On adding to the alkaline 
 liquid a solution of glucose, and heating it to the boil- 
 ing-point, the liquid is changed to a pale urine-yellow, 
 
 - 1 It is possible that the red substance dissolved by the paraffin oil is some 
 extraneous impurity, but the same behaviour occurs when ether is substituted, 
 and on separating the ethereal layer and shaking it with acid, a blue colouring 
 matter passes into the latter. Attempts to purify the commercial methylene- 
 blue by precipitating the colouring matter by potassium iodide or brine did 
 not affect its subsequent behaviour with paraffin oil. 
 
 2 The writer found approximately 5 per cent, of sugar by the methylene-blue 
 test in a sample of diabetic urine, which, by more accurate analysis, was proved 
 to contain i'd per cent. In this case 1 part of the urine was diluted to 100 
 by water. 
 
 3 Commercial safranine is a mixture of several homologous bodies, of which 
 C I9 H 17 N 4 C1, CaoHjg^Cl, and C 21 H 21 N 4 C1 are the chief. The second of these 
 has the constitution of a chloride of paramidophenyl-paramido-phenazonium, 
 and the following structural formula : — 
 
 ■■Uh 
 
 ,H 4 
 
 CI 'CHANE, 
 
84 SAFRANINE TEST FOR GLUCOSE. 
 
 and becomes turbid from the separation of the insoluble 
 leuco-derivative. If the glucose be not present in 
 large excess, the red colour returns on agitating or 
 exposing the solution to the air. 
 
 Safranine is not decolorised when heated in alkaline 
 solution with uric acid, creatine, creatinine, chloral, 
 chloroform, hydrogen peroxide, or salts of hydroxyl- 
 amine, and only slowly (but in the end completely) 
 by solutions of albumin. Hence any discharge of 
 colour produced by a sample of urine, preferably freed 
 from albumin, may confidently be set down to the 
 presence of sugar. The indifference of safranine to 
 1 uric acid and creatinine has been fully verified by the 
 author, who employs the reagent in the following 
 , manner : — Equal measures (2 c.c, or 30 minims) of the 
 urine, normal soda or potash (or liquor potassse, B.P.), 
 i and a solution of one part of safranine in 1000 parts of 
 ) water are mixed. The liquid is heated in a test-tube 
 1 till it boils freely, agitation being avoided as much as 
 possible. If the urine contain more than - l per 
 cent, of sugar the liquid will be decolorised, but other- 
 wise the red colour will remain intact or be only 
 1 partially discharged. If the colour be destroyed, the 
 i test may be repeated with twice or three times the 
 ' volume of safranine solution that is used of urine, 
 every additional measure of safranine which is found 
 ) to be decolorised representing roughly O'l per cent, of 
 sugar in the sample. If more than four or five 
 measures are required, the urine is distinctly diabetic, 
 and it is desirable to dilute a portion to ten times its 
 bulk with water, and employ equal measures of this 
 diluted urine, alkali, and safranine solution. Pellets 
 containing a definite weight of safranine may be used. 
 The safranine test has been fully investigated in 
 the author's laboratory byG. Bernard Brook. 
 \lt is one of the simplest and most certain clinical 
 
PHENYL-HYDRAZINE TEST. 85 
 
 tests for glucose, and deserves to come into general 
 use for that purpose. It possesses the advantage 
 over methylene-blue or picric acid of not being affected 
 by uric acid or creatinine, while its application is quite 
 as simple as that of either of these reagents. The rough 
 quantitative indications, obtainable as above indicated, 
 will often afford great assistance to the medical prac- 
 titioner. On the other hand, attempts to base on the 
 reaction an accurate quantitative method, by operat- 
 ing in the manner of Pavy, have been unsuccessful. 
 
 Reaction of Glucose with Phenyl-Hydrazine. 
 
 As all the methods of detecting sugar in urine which 
 are based on the reducing action of glucose are more 
 or less vitiated by the presence of other reducing 
 bodies, a special reagent for glucose has an exceptional 
 value. This reagent exists in phenyl-hydrazine, 
 which the classical researches of E. Fischer have 
 shown to react with all bodies of the aldehyde and 
 lactone classes, including glucose, with formation of 
 well-defined compounds which are generally readily 
 crystallisable, and insoluble, or only very sparingly 
 soluble, in water. Thus, with a single molecule of 
 phenyl-hydrazine, dextrose reacts to form dextrose- 
 phenyl-hydrazine, C 12 H 18 N 2 5 , a body which 
 forms fine crystals fusing at 144° to 145° C. But with 
 excess of phenyl-hydrazine, a body of the osazone class 
 is formed, dextrose yielding phenyl-dextros- 
 a z o n e, C 18 H 22 N 4 4 . Identically the same osazone 
 appears to be formed from lsevulose, so that it may 
 be conveniently called phenyl- glucosazone. 
 Similar compounds, differing in their melting-points, 
 are yielded by maltose and lactose, but not by cane- 
 sugar. The following table exhibits the distinguishing 
 characters of such osazones as are interesting in con- 
 nection with the examination of diabetic urine : — 
 
86 
 
 CHARACTERS OF OSAZONES. 
 
 Sugar, &o. 
 
 Compound (Osazone) with Phenyl -hydrazine. 
 
 Formula. 
 
 Appearance. 
 
 Melting- 
 point, ° C. 
 
 Dextrose, 
 Lsevulose, 
 
 Maltose, 
 
 Lactose, 
 
 Cane-sugar, . 
 
 Glycuronic acid, . 
 
 }C 18 H 22 N 4 4 
 C 24 H 32 A 4 9 
 
 C 2 4 H 32 N 4°9 
 
 No compound. 
 
 *-'42-°-48-'^loOlO 
 
 Needles. 
 
 Tables. 
 Wide prisms. 
 
 Yellow needles 
 
 or brown 
 resinous mass. 
 
 204 to 205. 
 
 190 to 191. 
 200. 
 
 115 to 115. 
 
 In applying Fischer's reagent in practice, the 
 phenyl-hydrazine is most conveniently used as the 
 hydrochloride, a salt which forms small, thin, lustrous 
 plates, of the formula C 6 H 5 .HN".NH 2 ,HC1. The crystals 
 are sparingly soluble in cold water, more readily in 
 hot. The salt is almost completely precipitated from 
 its aqueous solution by strong hydrochloric acid, 
 which behaviour furnishes a means of readily purify- 
 ing it from aniline hydrochloride and other impurities. 
 Much of the phenyl-hydrazine hydrochloride of com- 
 merce has a brownish tint, and should be purified in 
 the manner above indicated, followed by re-crystallisa- 
 tion from hot water. 1 
 
 To apply the phenyl-hydrazine test, E. von J a k s c h 
 (Zeits. din. Med., xi. 20) recommends that 50 c.c. of 
 the suspected urine, previously freed from albumin, 
 should be treated with 2 grammes of sodium acetate 
 and from 1 to 2 grammes of phenyl-hydrazine hy- 
 drochloride, 2 and the liquid heated to 100° C. for 
 
 1 C. W. Purdy states that phenyl-hydrazine hydrochloride must be handled 
 with great caution, as its contact with the skin is liable to produce eczema of 
 a malignant type. 
 
 2 Or 2 oz. of urine, 40 grains of sodium acetate, and from 20 to 30 grains 
 of phenyl-hydrazine hydrochloride. 
 
PHENYL-GLUCOSAZONE. 87 
 
 half an hour. On cooling, the phenyl-glucosazone 
 separates as an amorphous or crystalline precipitate 
 of a yellow or brick-red colour. If amorphous, the 
 precipitate should be dissolved in hot alcohol, and the 
 solution diluted with water and boiled, to expel the 
 greater part of the alcohol, when the glucosazone will 
 separate at once, or on cooling, in the form of charac- 
 teristic yellow needles, which melt at 205° C. with de- 
 composition. Phenyl-glucosazone is nearly insoluble 
 in cold water or alcohol, more soluble in hot water, 
 and moderately soluble in hot alcohol. It is dissolved 
 by glacial acetic acid to form a leevo-rotatory solution. 
 
 Instead of operating in the manner prescribed by 
 von Jaksch, the phenyl-hydrazine test may be 
 applied in the following simple manner, which is 
 substantially that recommended byC. Schwartz 
 (Pharm. Zeit., xxiii, 465) : — Ten c.c. of the urine is 
 heated to boiling and treated with half its volume, 
 or a sufficiency, of a 10 per cent, solution of neutral 
 lead acetate. The liquid is boiled and filtered hot. 
 Solution of caustic soda, in amount sufficient to 
 redissolve the precipitate which first forms, is added 
 to the filtrate, and then a little (as much as will lie on 
 the point of a pen-knife) phenyl-hydrazine hydro- 
 chloride is dropped in. The liquid is then boiled 
 for some minutes, and then strongly acidulated with 
 acetic acid. In presence of much sugar, an immediate 
 yellow turbidity or precipitate will be formed, but 
 if only minute traces be present a yellow coloration 
 is first produced, which, on cooling and standing, 
 changes to a turbidity. In all cases, considerable 
 time is required for the complete separation of the 
 glucosazone, but the qualitative indication is readily 
 and quickly obtained. 
 
 Unfortunately, the phenyl-hydrazine test does not 
 appear susceptible of being applied quantitatively, 
 
88 PHENYL-HYDRAZINE TEST. 
 
 though, of course, the intensity of the reaction and 
 the amount of precipitate afford a fair indication of 
 the proportion of sugar present. As little as 0'005 
 per cent. ( = ^o grain per ounce) of glucose can be 
 detected in urine by the phenyl- hydrazine reaction. 
 
 Uric acid, urea, xanthine, and creatinine in no way 
 simulate the reaction of glucose with phenyl-hydrazine. 
 The only bodies which can cause confusion are gly- 
 curonic acid and its derivatives (page 38). Glycuron- 
 ates give with Fischer's reagent a yellow, crystalline 
 compound resembling phenyl-glucosazone, but melting 
 at 150°. When the osazone is produced in an acid 
 solution, as in von Jaksch's modification of the test, 
 the glycuronic acid compound is precipitated as a 
 dark brown resinous mass, in which form it can cause 
 no confusion. According to H i r s c h 1 (Zeit. physiol. 
 Chem., xiv. 377), in applying the phenyl-hydrazine 
 test to urine, the liquid should be heated on the 
 water-bath for at least an hour. The fine, bright 
 yellow needles of phenyl-glucosazone, melting at 
 204° C, are readily distinguished from the brownish 
 amorphous compound, melting at 150°, produced by 
 glycuronic acid ; but if the heating be not continued 
 for the prescribed time, the latter compound may be 
 deposited in a crystalline state, and be mistaken for 
 glucosazone. Hirschl believes that traces of glycuronic 
 acid compounds are normally present in human urine. 
 
 In all doubtful cases the indications furnished by 
 the production of a turbidity or precipitate with 
 phenyl-hydrazine should be confirmed by obtaining 
 the osazone in a crystalline form, and examining it 
 under the microscope. If possible, the melting-point 
 should be observed, and in making this determination 
 it is essential that the heating should be conducted as 
 rapidly as possible, as the compound readily undergoes 
 decomposition at a temperature approaching its melt- 
 
PHENYL-HYDRAZINE TEST. 89 
 
 ing-point. The coloration obtained on moistening a 
 crystal of the glucosazone with a drop ,of concentrated 
 sulphuric acid is also an important means of identifi- 
 cation. Glucosazone acquires an olive-green colour, 
 and on subsequently adding a minute quantity of 
 manganese dioxide a fine purple colour is obtained, 
 closely resembling that yielded by strychnine under 
 similar treatment, but not undergoing the subsequent 
 change to cherry-red which characterises the alkaloid 
 (A. H. Allen). 
 
 The author has observed that phenyl-glucosazone 
 is readily dissolved by ether from its acidulated 
 aqueous solutions. On separating and evaporating 
 the ether, the glucosazone can be dissolved in alcohol, 
 and crystallised by adding water and evaporating, as 
 already described. 
 
 According to E o o s, human urine always gives an 
 affirmative reaction with the phenyl-hydrazine test, and 
 the same is said to be true of dogs' urine, while the 
 excretion of rabbits gives especially well -formed 
 crystals. By the application of the phenyl-hydrazine 
 test to a limited number of samples of urine from 
 persons apparently in perfect health, the author 
 satisfied himself of the presence of minute traces of 
 sugar in each case. 
 
 According to von J a k s c h, no sugar can be de- 
 tected by the phenyl-hydrazine test in the urine of 
 persons poisoned by arsenic, potash, or sulphuric acid, 
 but it always indicates the presence of sugar in the 
 urine of those poisoned by carbonic oxide or other 
 irrespirable gases. It is not improbable that the 
 inhalation of the products of the imperfect combustion 
 of coal-gas, as evolved from the faultily constructed gas- 
 stoves now so commonly used, may account for some 
 of the cases in which traces of sugar have been found 
 in the urine of persons apparently in perfect health. 
 
90 BENZOYL CHLORIDE TEST. 
 
 Reaction of Glucose with Benzoyl Chloride. 
 
 If a solution, of glucose be shaken with a little 
 benzoyl chloride, C 6 H 5 .C0C1, white crystals of 
 a compound of the two bodies gradually separate. 
 Wedinski has employed the reaction to demon- 
 strate the existence of traces of glucose in normal 
 human urine ; but the behaviour of co-occurring sub- 
 stances must be studied more fully than has hitherto 
 been done before the indications of the test can be 
 fully accepted. 
 
 ACETONURIA. 
 
 j The later stages of diabetes are generally charac- 
 terised by the appearance in the urine of certain 
 
 , substances which are evidently products of incomplete 
 oxidation. Among these are hydroxybutyric 
 acid, acetone, and aceto-acetic ether. 
 These bodies probably have their origin in the proteids 
 of the food, for their amount is independent of any 
 addition of carbohydrates to the diet but becomes 
 greater with increased proteid metabolism. They do 
 not occur in all cases of diabetes, but generally in the 
 more severe forms of the disease, in which the destructive 
 metabolism of proteids is augmented. In the last stage 
 of diabetes, when coma sets in, the amount of hydroxy- 
 butyric acid increases, while that of acetone diminishes. 
 The occurrence of hydroxybutyric acid, aceto-acetic 
 ether, and acetone is jiot confined to diabetes, but has 
 been observed in many other maladies, including 
 fever, however caused ; cancer, under certain condi- 
 tions, and in acetonsemia, apparently produced by 
 ingestion of alcohol. Hence the formation of acetone 
 and the allied anomalous products of metabolism may 
 not improbably be a consequence of certain complica- 
 tions which accompany diabetes, and not of the disease 
 
DERIVATIONS OP ACETONE. 91 
 
 itself. The occurrence of aceto-acetic acid or its ethyl 
 ester has been noticed in the urine of healthy persons 
 after prolonged fasting, and minute traces of acetone 
 exist in normal urine. The usual proportion of 
 acetone excreted is stated to be O'OIO gramme per 
 diem, but this is said to be increased to O^OO gramme 
 per diem, or even more, in marked cases of acetonuria. 
 The relationship existing between acetic acid, alde- 
 hyde, acetone, aceto-acetic acid, and hydroxybutyric 
 acid is shown by the following formulae : — ■ 
 
 Acetic acid, .... CH 3 — CO— OH. 
 
 Aldehyde, .... CH S — CO— H. 
 
 Acetone (dimethylketone), . CH 3 — CO — CH 3 . 
 
 Aceto-acetic acid (diacetic acid), CH 3 — CO — CH, — CO. OH. 
 
 Hydroxybutyric acid; . . CH 3 — CH(OH)— CH 2 — CO.OH. 
 
 Acetone. Dimethyl-ketone. CH 3 .CO.CH 3 . 
 
 Acetone results from the dry distillation of acetates, 
 especially those of barium and calcium, and may be 
 obtained in various other ways. It is a colourless, 
 limpid liquid of peculiar ethereal odour and burning 
 taste. It is miscible in all proportions with water, 
 alcohol, and ether, but is insoluble in a saturated 
 solution of calcium chloride. 
 
 Acetone Las many of the characters of the aldehydes, 
 and like them unites with the acid sulphites of the 
 alkali - metals to form crystalline compounds. On 
 adding to a tolerably strong solution of acetone a cold 
 saturated solution of bisulphite of sodium, the soda 
 compound separates out in shining scales. 
 
 When treated in solution with diazobenzene- 
 sulphonic acid and a little sodium amalgam, acetone 
 yields a dark red coloration. Aceto - acetic ether 
 behaves similarly, while aldehyde gives a violet colour. 
 
 B a e y e r and D r e w s e n have proposed a test for 
 acetone based on the formation of indigo-blue. A few 
 crystals of the highly explosive ortho-nitro-benzalde- 
 
92 EEACTIONS OP ACETONE. 
 
 hyde are dissolved by the aid of heat in the suspected 
 urine. On cooling, the reagent separates as a white 
 cloud. The liquid is now made alkaline with dilute 
 caustic alkali, when a yellow colour, changing in a few 
 minutes to green and indigo-blue, will be developed if 
 acetone be present. 
 
 The test is more satisfactory and delicate if applied 
 to the first portion of the distillate from the suspected 
 urine. To this, a solution of the nitro-benzaldehyde 
 should be added, and the liquid made alkaline. If the 
 change of colour from yellow to green and blue occurs 
 tardily, the liquid may be shaken with chloroform, 
 which dissolves the nascent colouring matter. 
 
 Acetone reduces Fehling's solution. If the test be 
 applied to a distillate from the urine, of course all 
 interference from glucose and other fixed reducing 
 agents will be avoided. 
 
 P. Chautard (Bull. Soc. Chem., xlv. 83) recom- 
 mends for the detection of acetone that 200 c.c. of 
 urine should be distilled at a gentle heat, and the first 
 15 c.c. of the distillate treated with a solution of ros- 
 aniline acetate previously decolorised with sulphurous 
 acid. 1 If acetone be present, a fine reddish-violet 
 coloration is produced, the intensity of which varies 
 with the amount. It is stated that less than 0"01 per 
 cent, of acetone can be detected in urine in this manner. 
 
 VilHersandFayolle {Bull. Soc. Chem., 1894, 
 page 693, and Jour. Chem. Soc, lxvi. 2, page 489) state 
 that the crimson reaction with the rosaniline reagent 
 
 1 The reagent is prepared by mixing 30 c.c. of a solution of magenta (ros- 
 aniline hydrochloride) containing 1 gramme per litre ; 20 e.c. of a solution of 
 sodium bisulphite of 1*31 sp. gr. ; 3 c.c. of strong sulphuric acid and 200 c.c. 
 of water. Villiers and Fayolle prepare their reagent by adding to a solution of 
 magenta such an amount of sulphurous acid as will just decolorise it after long 
 standing in a closely stoppered bottle. This reagent is very sensitive to all 
 oxidising agents. It gives a red colour even on prolonged exposure to air. 
 Glucose, galactose, and invert sugar give a coloration as intense as aldehyde, 
 but Isevulose, cane-sugar, maltose, and lactose give no reaction until the lapse 
 of some days. 
 
DETECTION OF ACETONE. 93 
 
 is not produced by pure acetone, but is due to alde- 
 hyde, which they state is almost always formed simul- 
 taneously with acetone. It seems probable that the 
 reaction is common to both bodies, but is capable of 
 detecting a much smaller amount of aldehyde than of 
 acetone, since Mohler found that O'Ol gramme per 
 litre of aldehyde but not less than 0"5 gramme of 
 acetone could be detected in spirits by this test. 
 
 For the detection of acetone in urine, Legal 
 (Jour. Pliarm., [5], xviii. 206) adds some drops of 
 a concentrated solution of sodium nitroprusside, and 
 makes the liquid slightly alkaline with caustic potash. 
 When the red coloration first produced has disappeared, 
 a little acetic acid is added, which in presence of 
 acetone will produce a deep violet coloration. 
 
 A somewhat modified form of the above test has been 
 described byLeNobel( Chem. Centralb. ,1884, page 
 626). He adds to the urine a solution of sodium nitro- 
 prusside, previously made alkaline by caustic soda and 
 so dilute as to have only a slight red tint. If acetone 
 be present, even to the small extent of - 0002 gramme, 
 a ruby-red colour will be produced, changing in a few 
 moments to yellow, and on acidulating the liquid and 
 boiling, a greenish-blue or violet colour is developed. 1 
 
 The most delicate test for acetone is that based on 
 its ready conversion into iodoform when treated with 
 caustic alkali and a solution of iodine. The following 
 equation represents the final reaction, but the highly 
 unstable potassium hypoiodite is probably formed as 
 a first product. 
 C 3 H 6 0+4K0H+3I 2 = 3KI+KC 2 H 3 2 +CHI 3 + 3H 2 0. 
 
 Acetone. Acetate. Iodoform. 
 
 To apply the iodoform test, 10 c.c. of the suspected 
 
 1 The nitroprusside test has been applied very successfully by A. Ashby 
 to the detection of acetone in methylated spirit and liquids containing it. 
 The orange-yellow colour given by aldehyde can scarcely be mistaken for the 
 far more intense colour produced by acetone. 
 
94 DETECTION OP ACETONE. 
 
 liquid should be treated with five or six drops of a 10 
 per cent, solution of caustic potash or soda, and the 
 liquid warmed to about 50° C. A solution of potassium 
 iodide, fully saturated with free iodine, is next added 
 drop by drop, with agitation, until the liquid becomes 
 permanently yellowish-brown, when it is carefully 
 decolorised by a further cautious addition of the 
 caustic alkali solution. Any iodoform will now be 
 apparent as a yellowish turbidity, gradually deposit- 
 ing at the bottom of the liquid as a yellow dust, 
 which, when examined with a lens, is seen to be 
 crystalline. Under a power of 300 diameters, its 
 appearance is very characteristic, the usual forms 
 being hexagonal plates, stars, and rosettes. The 
 formation of iodoform is pre-eminently a test for 
 acetone, but it is not peculiar to that substance, 
 being also produced by alcohol and its homologues, 
 aldehyde, various ethers, turpentine, sugar, &c. On 
 the other hand, it is not given by pure methyl or 
 amyl alcohol, chloroform, chloral, ether, or glycerin; 
 by acetic, formic, or oxalic acid; nor by acetal till 
 after the addition of acid. With acetone the reaction 
 occurs immediately and in highly dilute solutions, 
 whereas alcohol and most of the other above-named 
 bodies are converted into iodoform only slowly and in 
 concentrated solutions. 
 
 For the quantitative determination of acetone in 
 urine by the iodoform reaction, H. Huppert {Zeits. 
 anal. Chem., xxix. 632) recommends a method devised 
 by Messinger (Berichte, xxi. 3366). To get 
 rid of interfering bodies, such as phenol, ammonia, 
 and nitrous acid, 100 c.c. of the urine should be 
 previously distilled with 3 c.c. of acetic acid, the 
 distillate redistilled with 1 c.c. of dilute sulphuric 
 acid, and if nitrous acid be present a third distillation 
 with some urea is directed. 
 
DETERMINATION OF ACETONE. 95 
 
 Messinger's method for the estimation of acetone, 
 as modified by Collischonn (Zeits. anal. Chem., 
 xxix. 562), is as follows : — Twenty c.c. of normal caustic 
 soda, free from nitrites, is added to the distillate obtained 
 as above, and 30 c.c. of ^ normal iodine solution 
 added with constant agitation. The shaking is con- 
 tinued for four or five minutes, while the liquid is 
 warmed to a temperature not exceeding 60° C. The 
 liquid is then acidified with dilute hydrochloric acid 
 (sp. gr. 1"025), an excess of decinormal sodium thio- 
 sulphate added, and the solution finally titrated back 
 with the iodine solution, starch being used as an 
 indicator. 
 
 Every 762 parts of iodine which enter into com- 
 bination represent 70 parts of acetone previously 
 present. 
 
 Instead of estimating the iodoform volumetrically, 
 the excess of iodine may be got rid of by adding thiosul- 
 phate solution of unknown strength, and the iodoform 
 extracted by agitating the liquid with ether, which is 
 then separated, allowed to evaporate spontaneously, 
 and the residue of iodoform weighed. 142 parts of 
 iodoform represent 70 of acetone. The result is liable 
 to be low, owing to the volatility of iodoform even at 
 ordinary temperatures. 
 
 A. Schwicker (Chem. Zeit., xv. 914) employs 
 ammonia instead of fixed caustic alkali in making the 
 iodoform test. Alcohol then in no way interferes, 
 but aldehyde must be absent. The first fraction of 
 the distillate from the suspected urine is mixed with 
 a few drops of strong ammonia, and a few drops of 
 decinormal solution of iodine then added. A black 
 precipitate of nitrogen iodide is first formed, but this 
 disappears on shaking and warming the liquid. If 
 acetone be present in not too small a quantity, iodo- 
 form now makes its appearance, and increases on 
 
96 ACETONE IN URINE. 
 
 adding a little more iodine. The characteristic odour 
 is not greatly obscured by that of the ammonia. If 
 the nitrogen iodide does not readily disappear, it may 
 be got rid of by cautiously adding a few drops of very 
 dilute sodium thiosulphate (hyposulphite) solution. 
 If only traces of acetone are present, more time 
 should be allowed before removing the nitrogen iodide, 
 and the iodine solution used should be very dilute. 
 One part of acetone in 5000 of water is said to be 
 recognisable by this modification of the test. To- 
 gether with iodoform, there is formed a substance 
 which is characterised by a pungent smell and the 
 property of exciting lachrymation. It is probably an 
 iodine-substitution-product of acetone. 
 
 E. Salkowski states that sugar, when present in 
 urine, gives rise to a substance of an aldehydic nature 
 (probably acetaldehyde), which on distillation simu- 
 lates many of the reactions of acetone, including the 
 formation of iodoform. 
 
 The breath and urine of persons in which acetone 
 can be found by the iodoform and other chemical tests 
 often have an odour of acetone. Though the occur- 
 rence of acetone in the urine often precedes the coma 
 which ushers in the last stage of diabetes, acetone 
 is not itself a toxic agent. Given in large doses to 
 men and animals, even when diabetic, it produces no 
 coma, and it is met with in many febrile diseases 
 in which no coma occurs. 
 
 Von Jaksch found minute traces of acetone in 
 the urine of healthy persons, the amount excreted 
 being in some cases as high as O'Ol gramme ( = - 15 
 grain) in the twenty-four hours. For its detection in 
 such minute quantities a large quantity {e.g., 10 
 gallons) should be acidulated with dilute sulphuric 
 acid and distilled, the first fractions redistilled, and the 
 distillate saturated with fused calcium chloride, which 
 
ACETO-ACETIC ETHER. 97 
 
 unites with the alcohol while leaving the acetone un- 
 changed. The liquid is distilled on the water-bath, 
 the distillate again treated with calcium chloride, and 
 the process repeated until the acetone is concentrated 
 in a comparatively small bulk of fluid, when it may- 
 be detected and determined by the methods already 
 detailed. 
 
 AcetO-Acetic Acid. Diacetic Acid. Acetone- 
 Carboxylic Acid. CH 3 .CO.CH 2 .COOH. 
 
 This body has been obtained synthetically by treat- 
 ing aceto-acetic ether in the cold with a solution of an 
 equivalent quantity of caustic potash, and after twenty- 
 four hours acidifying the liquid and agitatingwith ether, 
 which extracts the free aceto-acetic acid. On evapora- 
 tion, the acid is obtained as a syrupy liquid, miscible 
 with water and decomposing at 100° C. into acetone 
 and carbon dioxide : — 
 
 CH 3 .CO.CH 2 .COOH = CH 3 .CO.CH 3 +C0 2 . 
 
 Aceto-acetic acid and its salts give a violet or claret- 
 red coloration with ferric chloride. The barium salt 
 is amorphous and very soluble in water. The copper 
 salt is also amorphous. 
 
 Ethyl Aceto-acetate, Aceto-acetic Ether, or Di- 
 acetic Ether, is the ethyl ester of aceto-acetic acid. Its 
 composition is CH 3 .CO.GH 2 .CO.O(C 2 H 5 ). It is pre- 
 pared by the action of sodium on perfectly dry ethyl 
 acetate. Aceto-acetic ether is a liquid of an agreeable 
 fruity odour, having a specific gravity of T031T at 
 15° C, compared with water at the same temperature. 
 It boils at 181° C, is slightly soluble in water, and the 
 solution gives a violet or claret coloration with ferric 
 chloride. Aceto-acetic ether resembles aldehydes and 
 ketones in combining with the acid sulphites of the 
 alkali-metals to form crystalline compounds, which are 
 decomposed into their constituents by treatment with 
 
 G 
 
98 ACETO- ACETIC ETHER. 
 
 sodium carbonate. The formation of the compound 
 with acid sodium sulphite may be conveniently used 
 for purifying the ether. 
 
 Aceto-acetic ether, like acetone, gives, with diazo- 
 benzene-sulphonic acid and a little sodium amalgam, 
 a dark red coloration. 
 
 Aceto-acetic ether behaves like a monobasic acid, and 
 forms salts. The sodium compound is produced by the 
 action of sodium or sodium ethylate on aceto-acetic 
 ether in the cold, and has the following formula : — 
 CH 3 .CO.CHNa.CO.O(C 2 H 5 ). 
 
 By the action of alkalies, aceto-acetic ether under- 
 goes decomposition into acetone, alcohol, and carbonic 
 acid. The sodium salt reacts similarly, the change in 
 this case being represented by the equation : — 
 
 CH 3 .CO.CHNa.CO.O.C 2 H 5 + 2H 2 = CH 3 .CO.CH s + 
 H.O.C 2 H 5 + NaHC0 3 . 
 
 This decomposition probably occurs in the blood and 
 urine in cases in which acetone is present. 
 
 The presence of aceto-acetic acid or its derivatives 
 in urine is an unfavourable symptom often preceding 
 the onset of diabetic coma and death. Such urine 
 often has an odour of acetone, and gives a violet or 
 claret-red coloration with ferric chloride. The colour 
 is destroyed by boiling, and is not produced at all if 
 the urine has been boiled previously. Ferric chloride 
 produces a somewhat similar coloration in the urine 
 of persons who have taken salicylic acid, phenol, 
 antipyrine or thalline, but the reaction due to these 
 drugs is not affected by heat. 
 
 By the distillation of diabetic urine, which gives the 
 above red or violet reaction with ferric chloride, a dis- 
 tillate is obtained containing acetone and alcohol. 
 The products are usually attributed to the decom- 
 position of ethyl aceto-acetate, which would split 
 up with formation of equal molecules of alcohol and 
 
HYDROXYBUTYRIC ACID. 99 
 
 acetone, thus:— CH 3 .CO.CH 2 .CO.O(C 2 H 5 ) + H 2 = 
 CH 3 .CO.CH 3 +C0 2 + HO(C 2 H 5 ). But in a case in- 
 vestigated by To liens no alcohol appeared in the 
 distillate, although much wine was taken. By treat- 
 ing the distillate with soda and iodised potassium 
 iodide, and weighing the iodoform produced, the 
 acetone in the urine was found to vary from 0*093 
 to 0"147 per cent. From the production of the 
 iodoform reaction by the distillate, and the absence 
 of alcohol therein, T o 1 1 e n s concluded that the body 
 producing the reaction with ferric chloride was free 
 aceto-acetic acid, and not its ethyl ester. 
 
 Tollens further found that urine giving the ferric 
 chloride reaction, when distilled slowly in presence of 
 hydrochloric acid, yielded distillates which gave the 
 iodoform reaction ; while the residual liquid, even to 
 the end, continued to show the ferric chloride reaction. 
 The original urine yielded to ether a mere trace of the 
 body giving the violet reaction, but after acidification 
 with sulphuric acid ether extracted it readily. 
 
 Hydroxybutyric Acid. CH.,CH(OH).CH„COOH. 
 
 The kind of hydroxybutyric acid present in urine, 
 and represented by the foregoing formula, is the (3-modi- 
 fication. It was first discovered in the urine by M i n- 
 kowski, and first obtained synthetically by acting 
 on ethyl aceto-acetate with water and sodium-amalgam. 
 It is also formed when propylene chlorhydrin is heated 
 to 100° with alcohol and potassium cyanide, and the 
 nitril thus produced decomposed by caustic potash. 
 The resultant liquid is acidulated with sulphuric acid 
 and shaken with ether. The ethereal layer, when eva- 
 porated, leaves the hydroxybutyric acid as a viscid 
 deliquescent syrup, volatile in a current of steam, but 
 converted, when heated alone to 130° C, into a-crotonic 
 acid, with loss of the elements of water. As prepared 
 
100 HYDROXYBUTYRIC ACID. 
 
 from diabetic urine, by an adaptation of the foregoing 
 process, hydroxybutyric acid bas a lsevo-rotatory power 
 of —23 "4° for tbe sodium ray. The sodium salt 
 forms very deliquescent needles. Tbe zinc salt is 
 amorphous. 
 
 Hydroxybutyric acid is poisonous when introduced 
 into the circulation. With ferric chloride it yields a 
 claret or violet-red colour similar to that produced by 
 aceto-acetic ether. 
 
 The formation of j3-hydroxybutyric acid by the action 
 of nascent hydrogen on aceto-acetic acid bas already 
 been referred to ; the quantities of the two bodies 
 present in diabetic urine are stated by W o 1 p e to 
 rise and fall together. 
 
ALBUMINOUS URINE. 
 
 Normal urine is almost, if not entirely, free from any 
 trace of albumin 1 or other of the allied substances 
 classed together under the generic name of " proteids." 2 
 
 1 The term "albumen" should be limited to its original signification, 
 namely, the white of egg ; the word "albumin" being applied to the most 
 characteristic constituent thereof, and extended to other analogous substances 
 contained in blood-serum, &c. The terms albumen and albumin will then 
 have the same relation to each other as benzol and benzene. In pronouncing 
 the word albumin, it is correct to accentuate the penultimate, but of late 
 years extensive custom has justified the accentuation of the first syllable. 
 
 2 The bodies called proteids are highly complex and, for the most part, 
 uncrystallisable compounds of carbon, hydrogen, nitrogen, oxygen, and 
 sulphur, having an ultimate composition ranging, according to Hoppe- 
 S e y 1 e r, within the following limits : — 
 
 Carbon. Hydrogen. Oxygen. Nitrogen. Sulphur. 
 From 51-5 6"9 20-9 15'2 0"3 per cent. 
 
 To 54-5 7-3 23-5 17"0 2'0 „ 
 
 Proteids occur in a solid viscous state or in solution in nearly all the solids 
 and fluids of the animal economy. The different individuals of the group 
 present differences in physical and, to some extent, in chemical properties ; 
 but all possess many chemical reactions in common, and they are united in a 
 close genetic relationship. 
 
 "W. D. Halliburton (Watts' Diet. Chem., 1894, iv. 330) classifies 
 proteids as follows : — 
 
 I. Albumins. Soluble in water, and not precipitated by saturating their 
 solutions with common salt or magnesium sulphate. Coagulated by heat. 
 Egg-albumin, serum-albumin, and lact-albumin are the most important 
 members of the group. 
 
 II. Globulins, represented by serum-globulin, fibrinogen, myosin, crystallin, 
 and globin. They are insoluble in water, but soluble in dilute solutions of 
 neutral salts, and are precipitated in an uncoagulated condition by saturating 
 these solutions with common salt or magnesium sulphate. Globulins are 
 coagulated by heat. 
 
 III. Albuminates, represented by alkali-albumin, acid-albumin or syntonin, 
 and metallic compounds of proteids. Acid- and alkali-albumin are insoluble 
 in water and solutions of neutral salts, but readily soluble in dilute acids and 
 alkalies. They are not coagulated by heat. 
 
 IV. Proteoses are proteids which are not coagulable by heat, but are mostly 
 precipitated by certain neutral salts. They are precipitated in the cold by 
 nitric acid, the precipitate dissolving on application of heat, and reappearing 
 
102 ALBTTMINUEIA. 
 
 But under particular conditions of fatigue or disease, 
 albumin may appear in the urine. 
 
 Temporary albuminuria is sometimes induced by a 
 cold bath, especially in persons prone to kidney-disease, 
 and it has been observed after excessive muscular exer- 
 cise, as in the urine of soldiers after a prolonged march. 
 
 Any cause which leads to increased blood-pressure 
 i in the kidneys tends to induce albuminuria, and many 
 of the cases in which it is the result of disease may 
 be traced to this cause. Albuminuria is a constant 
 accompaniment of the nephritis following scarlet fever, 
 and may occur to a less marked extent in pneumonia, 
 typhoid, and diphtheria. It may also occur in dia- 
 betes, and is then a highly unfavourable symptom. 
 
 The proportion of albumin present in pathological 
 urine varies greatly. In certain diseases the urine some- 
 times becomes so highly albuminous that on heating 
 it will undergo coagulation in a manner similar to 
 white of egg. More frequently, comparatively small 
 amounts of albumin are found, and in the cases of con- 
 valescent patients it is often of importance to ascertain 
 the presence or absence of mere traces of albumin. 1 
 
 as the liquid cools. Proteoses are intermediate in characters between the 
 proteids of Groups I., II., and III. and the peptones. (Compare page 120.) 
 
 V. Peptones are the ultimate products of the action of proteolytic ferments 
 on the bodies of the preceding groups. They are very soluble in water, not 
 precipitated by neutral salts or nitric acid, and not coagulated by heat. They 
 give the biuret reaction, are incompletely precipitated by picric, phospho- 
 tungstic, and phospho-molybdic acids, but completely by tannin, potassio- 
 mercuric iodide, and excess of absolute alcohol. Peptones are divided into 
 hemipeptones, which yield leucine and tyrosine as the further result of pan- 
 creatic digestion, and antipeptones which do not give these products. 
 
 VI. Insoluble proteids. This class includes a number of bodies not classified 
 above, but which resemble each other in their extreme insolubility in various 
 reagents. Among them are fibrin, coagulated albumin and casein, lardacein, 
 antialbumid, and gluten. 
 
 1 In cases of Bright's disease, the urine rarely contains more than 1 per 
 cent, of proteids ; but taking the volume of urine at the moderate estimate of 
 50 oz. daily, this corresponds to a loss of about 220 grains, or 1 4 grammes per 
 diem. F r e u n d has pointed out that the blood of the average body contains 
 only about 450 grammes of albumin, so that if only 7 to 8 grammes of this be 
 lost daily the condition is alarming. 
 
REACTIONS OF URINARY PROTEIDS. 
 
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104 COLLECTION OF URINE. 
 
 Detection of Albumin in Urine. 
 
 Until recently, the proteids liable to occur in urine 
 were classed together under the general name of 
 " albumin," but it is now recognised that several 
 forms are of common and simultaneous occurrence, 
 and apparently have a varied pathological significance. 
 
 The table on page 103 exhibits in a convenient 
 form the chief reactions of solutions of the proteids 
 which are liable to occur in urine. 
 
 For clinical use and medical purposes generally, it 
 is necessary to employ simple but fairly delicate tests 
 for the detection of albumin in urine, and many 
 attempts have been made to fulfil the requisite con- 
 ditions. The following are among those tests which 
 experience has shown to be most generally available 
 and reliable for the purpose, but the recognition of 
 mere traces of albuminous matters in urine is often of 
 great importance, and to effect this with certainty the 
 tests must be applied with care and skill. 
 
 Urine to be examined for albumin should, by 
 preference, be the mixed excretion of the previous 
 twenty-four hours ; but it is easy to lay too much 
 stress on this desideratum. It is better to have a 
 carefully collected sample of the urine passed at one 
 time than a sample of mixed urine collected under 
 conditions open to exception. Thus, in collecting a 
 sample of urine to be examined for albumin, it is 
 important to reject the first ounce or two passed, in 
 order to wash casual discharges out of the urethra. 
 The urine which follows should be passed direct into 
 the sample-bottle, which must be scrupulously clean 
 and may conveniently contain six ounces. 
 
 Before applying any of the following tests for 
 albumin it is essential that the urine to be examined 
 {should be filtered, so as to obtain an absolutely clear 
 liquid, and to ensure its freedom from casual contami- 
 
HEAT TEST FOR ALBUMIN. 105 
 
 nation with semen, mucus, epithelial cells, or other 
 debris from the urinary passages. 1 
 
 Previous to filtration, it is important to observe the 
 reaction of the urine. If a slip of blue litmus-paper be 
 promptly reddened when dipped for an instant into 
 the urine, the liquid may at once be filtered. Urine 
 passed during the so-called alkaline tide (page 9) may 
 fail to redden blue litmus, or may even restore the 
 blue colour to reddened litmus-paper. In such case, 
 the urine, before filtration, should be acidulated by 
 adding dilute acetic acid, 2 drop by drop, with frequent 
 agitation to ensure perfect homogeneity, until the 
 attainment of a proper degree of acidity is marked by 
 the prompt reddening of a slip of immersed litmus- 
 paper. The urine thus treated will, on filtration, 
 yield a perfectly bright filtrate, which practically is 
 the true urinary excretion, will give no trouble from 
 the presence of mucin, and will yield no precipitate 
 of earthy phosphates or other salts on boiling. 
 
 Heat Test. — One of the simplest, and in many 
 cases most satisfactory, tests for the presence of 
 albumin in urine is that of heat. About 10 c.c. (or 
 J oz.) of the sample, previously filtered and, if neces- 
 sary, acidulated, as above described, is boiled for about 
 one minute in a test-tube. If albumin be present, as 
 the boiling point is approached a cloud is seen to form 
 at the top of the liquid, and as the urine is boiled 
 the whole of the -albumin separates as a soft, white, 
 opaque precipitate, more or less dense according to the 
 
 1 To ensure perfect filtration, which, when practicable, should be performed 
 on the fresh, warm excretion, a fine close filter-paper should be employed. 
 The importance of operating on carefully filtered urine has been pointed out 
 and insisted on by James Edmunds {Lancet, November 9th, 1889, page 978). 
 
 2 The use of too strong an acid should be avoided. Dilute acetic acid of the 
 Pharmacopoeia contains 4 - 27 per cent, of real acetic acid, and is a suitable 
 reagent for the purpose ; or normal acetic acid containing 6 per cent, of real 
 acid, which can be obtained approximately by diluting 2 fluid ounces of 
 Acetic Acid, B.P., with 11 fluid ounces of distilled water, may be employed. 
 
106 HEAT TEST FOR ALBUMIN. 
 
 proportion which may be present. On standing for 
 a few minutes this precipitate aggregates into distinct 
 flocculi, and these gradually sink to the bottom of the 
 test-tube, leaving the supernatant liquid clear. After 
 standing for twenty-four hours, the volume occupied 
 by the precipitate will afford a rough indication of the 
 proportion of albumin present. (Compare Esbach's 
 test, page 111.) In highly albuminous urines the 
 precipitate is occasionally so voluminous as to cause 
 the coagulation of the entire liquid. 
 
 By the foregoing simple mode of procedure, any 
 proportion of albumin greater than traces will be 
 readily detected. For the detection of smaller 
 quantities, equal measures of the carefully filtered 
 acidulous urine should be placed in two exactly 
 similar test-tubes. The liquid in one tube is then 
 boiled, when, on comparing its appearance with that 
 in the other tube, placed side by side with it, the 
 faintest opalescence will be readily perceived, especially 
 if the tubes be observed in a proper light, with a 
 black background for the line of vision. An alterna- 
 tive plan is to boil the upper part of a column of urine 
 in a somewhat long test-tube by means of a small 
 flame. Any albumin in the upper heated portion of 
 the liquid will thus be coagulated, and will present 
 a marked contrast to the pellucid lower layer. 
 
 If not in a distinctly acidulous condition, human 
 urine, on boiling, often yields a precipitate of earthy 
 phosphates, while calcium carbonate is sometimes 
 thrown down from the urine of herbivorous animals. 
 These precipitates are readily and completely re-dis- 
 solved on adding a few drops of acetic acid, with 
 agitation between each addition, while a precipitate 
 of albumin will remain unchanged under such treat- 
 ment. A pulverulent precipitate of earthy phosphates, 
 usually CaHP0 4 , is easily distinguished from the fine 
 
NITRIC ACID TEST. 107 
 
 floceuli into which an albuminous precipitate soon 
 aggregates. From alkaline urine, albumin is not 
 thrown down by boiling. 
 
 In conducting the heat test for albumin, one of the 
 essentials of success is to have the liquid acidulated, 
 as already stated, to a suitable extent. The urine 
 should sharply redden blue litmus-paper, but excess 
 of acid must be avoided. Occasionally it is desirable 
 to make several tests on portions of the sample to 
 which gradually increased quantities of acetic acid 
 have been respectively added. 
 
 According to Tyson, the addition of a few drops of 
 acetic acid may diminish an albuminous precipitate, but 
 on adding more re-precipitation occurs. A large excess, 
 especially if the liquid be boiled, will permanently 
 dissolve the precipitate of albumin. Nitric acid is pre- 
 ferred to acetic acid by some operators, but even more 
 care is necessary to avoid the use of an excess, and 
 the reagent is unsuited for the study or bedside. 
 
 C. W. Purdy (Practical Uranalysis, 1894) recom- 
 mends that the urine should be treated with sufficient 
 of a saturated filtered solution of common salt to raise 
 the gravity to about 1035. One or two drops of acetic 
 acid should then be added and the upper portion of 
 the liquid boiled, as already described. The addition of 
 the brine is stated to prevent any precipitation of mucin, 
 and hence to avoid the confusion thereby occasioned. 
 But Purdy, apparently, omits to filter the acidulous 
 urine, which would practically remove the mucin. 
 
 Nitric Acid Test. — Another delicate and simple test 
 for albumin in urine is due to Heller, and is based 
 on its coagulation by cold nitric acid. The simple 
 addition of some of the urine to strong nitric acid 
 contained in a test-tube, in such a manner as to 
 prevent the liquids from mixing, suffices for the 
 detection of notable quantities of albumin, but the 
 
108 NITRIC ACID TEST. 
 
 reaction is much, increased in delicacy and reliability 
 by operating in the following manner, devised by 
 Sir William Roberts. A saturated solution of mag- 
 nesium sulphate is prepared by dissolving 10 parts 
 of the crystallised salt in 13 of hot water and filtering 
 the liquid To 5 measures of this solution, 1 of nitric 
 acid of 1 -42 specific gravity is added. This reagent 
 is so dense that it is easy to avoid admixture with the 
 urine to be tested. Some of the acid mixture is 
 placed in a test-tube, and an equal or larger measure 
 of the filtered urine allowed to flow gently on to the 
 surface, carefully avoiding any mixing of the two 
 layers. 1 In presence of much albumin a more or less 
 opalescent zone is immediately formed at the junction 
 of the two liquids ; but when only traces are present a 
 longer time, sometimes extending to a quarter of an 
 hour, is requisite for the development of the band. 
 The turbidity due to albumin occurs at the bottom of 
 the layer of urine, just above the line of demarcation, 
 while any cloudiness due to mucin 2 always appears 
 as a diffused haze towards the upper part of the liquid, 
 and therefore quite distinct from the albumin ring. 
 A stratum of uric acid occasionally separates in apply- 
 ing this test, but it disappears on warming. With 
 some urines a crystalline precipitate of nitrate of urea 
 may form, but this cannot be mistaken for albumin. 
 
 Ferrocyanide Test. — Another very delicate test for 
 albumin is to treat the suspected urine with excess 
 of acetic acid, and then add an aqueous solution of 
 
 1 J. E. Saul (Pharm. Jour., [3], xvii. 857) suggests the use of a small 
 glass syringe instead of a test-tube. About an inch of the clear urine is first 
 drawn up into the syringe, and then a sufficiency of the reagent. In this 
 manner the danger of inadvertently mixing the two layers is much lessened. 
 
 2 After taking copaiba balsam or sandal oil, the urine may contain resin acids, 
 precipitable by nitric acid, and therefore liable to be mistaken for albumin. 
 In such cases, Alexander recommends that 10 c.c. of the urine should be 
 treated with two or three drops of hydrochloric acid, which will precipitate the 
 resin acids. If, on adding acetic acid, a precipitate is formed, insoluble in 
 excess of the reagent, this consists of mucin. 
 
NITRIC ACID TEST. 109 ■ 
 
 potassium ferrocyanide. A white precipitate will 
 form immediately, or after a short interval, if any 
 trace of albumin be present. 1 The reaction is delicate, 
 and produced only by coagulable proteids, which is 
 important, since peptones may be present in urine 
 without albumin. If true albumin be present, there 
 will be paraglobulin and myosin as well. This is 
 stated to occur in amyloid degeneration of the kidney. 
 F. "W. Pavy employs tabloids containing citric 
 acid and potassium ferrocyanide, instead of using 
 solutions of the reagents. 2 For clinical purposes, this 
 plan is very convenient. 
 
 G-. Oliver, of Harrogate, has proposed to employ 
 strips of filter-paper impregnated with the reagents. 
 By immersing one of the potassium ferrocyanide 
 papers and another of the citric acid papers in a little 
 (5 c.c.) distilled water, the reagent can be prepared 
 in a few minutes. 
 
 C. "W. P u r d y {Practical Uranalysis) insists 
 strongly on the importance of adding the acetic acid 
 and ferrocyanide solution simultaneously, or at any 
 rate the latter reagent before the acid. In this way 
 he states that any precipitation of mucin is wholly 
 avoided. To a test-tube half filled with the sample of 
 
 1 A yellow coloration is sometimes produced on adding the ferrocyanide solu- 
 tion to urine. According to J. P. Karplus (Ohem. Centr., 1893, ii. 496) this 
 reaction is due to nitrites, which he states are often present in urine which 
 has been kept more than twenty-four hours, but not in the fresh excretion. 
 
 2 Pavy directs that a pellet of citric acid should be dropped into about a 
 drachm [ = 3'5 c.c.) of the urine, and the liquid agitated till solution occurs. 
 A ferrocyanide pellet is then added, and the fluid again agitated. An 
 immediate precipitate occurs if albumin be present. If the urine be turbid 
 from the presence of urates, it must be previously filtered or clarified by 
 heating, but otherwise heating is unnecessary. A turbidity on adding the 
 citric acid only may be due to either uric acid or mucin. In the former case, 
 previous dilution of the sample with an equal measure of water prevents its 
 formation ; in the latter, the density of the precipitate produced after adding 
 the ferrocyanide should be compared with that produced by citric acid alone. 
 The ferrocyanide test is less delicate when citric acid is employed than when 
 acetic acid is used, but Pavy's modification is very useful for clinical purposes. 
 The pellets may be obtained from Mr Cooper, 66 Oxford Street, London. 
 
110 NITRIC ACID TEST. 
 
 urine, Purdy adds " a drachm or so " of a 5 per cent, 
 solution of potassium ferrocyanide, and after agitating 
 adds from 1 to 15 drops of acetic acid. No reaction 
 is said to be produced by mucin, peptones, urates, phos- 
 phates, alkaloids, or resin acids. Any precipitate is 
 due to albumin and nothing but albumin. 
 
 Zouchlos proposes to substitute potassium 
 
 ihiocyanate (sulphocyanide) for the ferrocyanide. 
 
 / He prepares the reagent by mixing 10 c.c. of a 
 
 10 per cent, solution of the salt in water with 2 c.c. 
 
 of acetic acid. 
 
 A. Ollendorff (Zeitschr. anal. Chem., xxxiii. 
 120) confirms the value of Zouchlos' test, and states 
 that it is capable of detecting 0'005 per cent, of 
 albumin, while other constituents of urine, with the 
 exception of propeptone, have no disturbing influence. 
 Picric Acid Test. — A valuable reagent for the 
 detection of albumin in urine is a cold saturated 
 aqueous solution of picric acid, first proposed by 
 Braun, and since strongly advocated by Sir G. 
 Johnson and others. {Brit. Med. Jour., Oct. 11, 
 1884; Analyst, ix. 206.) This may be added either 
 to the original (filtered) urine, or to a portion in which 
 acetic acid has failed to give a precipitate, and either 
 the cold or hot urine may be employed. The reagent 
 may either be allowed to mix with the urine, or the 
 junction of the two layers may be observed, when a 
 turbidity will be produced if any trace of albumin be 
 present. For clinical purposes, a minute quantity of 
 powdered picric acid may be substituted for the 
 solution of the reagent. Peptones, alkaloids, pipera- 
 zine, and urates (when in large excess) are liable to 
 give precipitates with picric acid ; but these pre- 
 cipitates are readily distinguished from that due to 
 albumin by their disappearance on heating the liquid. 
 In the absence of acetic or other added acid, picric 
 
esbach's test for albumin. Ill 
 
 acid does not precipitate mucin, and this fact forms a 
 valuable distinction between that body (said by Sir G. 
 Johnson to be always present in traces even in normal 
 urine) and true albumin. 
 
 Esbach's Test. — A very fair approximation to the 
 quantity of albumin contained in urine is said to be 
 obtainable in the manner devised by E s b a c h, who 
 performs the test in a graduated tube, about 
 6 inches in length and 0"6 inch in diameter. 
 Esbach's reagent is prepared by dissolving 
 10 grammes of picric acid and 20 of citric acid 
 in about 900 c.c. of boiling water, and after 
 cooling making up the volume to 1 litre by 
 adding water. (Or ^ oz. of picric and -| oz. of 
 citric acid may be dissolved in 25 oz. of 
 water.) The tube is filled to the mark " U " 
 with the urine to be tested, and the reagent 
 added up to the mark " R." The liquids 
 are mixed by cautiously inverting the closed 
 tube several times, avoiding strong agitation, Fig- 6 ._ 
 and then left at rest for twenty -four hours. Esbach's 
 The volume of the precipitate is then ob- TuBE- 
 served, each degree corresponding to O'l per cent, of 
 albumin. 1 The process is inapplicable to urine con- 
 taining less albumin than 0"1 per cent., and samples 
 containing more than 0*7 per cent, must be previously 
 diluted with their own or twice their measure of 
 water, as the tubes are not graduated for more highly 
 albuminous urines. 
 
 1 The graduation of the tubes is purely empirical. The results are com- 
 monly declared to be quite accurate enough for all clinical requirements. In 
 the experience of the author, the results are fairly comparable if the same 
 conditions as to time, &c. , are observed, but the absolute quantities of albumin 
 indicated leave much to be desired in point of accuracy, and wide discrepancies 
 occur if the reading be taken much before or after the twenty-four hours 
 prescribed. 
 
 Esbach's tubes are obtainable from E. Cetti, 36 Brooke Street, Holborn, 
 London ; A. Gallenkamp & Co., Cross Street, Finsbury, London ; Southall 
 & Son, Birmingham ; or Gibbs, Cuxson, & Co., Wednesbury. 
 
112 IMPROVED ESBACHS TUBE. 
 
 A greatly improved form of Esbach's tube has 
 been devised by C. W. Purdy, of Chicago. 1 The 
 tube is drawn out into a blunt cone at the closed end, 
 so that much smaller quantities of albumin can be 
 measured. Dr Purdy makes the further great im- 
 provement of placing the tube in a centrifugal machine 
 (such as the Leffmann-Beam apparatus for estimating 
 fat in milk), whereby complete separation of the 
 albumin can be effected in a few minutes, instead of 
 after standing twenty-four hours. 
 
 Trichloracetic acid, CCl 3 .COOH, is a reagent for 
 albumin proposed by Raabe, and a test for which 
 special advantages are claimed. The reagent does not 
 precipitate peptones nor coagulate mucin. It is said to 
 precipitate a form of albumin not indicated by either 
 of the previously described tests, which form appears 
 to be specially characteristic of the presence of granular, 
 epithelial, or hyaline casts. Trichloracetic acid used 
 in saturated solution, and poured on the cold urine, 
 will detect 1 part of albumin in 100,000 of liquid. In 
 presence of quinine or other alkaloids a small addition 
 produces a dense white precipitate, soluble on heating 
 or on adding a large excess of the acid. 
 
 Salicyl-sulphonic acid 2 is recommended by M. 
 Roch (Archiv. des Pharm., xxvii. 998) and J. A. 
 MacWilliam (Brit. Med. Jour., i., 1891, page 
 837) as a precipitant of all varieties of proteids in 
 urine. In applying the test it is simply necessary to 
 add a few crystals of the reagent to a small quantity 
 of the clear urine and agitate, when the appearance 
 of a turbidity or an actual precipitate will indicate 
 the presence of albumin. The precipitate produced 
 
 1 Obtainable from Messrs Eimer & Amend, 205 Third Avenue, New York. 
 
 2 Salicyl-sulphonic acid or sulpho-salicylie acid is prepared by heating 
 salicylic acid with twice its weight of sulphuric anhydride at 100° C. until 
 dissolved. On cooling and standing brownish crystals of the compound 
 separate. These are purified by recrystallisation from boiling water. 
 
METAPHOSPHORIC ACID TEST. 113 
 
 by albumins and globulins is not affected by heat, 
 while that due to albumoses and peptones dissolves, 
 reappearing as the liquid cools. No normal or 
 abnormal constituent of urine other than proteids 
 is precipitated by the reagent, while these are very 
 completely separated. One part of egg-albumin in 
 20,000 of water can be detected. 
 
 Metaphosphoric acid, readily obtained by dissolv- 
 ing glacial phosphoric acid in cold water, is recom- 
 mended by C. Hindenlang (Chem. Centralb., 
 1881, page 471) for the detection of albumin in urine. 
 To obviate the inconvenience caused by the ready con- 
 version of the meta- into ortho-phosphoric acid, L. 
 Blum [Chem. Centralb., 1887, page 345) recom- 
 mends the following reagent : — From 0"03 to - 05 
 gramme of manganous chloride is dissolved in a little 
 water, acidulated with a few c.c. of dilute hydro- 
 chloric acid, and treated with 100 c.c. of a 10 per 
 cent, solution of sodium metaphosphate (best pre- 
 pared by igniting microcosmic salt in platinum). 
 Lead dioxide is then added, in small quantities at 
 a time, with constant agitation, the liquid is allowed 
 to settle, and filtered. The resulting pink solution 
 of manganic metaphosphate is used as an indicator 
 of albumin. The reagent should be placed in a test- 
 tube and the urine to be examined filtered into it. 
 The solution is said to keep well, and to give no 
 reaction with other constituents of urine. 
 
 Spiegler's test (Ber., xxv. 375) consists of a solu- 
 tion of 8 grammes of mercuric chloride, 4 of tartaric 
 acid, and 20 of sugar, in 200 c.c. of water. On running 
 onto the surface of this reagent some of the urine 
 (previously acidulated with a little strong acetic acid 
 and filtered if necessary), a distinct white ring is 
 formed at the line of demarcation if albumin be 
 present. Globulin and hemi-albumose behave simi- 
 
 H 
 
114 TANKETS REAGENT. 
 
 larly, but peptones give no reaction. The addition of 
 sugar to the reagent is simply for the purpose of 
 increasing the density, and so avoiding mixture with 
 the stratum of urine. 
 
 PoTASSio-MERCUBic iodide has been recommended by 
 Tanret as a precipitant of albumin. 1 The reagent 
 is prepared by dissolving 1*35 gramme of mercuric 
 chloride and 3 "32 of potassium iodide in 64 c.c. of 
 water, and adding 20 c.c. of acetic acid. Mehu 
 states that this reagent precipitates mucin. Brasse 
 found peptones and alkaloids to be thrown down, but 
 the precipitates due to these bodies dissolve on heat- 
 ing, leaving that due to albumin. The alkaloidal 
 precipitate is soluble in ether, the peptone precipitate 
 insoluble. Bile-salts gave a precipitate not dissolved 
 by heat, but distinguishable from that due to albumin 
 by its solubility in ether. 
 
 No reaction with Tanret 's reagent is produced by 
 creatine, creatinine, xanthine or hypoxanthine. 
 
 Various other reagents have been proposed for the 
 
 1 Tanret has proposed to determine the albumin volumetrically by 
 acidulating the urine with acetic acid and adding a standard solution of 
 potassio-mercuric iodide until a drop of the liquid yields a precipitate on 
 addition of mercuric chloride. The method is said to give fair clinical results. 
 
 F. Ven turini suggests a modification of Tanret' s process based on the 
 fact that mercuric chloride precipitates albumin from urine acidulated with 
 acetic acid before reacting with potassium iodide. A standard solution of 
 mercuric chloride, containing 10 grammes per litre, is prepared. Each 1 c.c. 
 of this solution precipitates 0245 gramme of albumin. To 5 c.c. of the 
 urine, 6 c.c. of a 5 per cent, solution of potassium iodide is added, together 
 with a few drops of acetic acid, and the standard mercuric chloride then 
 added drop by drop, until a permanent yellowish-red coloration is obtained. 
 From the volume used a deduction of 1 o.c. is made for the excess required to 
 show the coloration, and the difference is multiplied by the factor 0"0245 to 
 obtain the amount of albumin. 
 
 Georges {Jour. Pharm., [5], xiii. 353) utilises these facts for the detection 
 of peptones in the following manner. The coagulable albumin is first pre- 
 cipitated by heating the urine. The filtrate is precipitated by Tanret's 
 reagent, and the precipitate washed on the filter with cold water charged with 
 acetic acid to the same extent as the urine. It is then washed with the same 
 acidulated water heated to boiling, the washings being kept separate. The 
 clear liquid thus obtained gives a precipitate on cooling if any trace of peptone 
 has been dissolved. 
 
RELATIVE DELICACY OP ALBUMIN TEST. 115 
 
 detection of albumin in urine, but their behaviour 
 with co-occurring bodies has either been incompletely 
 studied or they present no tangible advantages over 
 the tests already described. 
 
 Grainger Stewart (Edin. Med. Jour., May 
 1887) is of opinion that picric acid is the most delicate 
 of all reagents for albumin, Tanret's reagent ranking 
 second. U. V e 1 1 e s e n, of Christiania, represents 
 the relative delicacy of the various tests by the 
 following figures : — Nitric acid, 85 ; trichloracetic 
 acid, 82 ; potassium ferrocyanide and acetic acid, 82 ; 
 metaphosphoric acid, 72 ; picric acid in solution, 36 ; 
 sodium sulphate and acetic acid, 25. D. C a m p b e 1 1 
 B 1 a c k ( Urine and Urinary Analysis), as the result of 
 careful experiments, considers Tanret's reagent the most 
 delicate ; heat, nitric acid, and the aceto-picric solu- 
 tion following in the order named ; and ferrocyanide 
 being one of the least delicate of the tests tried. 1 
 
 1 Eecent experiments by E. J. E v a n s {Pharm. Jour., [3], xxv. 913), with 
 a view of comparing the relative delicacy of different reagents, gave the follow- 
 ing results with solutions of egg-albumin. Solution No. 1 had a strength of 
 
 1 in 40 ; solution No. 2 of 1 in 200 ; while solution No. 3 contained 1 part of 
 albumin in 1000 of water. Half an ounce (8 c.c.) was employed for each 
 experiment. No indication is given in the paper whether dry albumin or 
 fresh egg-albumen was employed. 
 
 On boiling, No. 1 showed coagulation and slight opalescence, but only 
 frothing was observed in the case of solutions 2 and 3. 
 
 On adding a few drops of acetic acid and heating, No. 1 coagulated ; No. 
 
 2 gave a white froth with slight coagulation ; while No. 3 showed a slight 
 froth, without any sign of coagulation or opalescence. 
 
 Heated with a few drops of nitric acid, solution 1 gave a cloudy precipitate, 
 which became denser on heating ; No. 2, a white cloud in the line of the drops, 
 and on shaking a white opalescence ; while No. 3 showed only a slight froth 
 without opalescence. 
 
 Picric acid gave with No. 1 a bulky yellow precipitate, soluble in excess of 
 ammonia ; with No. 2 a yellow opalescence, and after heating and standing 
 for some time a yellowish precipitate ; and No. 3 behaved somewhat similarly. 
 
 With a nitric acid solution of ammonium molybdate No. 1 gave a white pre- 
 cipitate, separating in flocks when heated ; No. 2 a white opalescence ; and No. 
 
 3 the same. 
 
 Uranium acetate produced with No. 1 a yellowish-white precipitate, curdling 
 on heating ; with No. 2 a yellowish-white opalescence, a precipitate separating 
 on heating ; and much the same with No. 3. 
 
116 DETERMINATION OF ALBUMIN. 
 
 Determination of Albumin in Urine. 
 
 The method of Esbach (page 111) affords quantita- 
 tive results which are somewhat rough but sufficiently 
 accurate for many purposes. More exact determina- 
 tions can be obtained by precipitating the albumin, 
 &c, by a suitable reagent, and drying and weighing 
 the washed precipitate, or estimating the nitrogen 
 contained in it. 1 From the nitrogen found, the pro- 
 teids are calculated by multiplying by the factor 6"3. 2 
 Some observers employ the factor 6 '25, and others 
 as high a factor as 6 - 37. 6 '3 is a fair average figure, 
 and sufficiently accurate for all purposes. Picric 
 acid and ferrocyanide of potassium are inapplic- 
 able as precipitants, since they contain nitrogen. 3 
 Tannin, carbolic acid, or trichloracetic acid may be 
 used ; or Tanret's solution of potassio-mercuric iodide 
 may be employed, bearing in mind the substances 
 other than albumin which are liable to be thrown 
 down. Of the available precipitants, carbolic acid 
 and tannin appear to be the best. 
 
 The following method of determining albumin in 
 urine is strongly recommended by M£hu. 100 c.c. 
 measure of the cold urine is rendered slightly acid 
 with acetic acid, 2 c.c. of concentrated nitric acid 
 added, and the liquid thoroughly agitated. Ten c.c. of 
 a mixture of 1 part by weight of crystallised carbolic 
 acid, 1 of commercial acetic acid, and 2 of rectified 
 spirit is next added, the liquid mixed thoroughly, and 
 filtered after a few minutes. The filtration proceeds 
 rapidly. The precipitate is washed with a cold 4 per 
 
 1 Kj eldahl's process is well-adapted for this purpose. The modification 
 of it described on page 128 is convenient when approximate results will suffice. 
 
 2 The employment of this factor is based on the composition of albumin 
 and its allies, which contain on the average 15 '88 per cent, of nitrogen ; and 
 100 4- 15-88 = 6-3. 
 
 3 The nature of the precipitates produced by these reagents in solutions of 
 albumin appears to be uncertain. According to some writers at least, the 
 precipitates are actual compounds of the reagent with the proteid. 
 
DETERMINATION OF ALBUMIN. 117 
 
 cent, solution of carbolic acid in water, 1 when it may 
 either be dried and weighed, or treated (paper and all) 
 by the modified Kjeldahl's method, and the nitrogen 
 found multiplied by 6'3 to obtain the weight of the 
 proteids precipitated. The presence of sugar or much 
 saline matter in no way affects the accuracy of this 
 process, but in the presence of a large proportion of 
 salts the addition of nitrie acid becomes unnecessary. 
 
 Van Nuys and Lyons (Amer. Chem. Jour., 
 xii. 336 ; Analyst, xv. 234 ; xvi. 7) have described a 
 method of determining albumin dependent on its 
 precipitation by a solution of tannin. This is pre- 
 pared, according to the method of Alm6n, by mixing 
 4 grammes of tannic acid, 8 c.c. of acetic acid (1 part 
 of glacial acid to 3 parts of water), and 190 c.c. of 
 50 per cent, alcohol. Ten c.c. of this solution and an 
 equal measure of the filtered urine are mixed well, 
 and the liquid passed through a dry filter. In 5 c.c. 
 of the filtrate the nitrogen is then determined by 
 Kjeldahl's process, and by the same method the 
 nitrogen is determined in 5 c.c. of the original urine. 
 The difference between the two results represents the 
 nitrogen precipitated by the tannin reagent, and this 
 amount multiplied by 6 - 3 gives the corresponding 
 weight of albumin and globulin in the precipitate. If 
 the urine contain more than 2 per cent, of albuminoids 
 it should be diluted with an equal or double measure 
 of water, and the calculation modified accordingly. 
 
 H. 0. G. E 1 1 i n g e r (Jour. Prakt. Chem., [2], xliv. 
 256) has described a method of determining albumin 
 in urine by means of an instrument closely allied to 
 Amagat and Jean's refractometer. The sample in its 
 
 1 According to Menu's original directions, the precipitate is to be washed 
 with boiling water containing 1 per cent, of carbolic acid. L. Ruisand 
 (Jour. Phcurm., [5], xxix. 364) finds that a very appreciable amount of albumin 
 is dissolved by this treatment, but that no appreciable solution occurs when 
 the washing is conducted as described in the text. 
 
118 PARAGLOBULIN. 
 
 original state is compared with the same urine from 
 which the albumin has been removed by heat and 
 acetic acid. The results agree somewhat roughly 
 with the gravimetric determination. 
 
 Distinction and Separation of Urinary Proteids. 
 
 Until recently it was not recognised that more than 
 one proteid body was liable to occur in urine, but 
 it is now known that serum-globulin (paraglobulin) 
 frequently co-exists with serum-albumin, and, accord- 
 ing to Senator, invariably accompanies the latter sub- 
 stance or even exists alone. Certain albumoses, 
 proteoses or propeptones may also be present, in addi- 
 tion to which true peptones sometimes occur either 
 with or without serum-albumin. Traces of mucin are 
 usually present, and fibrin and haemoglobin may 
 occur in certain septic and purpurous conditions. 
 
 Paraglobulin may be detected in urine, if present 
 in large amount, by diluting the liquid with two 
 measures of distilled water, rendering it faintly acid, 
 and passing a stream of carbon dioxide. On standing 
 for twenty-four hours or more, the globulin forms a 
 white flocculent precipitate. 
 
 Noel Paton (Brit. Med. Jour., 1890, page 197) 
 has described the following method of separating 
 and estimating paraglobulin and serum-albumin when 
 occurring together in urine. The total proteids 
 present are first determined by Esbach's method. 
 Fifty c.c. measure of the urine is then rendered faintly 
 alkaline, and powdered magnesium sulphate added 
 until the liquid is saturated. It is then allowed to 
 stand in a warm place for twenty-four hours, when the 
 globulin will be completely precipitated. The liquid 
 is then measured, filtered, and a portion again treated 
 by Esbach's method (page 111). The result now ob- 
 tained, after due allowance for the increased volume, 
 
SEPARATION OF PROTEIDS. 119 
 
 represents the albumin of the urine, and the difference 
 between this and the figure previously obtained will 
 be the globulin (plus any hemi-albumose) of the 
 urine. The globulin and hemi-albumose (" hetero- 
 proteose ") may be separated, if desired, by redis- 
 solving the precipitate produced by ammonium 
 sulphate in a small quantity of water, and treating 
 the solution obtained with ten times its measure of 
 absolute alcohol. The resultant precipitate is collected 
 and digested with cold absolute alcohol for a week 
 or ten days. The liquid is then filtered, when a 
 residue will consist of globulin, and the hemi-albumose 
 will be found in the filtrate. 
 
 In the urine of a person recovering from a prolonged 
 attack of diarrhoea, Paton found 2 per cent, of total 
 proteids, of which l - 92 was globulin; and in another 
 case 3*82 of total proteids, of which 3 '73 was globulin, 
 which was obtained in elongated rhombic crystals. 
 
 Instead of employing Esbach's method, A. s t 
 (Chein. Centralb., 1884, page 500) observes the op- 
 tical activity of the urine before and after saturation 
 with magnesium sulphate. 
 
 An alternative plan is to precipitate the globulin 
 and albumin together by boiling the faintly acidulated 
 urine, and estimate the nitrogen in the washed pre- 
 cipitate by the author's modification of KjeldahL's 
 method (page 128). In another portion of the urine 
 the globulin is precipitated by saturating the liquid 
 with magnesium sulphate, the precipitate collected and 
 washed with magnesium sulphate solution, and the 
 contained nitrogen estimated by the modified Kjeldahl's 
 process. The difference between the two results is the 
 nitrogen corresponding to the albumin of the urine. 
 
 According to Senator, paraglobulin occurs in 
 urine in cases of lardaceous disease of the kidneys, and 
 has also been found in excess in the intense hypersemia 
 
120 ALBUMOSES OR PROTEOSES. 
 
 resulting from poisoning by cantharides, and in 
 functional albuminuria associated with marked dis- 
 turbance of the digestive organs. The greater the 
 proportion of paraglobulin present, the more unfavour- 
 able appears the diagnosis in Bright's disease. When 
 blood is present in urine, as in nephritis after scarlet 
 fever, there is a large increase in the proportion of 
 paraglobulin. Noel Paton {Brit. Med. Jour., ii., 
 1890, page 196) finds the ratio of globulin to albu- 
 min to vary enormously (from 1 : 0'6 to 1 : 39). It 
 varies much during the day, and in such an erratic 
 manner that no conclusions can be drawn. 
 
 Albumoses or Proteoses. 
 
 Of late years a series of bodies, intermediate in 
 characters and composition between the albumins and 
 peptones, have been isolated, and some of these have 
 been proved to exist in certain forms of pathological 
 urine. Thus G e r r a r d has recently shown (Pharm. 
 Jour., [3], xxiii. 261) that in the milk-treatment 
 of albuminuria no albumin coagulable by heat exists 
 in the urine, but that nitric acid gives a precipitate 
 soluble in excess, or on warming, reappearing on 
 cooling ; and saturated brine a flocculent precipitate 
 increased by the addition of acetic acid. These 
 reactions are characteristic of the form of proteose 
 called hetero-albumose or hetero-proteose, the presence 
 of which in urine is said to be an indication of 
 approaching nephritis. The same substance has been 
 found in cases of osteo-malacia and atrophy of the 
 kidneys, and in the urine of persons who have been 
 rubbed with petroleum. 
 
 For the detection of hetero-albumose, Tyson acidi- 
 fies the urine with a few drops of acetic acid, and 
 adds one-sixth of its volume of saturated brine, boils, 
 and filters. Albumin and globulin are precipitated. 
 If the filtrate after cooling gives a precipitate on 
 
SEPARATION OF PHOTE1DS. 
 
 121 
 
 further addition of brine, which dissolves on heating 
 and reappears on cooling, the presence of albumose is 
 indicated. 
 
 For the distinction of the proteoses liable to occur in 
 urine, and their separation from co-occurring proteids, 
 the following plan may be used. It is taken, with 
 slight modifications, from W. D. Halliburton's 
 Text-booh of Chemical Physiology and Pathology. 
 
 Bring the uriue to a faintly acid condition by cautious addition of acetic 
 acid or dilute caustic alkali (as the original reaction to litmus may 
 indicate), boil for a minute or so, and filter. 
 
 Precipitate consists 
 of albumin and glo- 
 bulin. These may 
 be separated by 
 saturating another 
 portion of the 
 faintly acidulated 
 urine with powdered 
 magnesium sul- 
 phate, which pre- 
 cipitates globulin, 
 proto-albumose, and 
 hetero -albumose. 
 From the filtrate, 
 albumin will be 
 precipitated on boil- 
 ing, while deutero- 
 albumose and pep- 
 tones remain in 
 solution. 
 
 Filtrate, allow to cool, saturate thoroughly with powdered 
 ammonium sulphate, and filter. 
 
 Precipitate consists of albumoses. Wash 
 with a saturated aqueous solution of am- 
 monium sulphate. Redissolve the washed 
 precipitate in a minimum quantity of 
 water. Faintly acidulate the solution with 
 acetic acid, saturate it with common salt, 
 and filter. 
 
 Precipitate consists of 
 primary albumoses. 
 Wash with saturated 
 brine, redissolve by add- 
 ing water, and dialyse 
 the solution. 
 
 Precipitate 
 consists of 
 hetero-al- 
 buinose, 
 which may 
 be further 
 identified 
 by reac- 
 tions on 
 page 120. 
 
 Filtrate 
 may con- 
 tain proto- 
 albumose, 
 preeipitable 
 by excess of 
 alcohol 
 (10 : 1). 
 
 Filtrate may 
 contain deutero- 
 albumose, pre- 
 cipitated by 
 saturating the 
 solution with 
 ammonium sul- 
 phate, and re- 
 cognisable by 
 other reactions 
 on page 123. 
 
 Filtrate con- 
 tains peptones 
 only, recognis- 
 able by xan- 
 thoproteic and 
 biuret reac- 
 tions, as on 
 page 123. 
 
 Traces of albumoses are liable to be formed by the 
 action of the boiling acidulated liquid on the albumin 
 and globulin. A sharper separation can be effected 
 by treating the urine with ten times its volume of 
 
122 PEPTONES. 
 
 strong alcohol, which precipitates all the proteids. 
 The precipitate is rinsed off the filter with absolute 
 alcohol, and left in contact with the alcohol for five to 
 ten weeks. This treatment coagulates the albumin 
 and globulin without affecting the albumoses or 
 peptones. The supernatant alcohol is poured off, the 
 remainder evaporated at a temperature not exceeding 
 40° C. ( = 104° F.), and the residue treated with water, 
 which dissolves the albumoses and peptones, leaving 
 the albumin and globulin insoluble. 
 
 Peptones are now known to occur in the urine 
 under a great variety of pathological conditions, 
 especially in acute febrile diseases and nervous com- 
 plaints, and they probably exist in traces in normal 
 urine. The only reliable method of distinguishing 
 and separating peptones from co-occurring proteids 
 appears to be that of S. H. C. Martin {Brit. Med. 
 Jour., i., 1888, page 842). This consists in saturat- 
 ing the urine, faintly acidulated with acetic acid, with 
 ammonium sulphate. The powdered salt is added 
 gradually to the urine till no more is taken up. The 
 precipitate of proteids rises to the surface of the 
 liquid and can readily be separated by filtration. 1 By 
 this treatment any albumin, globulin, or proteose is 
 completely precipitated, whatever the reaction of the 
 liquid, while any peptone remains in solution. 
 
 Peptones present the closest similarity in their pro- 
 
 1 If the precipitate be washed twice with a cold saturated solution of 
 ammonium sulphate, and then redissolved on the filter in distilled water, a 
 solution is obtained in which the proteids are easily differentiated. Thus, on 
 saturating the liquid with magnesium sulphate, the globulin is precipitated, 
 while the albumin can be detected in the filtered liquid by its property of 
 coagulating when heated to 73° C, after slightly acidulating the solution with 
 acetic acid. Hemi-albumose is precipitated at 43° to 50° C. , the precipitate 
 being soluble in a few drops of a weak acid, and it is precipitated in the 
 absence of acids, which albumin and globulin are not. It also gives a pink 
 reaction with the biuret test, and with nitric acid a precipitate which dis- 
 solves on heating and reappears on cooling. It is likewise precipitated from a 
 solution faintly acidulated with acetic acid by potassium ferrocyanide, and 
 by saturating its solution with magnesium sulphate. 
 
ALBUMOSES AND PEPTONES. 123 
 
 perties and reactions to the proteoses, and especially 
 to the body called deutero-proteose or deutero- 
 albumose. Peptones differ from deutero-proteose in 
 not being precipitated on saturating the solution with 
 ammonium sulphate, and in giving no precipitate with 
 nitric acid under any conditions. Deutero-proteose, 
 on the other hand, is precipitated by nitric acid after 
 a considerable quantity of common salt has been 
 added to the liquid. This precipitate dissolves on 
 heating the liquid containing it, but reappears on 
 cooling. 
 
 Both peptone and deutero-albumose are precipi- 
 tated, but not coagulated, by alcohol. They are not 
 precipitated by boiling, nor by cupric sulphate, but 
 are precipitated by phospho-molybdic, phospho-tung- 
 stic, picric acid, tannin, or potassio-mercuric iodide. 
 They resemble other proteids in yielding a yellow 
 coloration on boiling with nitric acid, becoming 
 brownish on adding excess of ammonia (the " xantho- 
 proteic reaction ") ; and by the pink or rose-red 
 coloration obtained on adding excess of caustic 
 alkali, followed by a few drops of a very dilute solu- 
 tion of cupric sulphate (" biuret reaction"). 
 
 These two reactions can be employed for the de- 
 tection of peptones in the filtrate from the precipitate 
 produced by saturating the urine with ammonium 
 sulphate, as above described. 
 
 Where mucin and albumin are already absent, it 
 is stated that peptones may be detected by treating 
 50 c.c. of the original urine with 5 c.c. of hydro- 
 chloric acid and precipitating the warm liquid with 
 sodium phospho-tungstate. The supernatant liquid 
 is decanted, and the resinous precipitate washed twice 
 with water containing 0'5 c.c. of caustic soda solu- 
 tion of 1'16 specific gravity, which dissolves it. The 
 resultant solution is warmed till a greenish turbidity 
 
124 MUCIN. 
 
 is produced, allowed to cool, and a 1 per cent, solution 
 of cupric sulphate added drop by drop. In presence 
 of a peptone, a red coloration is produced, which is 
 rendered more evident by filtering the liquid. 
 
 Roux (Jour. Pharm., [5], xxv. 544) proposes to 
 determine the peptones in urine volumetrically. The 
 sample is freed from albumin and reducing com- 
 pounds, and a decinormal Fehling's solution added 
 until the colour changes through light blue, blue-violet, 
 lilac, and rose-purple, to a greyish tint. One c.c. is 
 said to represent - 004 gramme of peptone. 
 
 Mucin is the chief constituent of the mucus derived 
 from the renal and urinary passages, and is probably 
 the source of the so-called "animal gum " found in the 
 urine by Landwehr. It occurs very commonly (ac- 
 cording to Sir G. Johnson, invariably) in traces even 
 in normal urine, and in larger amount in urinary 
 catarrh and other affections of the urinary organs. 
 Mucin is slightly soluble in neutral or alkaline urine, 
 but is precipitated on adding acetic acid, and is 
 insoluble in excess of the precipitant. In many of its 
 reactions it resembles albumin, for which it is apt to 
 be mistaken, but is not coagulated by heat. It is 
 precipitated by alcohol, alum, dilute mineral acids, 
 and by certain organic acids, including acetic and 
 citric. If urine containing mucin be poured on to the 
 surface of acetic acid saturated with salt, or on to a 
 strong solution of citric acid, a cloud appears at the 
 junction of the two layers. On the other hand, 
 mucin is soluble in saline solutions of moderate 
 strength, and P u r d y actually adds brine to urine 
 in quantity sufficient to raise its density to 1035, in 
 order to prevent the precipitation of mucin when 
 acetic acid is subsequently added. In the nitric acid 
 test for albumin the haze due to mucin appears above 
 and distinct from that produced by albumin. 
 
BILE-PIGMENTS. 125 
 
 Salkowski and L e u b e test for mucin by treat- 
 ing the urine with two measures of nearly absolute 
 alcohol, separating the precipitate by nitration, and 
 redissolving it in water. The resultant solution gives 
 with acetic acid a cloud which is insoluble in excess, 
 but soluble in hydrochloric or nitric acid. Mucin 
 gives the violet biuret reaction with caustic alkali 
 and cupric sulphate, and is completely precipitated 
 by lead acetate. 
 
 Mucin may be separated from pus by precipitating 
 the latter by mercuric chloride. On adding acetic 
 acid to the filtrate the mucin is precipitated. Pus is 
 characterised by forming a gelatinous mass with 
 alkalies, which reagents give no reaction with mucin. 
 
 There are several varieties of mucin. They all 
 appear to have the constitution of glucosides, 
 being compounds of a proteid (probably variable, but 
 generally a globulin) with animal gum, which, by 
 boiling with dilute sulphuric acid, yields a reducing 
 unfermentable sugar. 
 
 In certain forms of liver-disease it has been found 
 that the urine gives precipitates which may be mis- 
 taken for those of albumin, but which are in reality 
 hile-pigments. It has been found, however, that these 
 can be got rid of by previous treatment with acetic 
 or a dilute mineral acid. G r o c c o, therefore, recom- 
 mends that all samples of urine likely to contain such 
 matters should be first treated with 2 or 3 per cent. 
 of concentrated acetic acid, set aside in a cool place 
 for two or three hours, and then filtered, before apply- 
 ing the ordinary tests. The precipitate thus formed 
 by the addition of acetic or dilute mineral acids is 
 soluble in alcohol, and does not give the biuret 
 reaction. 
 
THE NITROGENISED CONSTITUENTS OF 
 URINE. 
 
 The determination of the nitrogen contained in urine 
 is often of great physiological and pathological in- 
 terest, since the whole of the nitrogen contained in 
 the effete nitrogenised tissues and in the food digested 
 is ultimately eliminated by the kidneys. 
 
 About 90 per cent, of the total nitrogen contained 
 in normal human urine exists in the form of urea, 
 the remainder being divided between uric acid, 
 hippuric acid, xanthine, creatinine, &C. 1 
 In the urine of herbivorous mammals the uric acid is 
 replaced by hippuric acid, while the nitrogen of birds 
 and reptiles is eliminated chiefly in the form of uric 
 acid instead of as urea. 
 
 As urea is the predominant nitrogenous constituent 
 of normal human urine, it is evident that for many 
 purposes its determination will afford sufficient infor- 
 
 1 The result of a large number of observations by Russell and West 
 (Proc. Royal Soc, xxx. 439) on various cases of disease was to prove that the 
 relation of the ureal to the total nitrogen of urine is approximately constant, 
 except in rare cases of acute yellow atrophy of the liver ; and even in these it 
 is doubtful whether the observed replacement of the urea by leucine and 
 tyrosine is a constant phenomenon. In a case of acute fatty atrophy of the 
 liver the urea was still normally formed, while leucine and tyrosine were 
 absent. The following table shows the percentage of the total nitrogen 
 existent as urea, according to the observations of Russell and West : — 
 
 Pneumonia (6 cases), 90 per cent. ; jaundice (Case 1), 85 "7 ; jaundice (Case 
 2), 90"2 ; albuminuria (2 cases), 86 '0 ; collected cases, 93'8 ; dieted cases, 
 90 '1 ; and mean of all, 89 - 3 per cent. The mean, excluding the jaundice and 
 albuminuria cases, was 91 '3 per cent. 
 
kjeldahl's nitrogen process. 127 
 
 mation as to the amount of nitrogen passing away in 
 the urine. 
 
 Albumin and its allies are not present in normal 
 urine, at least not in estimable amount, and their detec- 
 tion and determination in the pathological excretion 
 have already been fully considered (page 105 et seq.). 
 
 Determination of the Total Nitrogen in Urine. 
 
 In certain cases it is desirable to ascertain the total 
 amount of nitrogen existing in urine, without differen- 
 tiating between the different modes of combination. 
 The total amount of nitrogen is best ascertained by 
 the well-known Kjeldahl method, which is based 
 on the fact that nearly all forms of nitrogenised 
 organic matter yield their nitrogen in the form of 
 ammonia when heated strongly with concentrated sul- 
 phuric acid. Urea readily undergoes this conversion, 
 and the decomposition of uric acid presents no difficulty. 
 Albumin is split up primarily into glycocine, leucine, 
 tyrosine, and probably other amido-compounds ; but 
 these, on further heating with strong sulphuric acid, 
 yield all their nitrogen in the form of ammonia. 
 Gunning finds the destruction of the organic 
 matter to be much facilitated by adding some solid 
 potassium sulphate, which raises the boiling point 
 of the sulphuric acid. 1 
 
 The conversion of the nitrogen into ammonia 
 having been effected by the foregoing treatment, 
 excess of caustic soda is added and the ammonia 
 distilled off; an addition of sulphide of alkali-metal 
 
 1 Potassium permanganate has been employed, but it is now recognised 
 that its use is unnecessary and liable to lead to loss from oxidation of the 
 ammonia formed. W. F. K. Stock effects rapid and complete oxidation 
 of the organic matter by adding manganese dioxide to the hot acid. This 
 plan is not available when the ammonia formed is to be decomposed with 
 hypobromite, as described in the text. The use of mercury or mercuric oxide, 
 which is a convenient means of effecting oxidation, results in the formation 
 of a mercur-ammonium compound not decomposed by the reagent. 
 
128 MODIFIED KJELDAHL'S PROCESS. 
 
 being made if mercury has been employed, in order to 
 ensure the decomposition of mercur-ammonium bases. 
 
 The ammonia in the distillate is finally determined 
 by titration with standard acid, and the amount 
 found calculated into its equivalent of nitrogen. 
 
 The estimation of nitrogen by this process requires 
 the observance of numerous precautions to ensure 
 accuracy. 
 
 In a modified process, which presents fewer diffi- 
 culties than the foregoing, and gives results suffi- 
 ciently accurate for the ordinary purposes of urinary 
 analysis, the nitrogen is first converted into ammonia 
 by strongly heating with sulphuric acid, and the 
 ammonia then decomposed by sodium hypobromite, 
 the nitrogen evolved being measured in the form of 
 gas. When treated with an alkaline hypobromite, 
 ammonia compounds are decomposed in accordance 
 with the following equation : — 
 
 3NaBrO + 2NH 3 = 3NaBr + 3H 2 + N 2 . 
 
 This reaction, which was first applied by W. K n o p, 
 under favourable conditions occurs very promptly and 
 completely ; but in order to adapt the method to the 
 requirements of urinary analysis, the manipulative 
 details require careful arrangement. They have been 
 thoroughly worked out in the author's laboratory, 
 and by operating in the following manner fairly good 
 determinations of the total nitrogen of urine can be 
 obtained in about one hour. 1 
 
 Twenty-five c.c. of the urine to be examined should 
 be treated in a porcelain basin with 10 c.c. of strong 
 sulphuric acid, and the liquid kept gently boiling 
 until the volume is reduced to about 10 c.c. and 
 
 i A process on the same lines has been described by Petit and Monfet 
 (Jour. Pharm. wnd Chem. , 1893, page 297), but their method of manipulating 
 is different in many respects from that employed by the author. Both 
 modifications are liable to give results below the truth. 
 
TOTAL NITROGEN IN UBINE. 129 
 
 white fumes of sulphuric acid are evolved. 1 The 
 liquid is then allowed to cool, and carefully trans- 
 ferred to a pear-shaped flask, the basin being rinsed 
 with a few drops of water. The flask is placed in 
 an inclined position, to prevent loss by spurting, and 
 the contents kept in gentle ebullition. If excessive 
 frothing occur, it may be moderated by adding a 
 small fragment of paraffin-wax (candle). When the 
 frothing has ceased, about 5 grammes of potassium 
 sulphate should be added and the flask heated 
 strongly until the liquid is colourless or only a very 
 pale yellow. The contents of the flask are then 
 allowed to become cold, when about 20 c.c. of water 
 is added very cautiously and a few drops at a time, 
 agitating the liquid by a rotatory movement between 
 each fresh addition. A highly concentrated solution 
 of caustic soda, made by dissolving the alkali in about 
 an equal weight of water, is now added gradually 
 with constant agitation, until the sulphuric acid is 
 nearly neutralised. This point may be ascertained 
 by means of litmus-paper, or a few drops of litmus 
 or phenol-phthalein solution may be added to the 
 contents of the flask. 
 
 The neutralised liquid, which will measure about 80 
 c.c, is now diluted to exactly 100 c.c. with water, and 
 thoroughly mixed by agitation. Ten c.c. of the solu- 
 
 1 As a rule, the quantity of sulphuric acid prescribed is amply sufficient for 
 the decomposition of the solids of 25 c.c. of urine. In the case of highly 
 saccharine urine, however, the sugar chars and forms a black pasty mass, 
 which cannot be readily transferred to the flask. In such a case, a further 
 addition of sulphuric acid (5 to 10 c.c.) shoulcl be made, and the heating 
 continued till the greater part of the carbonaceous matter is oxidised. It is 
 important in all cases to avoid the use of an excessive amount of sulphuric 
 acid, or so large an amount of soda must be employed to neutralise it, and so 
 large a volume of water added to retain the salts, in solution, that the 
 measure of the neutralised liquid cannot be kept within 100 c.c, or indeed 
 within any reasonable limits. On the other hand, less than 10 c.c. of acid is 
 an inconveniently small volume to heat and manipulate. Hence it is desirable 
 to adhere to the quantities of urine and acid prescribed in the text, and take 
 an aliquot part of the neutralised liquid for treatment with hypobromite. 
 
 I 
 
130 ESTIMATION OF TOTAL NITROGEN. 
 
 tion, representing 2"5 c.c. of the original urine, is now- 
 treated with the alkaline hypobromite reagent used 
 for the determination of urea (page 141). The mani- 
 pulation is the same as is described in the section on 
 " Urea," and one of the forms of apparatus therein 
 referred to may be employed. By far the most con- 
 venient apparatus for the purpose is, however, that 
 shown in fig. 7. Ten c.c. of the neutralised solu- 
 
 Fig. 7. — Nitrogen-Evolution Apparatus. 
 
 tion from the sulphuric acid treatment being placed 
 in the flask, 25 c.c. of the hypobromite reagent 
 should be poured into the separator, and the connec- 
 tions made as shown in the figure. The nitrometer 
 should be filled to the tap with water or (preferably) 
 brine, or, for very accurate experiments, mercury 
 may be advantageously used. The apparatus being 
 
TOTAL NITROGEN OF URINE. 
 
 131 
 
 adjusted, and the clip at the top of the nitrometer-cup 
 having been momentarily opened to equalise the pres- 
 sure in the cup with that in the flask, the tap of the 
 nitrometer is opened, and the hypobromite solution 
 then allowed to flow gradually into the flask. After 
 adding about 10 c.c, the separator-tap should be closed 
 and the flask agitated. A further addition of hypo- 
 bromite is then made, the flask again agitated, and this 
 treatment repeated until no further evolution of nitro- 
 gen takes place. As a rule, 10 c.c. of the reagent is 
 sufficient to complete the reaction, which occurs very 
 promptly and completely. 1 The flask is now allowed 
 to acquire the temperature of the room, when the 
 liquid in the nitrometer-tube is brought to the same 
 level with that in the reservoir-tube, and the volume of 
 nitrogen read off. If less than 20 c.c. of gas has been 
 evolved, the process may be advantageously repeated 
 on 20 c.c. or more of the neutralised liquid from the 
 sulphuric acid treatment. 
 
 From the number of cubic centimetres of nitrogen 
 evolved, v, the corresponding weight in milli- 
 grammes, W, may be calculated by the following 
 formula, in which p represents the barometric pressure 
 in millimetres ; w the tension of aqueous vapour at 
 
 1 The following figures, obtained in the author's laboratory by G. B. 
 B r o o k, show that the process gives tolerably good, but somewhat low, results 
 with solutions of pure ammonium sulphate, and that the reaction is fairly 
 complete unless the dilution is excessive. 
 
 
 Volumes of Solutions employed. 
 
 Percentage of 
 
 Total Nitrogen 
 
 evolved. 
 
 Soda. 
 
 Bromine. 
 
 Water. 
 
 Am. Sulphate. 
 
 Total. 
 
 A. 
 B. 
 0. 
 D. 
 
 25 co. 
 25 „ 
 25 „ 
 
 25 „ 
 
 2-5 C.C. 
 2-5 „ 
 2-5 „ 
 2-5 „ 
 
 none c.c 
 
 20 „ 
 30 „ 
 50 „ 
 
 5 c.c. 
 
 5 „ 
 5 „ 
 5 „ 
 
 32-5 c.c. 
 47-5 „ 
 62-5 ,,, 
 82-5 „ 
 
 100-0 
 98-1 
 97-1 
 927 
 
 If preferred, the hypobromite reagent may be placed in the flask and the 
 neutralised solution added to it. In this case, a layer of water should be floated 
 on the liquid in the separator, so as to rinse it completely into the flask. 
 
132 DETERMINATION OF TOTAL NITROGEN. 
 
 the temperature at which the gas was measured ; and 
 t the temperature in centigrade degrees :■ — 
 
 t,x(p- W ) 
 W_ 273 + * xiZiU - 
 
 When strictly accurate results are not required, the 
 corrections for temperature, pressure, and tension of 
 aqueous vapour may be omitted and the calculation 
 much simplified. Thus, the volume occupied by 28 
 milligrammes ( = 0*028 gramme) of moist nitrogen at 
 16° C. ( = 61°F.) and 762 millimetres ( = 30 inches) 
 pressure is 24 c.c. From this it follows that the 
 grammes of nitrogen contained in tOO c.c. of urine can 
 be calculated by the following equation, in which G 
 represents the number of c.c. of gas evolved, and U 
 the volume (in c.c.) of the original urine represented 
 by the neutralised liquid used : — 
 
 N = Gx 28x100 Gx7 
 ~Ux24xl000~Ux60' 
 
 Thus if the gas evolved from a measure of the 
 neutralised liquid corresponding to 5 c.c. of the original 
 urine measured 38 - 2 c.c, the sample contained 0*891 
 gramme of nitrogen per 100 c.c. 
 
 ,, 38-2x7 267-4 
 
 N = -5^60^ = W - ' 891 - 
 
 This figure, multiplied by 4 "375, will give the grains 
 of nitrogen per fluid ounce of the urine ; or, if divided 
 by the specific gravity of the sample (water = 1-000), 
 the actual percentage by weight of nitrogen contained 
 in the urine will be obtained. 
 
 It is evident that as Kjeldahl's process is applic- 
 able to the determination of the nitrogen of urea, uric 
 acid, creatinine, albumin, &c, as they exist in urine, it 
 is equally applicable to its determination in these sub- 
 stances when in an isolated state. Thus the nitrogen 
 
FORMS OP URINARY NITROGEN. 133 
 
 contained in a precipitate of uric acid may be ascer- 
 tained, and the weight of uric acid itself calculated 
 therefrom ; compounds of creatinine may be subjected 
 to the treatment and the base thus determined 1 ; or the 
 albumin may be precipitated from 100 c.c. of urine, 
 washed, and treated (together with the filter contain- 
 ing it) with strong sulphuric acid. 1 
 
 A volume of 24 c.c. of moist nitrogen, measured at the 
 ordinary pressure and temperature, corresponds to :— 
 
 o- 
 
 028 gramme of Nitrogen ; 
 034 ,, Ammonia; 
 
 060 
 084 
 358 
 164 
 
 Urea ; 
 Uric acid ; 
 Hippuric acid ; or 
 Albumin. 
 
 Urea. Carbamide. CH 4 N 2 ; or, CO(NH 2 ) 2 . 
 
 Urea exists ready-formed in the urine of mammals, 
 and in blood, milk, and other animal fluids. It 
 was first prepared synthetically by Liebig and 
 Wohler in 1828, being the first of the natural 
 organic bodies obtained by a synthetic process. 2 
 
 Urea forms transparent, colourless, four -sided, 
 anhydrous prisms (fig. 8). It is somewhat hygro- 
 scopic. Urea is odourless, and possesses a cooling 
 saline taste, resembling that of nitre. When heated 
 
 1 The evolution-method cannot be recommended in the case of creatinine. 
 Albumin requires prolonged treatment with sulphuric acid to effect complete 
 conversion of the proximate products of decomposition into ammonia. 
 
 2 This classical discovery affords an interesting example of re-arrangement 
 of the atoms in the molecule. Both ammonium cyanate and urea have an 
 elementary composition corresponding to the empirical formula : — CH 4 N 2 0. 
 On evaporating an aqueous solution of ammonium cyanate at the 
 temperature of boiling water, the salt suffers molecular change into urea, 
 according to the equation : — CN.O(NH 4 ) = CO(NH 2 ) 2 . The converse reac 
 tion occurs when an aqueous solution of urea is heated with silver nitrate. 
 A white precipitate of silver cyanate is formed, soluble in boiling water, while 
 the solution is found to contain ammonium nitrate : — CO(NH a ) 2 + AgN0 3 = 
 CN.OAg + (NH 4 )N0 3 , 
 
134 CHARACTERS OP UREA. 
 
 to 132° C. it melts, and at a higher temperature 
 decomposes with evolution of a m m o n i a and ammo- 
 nium cyanate, leaving a residue of cyanuric 
 acid, C 3 H 3 N 3 3 , which bears a much stronger heat 
 without change. 
 
 Urea is soluble in an equal weight of cold water, 
 and in a much less quantity of hot. It is also readily 
 soluble in alcohol, and dissolves in amy lie alcohol, but 
 it is nearly insoluble in ether, and quite so in chloro- 
 form and volatile oils. 
 
 At the ordinary temperature, an aqueous solution 
 of pure urea shows no tendency to change, and is not 
 decomposed by boiling ; but when heated with water 
 
 Fig. 8. — Crystals of Ukea — a, Quadrangular prisms ; b, Indefinite 
 crystals, as deposited from alcoholic solutions. 
 
 under pressure urea undergoes hydrolysis, with for- 
 mation of ammonium carbonate, CH 4 N 2 + 2H 2 = 
 (NH 4 ) 2 C0 3 . In the urine, where the urea is associated 
 with putrescible organic matter, it readily undergoes 
 a similar change, which is the cause of the alkaline 
 reaction of putrid urine. The ammoniacal fermenta- 
 tion of urine has been found to be due to the action 
 of an organised ferment (Torula urece) in the urine. 
 This change is set up by contact with the stomachs 
 of men, dogs, or rabbits, and has often been occa- 
 
UREA. NITRATE. 135 
 
 sioned in the bladder by the introduction of a septic 
 catheter. 
 
 Urea also yields ammonia when fused with caustic 
 alkali or ignited with soda-lime, a carbonate being 
 formed at the same time. When heated with a 
 strong mineral acid, urea similarly forms an ammonia- 
 cal salt, carbon dioxide being evolved. 
 
 Pure concentrated nitric acid combines with urea 
 without decomposing it, but if the acid contain 
 nitrous acid the urea is resolved into water, nitrogen, 
 and carbon dioxide, according to the following equa- 
 tion, the reaction, however, being far from complete : — 
 CH 4 N 2 + N 2 3 = 2H 2 + 2N 2 + C0 2 . 
 
 Chlorine, bromine, hypochlorites, and hypobromites 
 decompose solutions of urea with evolution of nitro- 
 gen. The best practical method of determining urea 
 in urine is based on this reaction (page 138). 
 
 The basic character of urea is well-marked, although 
 its solutions exhibit no alkaline reaction to litmus. It 
 forms a series of well-defined salts, some of which 
 crystallise readily. Many of them are decomposed 
 by water. 
 
 Urea Nitrate, CH 4 N 2 O.HN0 3 , separates in crystals 
 when moderately strong nitric acid is added to a 
 concentrated aqueous solution of urea, and the liquid 
 cooled. The compound forms brilliant white scales or 
 plates, or, if the deposition is slow, prismatic crystals. 
 When nitric acid and urea are brought together on a 
 microscope-slide, and the reaction observed under a 
 low power, the formation of obtuse rhombic octahedra 
 is first noticed, the angles being constantly 82°. 
 These octahedra change to rhombic and hexagonal 
 tables, either separate or superposed (see fig. 9, a), but 
 also having angles of 82°. For the formation of 
 nitrate of urea from normal urine, it is sufficient to 
 concentrate the liquid to about one-fourth of its 
 
136 
 
 UREA OXALATE. 
 
 volume, filter after cooling from the precipitated 
 urates, &c, and add nitric acid to the cold filtrate. 
 Nitrate of urea is unalterable in the air. It is readily 
 soluble in water, forming a solution of acid reaction 
 and taste. It is also soluble in alcohol, but only very 
 slightly in presence of nitric acid. Oxalic acid pre- 
 cipitates urea oxalate from concentrated solutions of 
 the nitrate. 
 
 Fig. 9. — a, Ueba Nitbate ; b, Ukea Oxalate. 
 
 Urea Oxalate, (CH 4 N 2 0)2.C 2 H 2 4 , is readily formed 
 on mixing concentrated solutions of urea and oxalic 
 acid. From urine it may be prepared by adding 
 oxalic acid to the concentrated and filtered liquid. 
 Urea oxalate forms thin crystalline plates (see fig. 9, b), 
 usually grouped together, but sometimes in well- 
 formed separate crystals. Its microscopic appearance 
 is not unlike that of the nitrate of urea, but the forms 
 are less characteristic, and the angles are different. 
 Oxalate of urea is soluble with difficulty in cold 
 water, but dissolves readily at a boiling heat. It is 
 less soluble in a solution of oxalic acid than in pure 
 water. The salt dissolves in 62 parts of alcohol, but is 
 quite insoluble in amylic alcohol. Hence, if a solu- 
 tion of urea in amylic alcohol (such as will result 
 
COMPOUNDS OF UREA. 137 
 
 from evaporating urine to dryness, heating the 
 residue with amylic alcohol, and filtering) be treated 
 with a cold saturated solution of oxalic acid in amylic 
 alcohol, urea oxalate is precipitated in small crystals. 
 By warming the liquid until the crystals are redis- 
 solved and allowing it to cool, the salt is obtained in 
 a state fit for microscopic examination. The process 
 may be modified by treating the solution of urea in 
 amylic alcohol with one of oxalic acid in anhydrous 
 ether. Precipitation takes place abundantly and 
 quickly, but the crystals are usually small and im- 
 perfect. The oxalic acid may be added in powder, 
 the liquid heated and thoroughly cooled, and the 
 excess of oxalic acid removed from the precipitate by 
 treatment with anhydrous ether. The method is 
 capable of being employed quantitatively. The 
 amylic alcohol used in the process must not develop 
 a red or brown colour with oxalic acid, and should be 
 free from water and ethylic alcohol. 
 
 Urea Phosphate, CH 4 N 2 0,H 3 P0 4 , forms large, very 
 soluble, rhombic crystals on evaporating pig's urine, or 
 mixed solutions of urea and phosphoric acid. 
 
 A compound of urea with sodium chloride, of 
 the formula CH 4 N 2 0,NaCl,H 2 0, separates in brilliant 
 rhombic crystals when mixed solutions of urea and 
 common salt are evaporated. It sometimes crystal- 
 lises from concentrated human urine. 
 
 On mixing a solution of urea with one of neutral 
 mercuric nitrate, a white flocculent precipitate is 
 obtained. This has a composition dependent on the 
 concentration of the liquid, containing, according to 
 the conditions of its formation, 1, 1^, or 2 molecules 
 of mercuric oxide to 1 of urea. If, however, the 
 addition of the mercuric nitrate be continued as long 
 as precipitation occurs, and sodium carbonate be 
 added from time to time to neutralise the nitric 
 
138 REACTIONS OF UREA. 
 
 acid set free, the precipitate has the composition 
 CH 4 N 2 0,2HgO. The end of the reaction is indicated 
 by the yellow colour developed from the formation of 
 basic nitrate of mercury. L i e b i g 's method of de- 
 termining urea, now chiefly of historical interest, was 
 based on this reaction. 
 
 Urea is not precipitated by a solution of mercuric 
 chloride. The addition of mercuric nitrate to a 
 soluble chloride results potentially in the formation 
 of mercuric chloride. As sodium chloride is present 
 in urine, mercuric nitrate produces no precipitate of 
 Liebig's compound in that liquid until sufficient has 
 been added to react fully with the chloride present. 
 On this fact Liebig based a method for determining 
 chlorides in urine. 
 
 Mercuric acetate gives no precipitate with urea in 
 the cold, and the separation is very incomplete on 
 boiling. 
 
 For the recognition of urea in a weak aqueous solu- 
 tion B 1 o x a m has suggested the following method : — 
 If a nitrate be present, add a few drops of ammonium 
 chloride solution, but if absent, acidulate the liquid 
 with hydrochloric acid. Evaporate the solution to 
 dryness in a watch-glass, and heat the residue 
 cautiously as long as thick white fumes are evolved. 
 Dissolve the cooled residue in a drop or two of 
 ammonia, add a drop of barium chloride, and stir. 
 If urea was present, crystalline streaks of barium 
 cyanurate will be formed in the track of the glass 
 rod. 
 
 Determination of Urea. 
 
 Various methods have been devised for the deter- 
 mination of urea in urine, but those dependent on 
 the measurement of the nitrogen gas evolved by its 
 decomposition are by far the most convenient, and 
 sufficiently accurate for ordinary purposes. They are 
 
DETERMINATION OP UREA. 139 
 
 based on the reaction between urea and a strongly- 
 alkaline solution of hypobromite of sodium, whereby 
 sodium bromide, water, carbon dioxide, and free nitrogen 
 are produced, according to the following equation : — 
 
 3NaBrO + CH 4 N 2 = 3NaBr+2H 2 0-f-C0 2 +N 2 . 
 
 The carbon dioxide (carbonic acid) gas is absorbed 
 by the excess of caustic alkali employed, so that, 
 under the conditions of the experiment, pure nitrogen 
 gas is evolved. 
 
 According to the great majority of observers, the 
 reaction of cold hypobromite solution of the strength 
 commonly employed results in the evolution of from 
 92 to 93 per cent, of the nitrogen existing in the urea 
 present. 1 The suppressed nitrogen has been found to 
 suffer conversion into cyanate. On heating the liquid, 
 some further evolution of gas occurs, but the theo- 
 retical production is never realised, and under some 
 circumstances there is a tendency to error from evolu- 
 tion of oxygen. In presence of cane-sugar, a more 
 complete evolution of the nitrogen occurs, and hence 
 C. Mehu {Bull. Soc. Chim., [2], xxxiii. 410, and 
 Jour. Chem. Soc, xxxviii. 681) has proposed always 
 to add 10 parts of cane-sugar for each 1 of urea 
 supposed to be present. Glucose induces a still more 
 perfect evolution of the nitrogen, but is said to be apt 
 to occasion the liberation of traces of gas even in the 
 absence of urea. In consequence of the peculiar action 
 of glucose, the evolution of nitrogen on treating dia- 
 betic urine with hypobromite reaches 99 per cent, of 
 the total nitrogen present in the urea. Even in 
 normal urine, the evolved nitrogen bears a greater 
 
 1 According to T. G. Wormley (Chem. News, xlv. 27), under favourable 
 conditions the whole of the nitr ogen of urea is evolved as gas. J. R. D u g g a n 
 (Arner. Chem. Jour., iv. 47, and Jour. Chem. Soc, xlii. 778) states that I 
 fully 99 per cent, of the nitrogen is evolved as gas if 5 c.c. of the urine be first 
 mixed with 20 c.c. of a solution of 20 grammes of caustic soda in 100 c.c. of 
 water, and 1 c.c. of bromine is subsequently added. 
 
140 DETERMINATION OE UREA. 
 
 j proportion to the total amount present in the urea 
 than is the case when pure solutions of urea are 
 operated on. This fact is commonly attributed to the 
 liberation of nitrogen from the uric acid, creatinine, 
 and other urinary constituents, which, though present 
 in but small amount relatively to the urea, are able to 
 exert a sensible influence on the proportion of gas 
 evolved. For ordinary purposes, the error due to this 
 cause is wholly unimportant, and it has even been 
 contended that, as the usual object of determining 
 urea is to obtain a measure of the nitrogenous waste, 
 all nitrogenised constituents of the urine should, as 
 far as possible, be determined. Of course this is 
 directly effected by determining the total nitrogen in 
 the manner described on page 128. 
 
 M£hu points out that the uric acid present in 
 urine is rarely more than 2 per cent, of the urea, and 
 the creatinine still less, and as these bodies only 
 evolve a portion of their nitrogen when treated with 
 hypobromite they are incompetent to produce the 
 whole of the effect ascribed to them. He attributes 
 the better yield of nitrogen obtained from normal 
 urine to the extractive matter present, which acts 
 more or less like sugar. Hence M^hu recommends 
 the addition of sugar in every case, so as to ensure the 
 evolution of practically the whole of the nitrogen in 
 the gaseous state. 
 
 G. Esbach {Bull. Soc. Chim., [2], xxiv. 632) 
 states that pure glucose solution, whether boiling or 
 not, evolves a small quantity of gas when treated 
 with the hypobromite reagent, but that cane-sugar 
 yields no gas. He finds that in aqueous solutions of 
 urea, the excess over the 92 per cent, normally evolved 
 varies with the quantity and nature of the sugar 
 added, the strength of the urea solution, and com- 
 position, especially the free alkali of the hypobromite 
 
HYPOBROMITE REAGENT. 141 
 
 reagent, the volume of gas being greater the more 
 alkali there is present For the proportions of glucose 
 present in diabetic urine, the excess of nitrogen over 
 92 per cent, is sensibly proportional to the mass of the 
 sugar, but this does not hold good for large quantities. 
 Urea added to a true diabetic urine is stated to evolve 
 only 92 per cent, of its nitrogen. Esbach concludes \ 
 that sugar should not be added to urine before apply- 
 ing the hypobromite process. 
 
 A very large number of determinations of urea by 
 the hypobromite method have been made in the author's 
 laboratory, the process being varied in many cases with 
 the view of ascertaining the best conditions under 
 which to work. The results have shown that the 
 method is by no means so constant as generally sup- 
 posed, and that it is liable to give low results from 
 undiscovered causes. In presence of glucose or cane-| 
 sugar great evolution of heat occurs, and it is probably 
 this phenomenon which is the cause of the higher 
 results obtained in presence of sugar. Dilution of 
 the hypobromite reagent with water, or the use of 
 less bromine, did not greatly affect the results, but 
 a large excess of bromine was prejudicial. 
 
 The hypobromite solution employed for the de- 
 composition of urea is prepared by dissolving 100 
 grammes of good caustic soda in 250 c.c. of water, 
 and thoroughly cooling the liquid ; 25 c.c. measure of 
 bromine is then added, and the resultant solution pre- 
 served in a cool place. 1 The reagent does not keep 
 very well, in consequence of the gradual occurrence of 
 the reaction: — 3NaBrO = 2NaBr+NaBr0 3 . Hence 
 it is preferable to prepare the solution, when required, 
 by mixing 25 c.c, of the caustic soda solution with 25 
 c. c. of bromin e. Considerable variations in the strength 
 of the reagent do not materially affect the results. 
 
 1 Or 3J oz. of caustic soda, 9 oz. of water, and If fluid ounce of bromine. 
 
142 
 
 GERRARDS AREOMETER. 
 
 Various forms of apparatus have been devised for 
 effecting the reaction and collecting the evolved nitro- 
 gen, and a suitable arrangement is easily extempor- 
 ised. Of the special forms, that devised by A. W. 
 Gerrard (Pharm. Jour., [3], xv. 464) is among the 
 best, when no extreme degree of accuracy is aimed 
 at. Gerrard's apparatus (fig. 10) consists of a gradu- 
 ated tube, which is connected with a second tube, 
 serving as a reservoir, by means of india-rubber 
 tubing. The top of the graduated tube is closed by a 
 
 Fig. 10. — Gebbard's Ureometer. 
 
 caoutchouc stopper, through which passes a T-tube, 
 one orifice of which is fitted with a short piece of 
 india-rubber tubing closed by a clip, while the other 
 communicates by a second piece of tubing with a 
 bottle fitted with a perforated cork. In making the test, 
 25 c.c. of the hypobromite reagent should be poured 
 into this bottle, and then a small test-tube contain- 
 ing 5 c.c. of the sample of urine cautiously placed in 
 
DETERMINATION OP UREA. 143 
 
 it in such a manner as to avoid any contact between 
 the urine and the reagent. The bottle is now con- 
 nected with the graduated tube in the manner shown 
 in the figure, the clip opened, and water poured into 
 the reservoir-tube until, on suitably adjusting its 
 height, the water stands at the zero-point in the 
 measuring tube and at the same level in the reser- 
 voir, taking care that when this is effected but little 
 water remains in the latter tube. 1 
 
 The clip is then closed, and the bottle is tilted in 
 such a manner as to allow the urine to mix gradually 
 with the hypobromite solution, the bottle being gently 
 agitated to promote the evolution of gas, which com- 
 mences immediately and is complete in a few minutes. 
 After five minutes, or preferably ten, the water in the 
 measuring and reservoir tubes are brought to the same 
 level by lowering the latter, when the volume of gas 
 is read off. 2 In Gerrard's apparatus the tube is so 
 graduated as at once to show the percentage of urea 
 contained in the urine. If the urine contain more 
 than 3 per cent, of urea, it is necessary to take 2 - 5 
 instead of 5 c.c, dilute it with an equal measure of 
 water, and double the result obtained. With normal 
 urine, the volume of nitrogen evolved under the fore- 
 going mode of treatment is only 92 per cent, of that 
 which would be yielded if the reaction formulated on 
 page 139 were the only one which took place ; but as a 
 fact, some 7^ to 8 per cent, is suppressed. Gerrard's 
 apparatus is so graduated as to allow for this loss, and 
 hence gives correct results when non-saccharine urine is 
 under examination. But, in the case of urine contain- 
 ing much sugar, fully 99 per cent, of the ureal nitrogen 
 
 1 This can be effected by raising the reservoir, and is necessary to make 
 room for the water displaced by the gas subsequently evolved. 
 
 2 In case the temperature of the room is greatly different from 15 '5° C. ( = 60° F. ) 
 the measuring tube should be immersed in water at that temperature, but for ordi- 
 nary purposes, and under ordinary conditions, this precaution is unnecessary. 
 
144 THE NITROMETER. 
 
 is given off, so that, to obtain the true amount of urea, 
 the result indicated by Gerrard's apparatus should be 
 multiplied by 92 -=- 99 = 0'93. 
 
 In the absence of any specially constructed apparatus 
 for the estimation of urea, a suitable arrangement can be 
 readily extemporised. Thus, if preferred, the nitrogen 
 may be simply collected in a graduated tube filled with 
 and standing over water, but it is more convenient 
 to employ a graduated tube open at the upper end, 
 which is drawn out so as to admit of being readily 
 connected with the india-rubber tube attached to the 
 evolution flask. An inverted Mohr's burette, of a 
 capacity of 50 c.c, immersed in a cylinder of water, 
 answers very well. As long ago as 1877, A. Dupre" 
 (Jour. Chem. Soc, xxxi. 534) recommended a tube of 
 this kind, but furnished with a side-tube to which a 
 clip was attached, which arrangement afforded some 
 facilities in manipulating. These devices have all 
 been superseded by the nitrometer, the employment 
 of which for the purpose was first proposed by the 
 author (Jour. Soc. Chem. Ind., iv. 179). 1 
 
 Fig. 1 1 shows the improved form of Lunge's nitro- 
 meter, furnished with a three-way tap, which enables 
 communication between the nitrometer-tube and either 
 the evolution-flask or the external air to be made at 
 will, and materially facilitates the manipulation. 
 The method of working is almost the same as with 
 Gerrard's form of apparatus. The hypobromite solu- 
 tion is placed in the flask, the tube charged with the 
 sample of urine is. carefully introduced, the stopper 
 firmly adjusted, and the india-rubber leading-tube 
 
 1 D u p r & ' s apparatus appears to have been the first ureometer in which the 
 principle of the nitrometer wits adopted. The same principle was utilised in 
 Gerrard's arrangement, the description of which was published a few months 
 before the appearance of the author's paper on "New and little-known 
 applications of the Nitrometer"; but the apparatus had been used and 
 sketched by the author some years previously. 
 
FORMS OF NITROMETER. 
 
 145 
 
 
 attached to the nose of the three-way tap. The tap 
 is then turned, so as to allow the nitrometer-tube to 
 communicate with the external 
 air, and the open reservoir-tube 
 raised until the liquid in the 
 graduated tube just rises to the 
 zero-point, whereon the tap is 
 closed. The tap is then turned 
 so as to connect the flask with 
 the nitrometer-tube, while cut- 
 ting off the external air. The 
 urine is then allowed to come 
 gradually into contact with the 
 reagent in the manner already 
 described. When the reaction is 
 complete, the liquid in the reser- 
 voir-tube is brought to the level 
 of that in the nitrometer, and 
 the volume of gas read off. 
 
 In the absence of a Lunge's 
 nitrometer, the simpler form of 
 apparatus devised by the author for assaying spirit 
 of nitrous ether (Pharm. Jour., [3], xv. 673), and 
 known as "Allen's nitrometer," will answer every 
 purpose. The arrangement is shown in fig. 12, but 
 there is no absolute necessity for the T-tube and spring 
 clip ; though in their absence it is difficult to adjust 
 the level of the liquid in the nitrometer-tube to the 
 zero-point, and it becomes necessary to observe the 
 position when the water in the open and graduated 
 tubes stand at the same level. 
 
 In the modified form of apparatus shown on page 
 130, a separating funnel is substituted for the sample 
 tube. This arrangement allows the urine and reagent 
 to be brought together with any desired rapidity, or the 
 bromine to be added to the soda and urine previously 
 
 K 
 
 Fist. 11. 
 
146 
 
 FORMS OF NITROMETER. 
 
 mixed together in the flask. 1 These and other varia- 
 tions in manipulation are very valuable in researches, 
 and in certain special applications of the nitrometer. 
 
 For all ordinary purposes, it is convenient and 
 sufficiently accurate to fill the nitrometer with water, 
 but mercury is sometimes used in research-work. 
 
 Another mode of using the nitrometer, and one 
 which does not necessitate the use of an instrument 
 
 Fig. 12. 
 provided with a three-way tap or equivalent arrange- 
 ment, has also been described by the author (loc. 
 cit.). For this purpose the nitrometer is filled to the 
 
 1 This mode of operating appears to be a valuable modification of the 
 ordinary plan, since a sensibly more complete evolution of the ureal nitrogen 
 is obtained, as was proved by experiments made in the author's laboratory by 
 A. R. Tankard. Twenty-five c.c. of a 40 per cent, solution of caustic soda is 
 placed in the flask, and 5 c. c. of the sample of urine added. The cork is then 
 adjusted and a solution of bromine in aqueous bromide of potassium (20 per 
 cent, solution) added gradually from the tapped separator. In each case 
 the evolution of nitrogen occurred very promptly. 
 
DETERMINATION OF UREA. 147 
 
 tap with strong brine, and the tap closed. Five c.c. of 
 the sample of urine 1 should then be placed in the cup 
 by means of a pipette, and allowed to flow into the 
 tube by opening the tap cautiously. The last traces 
 are rinsed in by a few drops of water. Ten c.c. of 
 the hypobromite solution, previously diluted with an 
 equal measure of water, is next added through the tap. 
 The greater part of the nitrogen is liberated as soon as 
 the reagent comes into contact with the urine. When 
 the reaction, has somewhat abated, a clip is placed on 
 the india-rubber connecting-tube, and the nitrometer- 
 tube vigorously shaken. When the reaction is com- 
 pleted, the clip may be removed, the liquids in the 
 nitrometer- and reservoir-tubes brought to the same 
 level, and the volume of gas observed. If the read- 
 ing be rendered difficult from the formation of a 
 persistent froth, this may be instantly destroyed by 
 introducing a few drops of alcohol through the tap. 
 
 Instead of measuring the volume of gas evolved, it 
 is sometimes convenient to measure the water dis- 
 placed by it. This can be effected very simply by an 
 arrangement devised by E. R. Squibb {Ephemeris, 
 ii. 449). A still simpler device, and one which can 
 be readily extemporised, has been devised by the 
 author. It consists simply of the apparatus shown 
 in fig, 3, page 47, with the addition of a test-tube 
 to contain the sample of urine. One fluid ounce of the 
 hypobromite mixture is placed in the feeding-bottle, 
 and in the test-tube 2 fluid drachms ( = 7'1 c.c.) of 
 the sample of urine, together with a little simple 
 syrup. The test-tube is placed inside the feeding- 
 bottle, the stopper adjusted, and the contents of the 
 tube allowed to mix with the hypobromite solution, 
 just as when operating with the forms of apparatus 
 
 1 If the volume of gas evolved exceed 40 c.c., a smaller measure of urine 
 should be employed. 
 
148 DETERMINATION OP UREA. 
 
 already described. The displaced water may be 
 conveniently collected in a 2-oz. measure. Operat- 
 ing with £ oz. of urine, as prescribed above, each 
 fluid ounce of water displaced represents 7 grains 
 of urea per fluid ounce of the sample. Hence, if 
 the measure collected were If oz. ( = 14 drachms), 
 the urine contained 12^ grains per fluid ounce. The 
 percentage of urea may be found by dividing 12^ 
 by 4-375, or multiplying it by - 2286, which in the 
 example taken would give 2 '80 per cent. 
 
 In interpreting the results obtained by any of the 
 modifications of the hypobromite method of estimating 
 urea, it is unnecessary for ordinary purposes to take 
 into account the barometric pressure, tension of 
 aqueous vapour, or temperature at the time of making 
 the experiment. By a fortunate coincidence, the in- 
 crease in the volume of the gas, due to its being 
 measured in a wet condition and at a temperature of 
 18° C. ( = 65 - 4° F.), almost exactly compensates for 
 the *1\ or 8 per cent, of the total nitrogen ordinarily 
 suppressed in the reaction. Thus Russell and 
 West (Jour. Chem. Soc, xxvii. 749) found that 
 0"100 gramme of urea gave off 37*1 c.c. of moist 
 nitrogen at a temperature of 65° F. ( = 18"3° C). 
 This, if dry, and at the standard pressure and tem- 
 perature, would have measured 34"05 c.c, whereas the 
 total nitrogen contained in the same amount of urea 
 would measure 37"2 c.c. under the standard conditions, 
 or nearly the volume actually obtained at the ordinary 
 temperature. Hence 37 '1 c.c. of gas, measured under 
 the ordinary conditions of experiment, may be taken 
 to represent O'l gramme of urea. Thus, if the cubic 
 
 centimetres of gas evolved be multiplied by _, =2*7 
 
 OIL 
 
 (or, more exactly, 2"696) the product will be the 
 number of milligrammes of urea in the volume of 
 
RELATION OF NITROGEN TO UREA. 
 
 149 
 
 urine employed ; or, if 5 c.c. of urine were taken for 
 the experiment, the volume of gas evolved, multiplied 
 by 0"054 (or, more exactly, 0*0539) will be the grammes 
 of urea (so-called " percentage ") in 100 c.c. of the 
 urine. The percentage thus found, multiplied by 
 4"375, will be the number of grains of urea contained 
 in 1 fluid ounce of the urine. 
 
 If preferred, the above calculations can be combined, 
 and the grains of urea per fluid ounce of the sample found 
 at once by multiplying the measure of gas evolved from 
 5 c.c. of the sample by 0'236 (or, more exactly, 0'23585). 
 
 Instead of employing the foregoing factors, the con- 
 tent of urea per 100 measures and per fluid ounce can be 
 found by reference to the following table, compiled by 
 S. Henry Smith (Pharm. Jour., [3], xxi. 294): — 
 
 
 Urea. 
 
 
 Urea. 
 
 Volume of Nitrogen 
 in c.c. evolved from 
 
 
 
 Volume of Nitrogen 
 
 in c.c. evolved from 
 
 5 c.c. of Urine. 
 
 
 
 
 
 
 
 5 c.c. of Urine. 
 
 Per cent. 
 
 Grains per 
 oz. 
 
 Per cent. 
 
 Grains per 
 oz. 
 
 1 . 
 
 ■053 
 
 •231 
 
 29 . 
 
 1-56 
 
 6-82 
 
 2 . 
 
 
 
 •107 
 
 •468 
 
 30 . 
 
 
 1-61 
 
 7-04 
 
 3 . 
 
 
 
 •161 
 
 •704 
 
 31 . 
 
 
 1-67 
 
 7-30 
 
 4 . 
 
 
 
 •215 
 
 •94 
 
 32 . 
 
 
 1-72 
 
 7-52 
 
 5 . 
 
 
 
 •269 
 
 1-17 
 
 33 . 
 
 
 1-77 
 
 7-74 
 
 6 . 
 
 
 
 •323 
 
 1-41 
 
 34 . 
 
 
 1-83 
 
 8-00 
 
 7 . 
 
 
 
 •377 
 
 1-64 
 
 35 . 
 
 
 1-88 
 
 8-22 
 
 8 . 
 
 
 
 •431 
 
 1-88 
 
 36 . 
 
 
 1-94 
 
 8-48 
 
 9 . 
 
 
 
 •485 
 
 2-12 
 
 37 . 
 
 
 1-99 
 
 8-70 
 
 10 . 
 
 
 
 •539 
 
 2-35 
 
 38 . 
 
 
 2-04 
 
 8-92- 
 
 11 . 
 
 
 
 •592 
 
 2-59 
 
 39 . 
 
 
 2-10 
 
 9-18 
 
 12 . 
 
 
 
 •646 
 
 2-82 
 
 40 . 
 
 
 2-15 
 
 9-40 
 
 13 . 
 
 
 
 ■700 
 
 3-06 
 
 41 . 
 
 
 2-21 
 
 9-66 
 
 14 . 
 
 
 
 •754 
 
 3-29 
 
 42 . 
 
 
 2-26 
 
 9-88 
 
 15 . 
 
 
 
 •808 
 
 3-53 
 
 43 . 
 
 
 2-31 
 
 10-10 
 
 16 . 
 
 
 
 •886 
 
 3-87 
 
 44 . 
 
 
 2-37 
 
 10-36 
 
 17 . 
 
 
 
 •916 
 
 4-00 
 
 45 . 
 
 
 2-42 
 
 10-58 
 
 18 . 
 
 
 
 •975 
 
 4-26 
 
 46 . 
 
 
 2-47 
 
 10-80 
 
 19 . 
 
 
 
 1-02 
 
 4-46 
 
 47 . 
 
 
 2-53 
 
 11-06 
 
 20 . 
 
 
 
 1-07 
 
 4-68 
 
 48 . 
 
 
 2-58 
 
 11-28 
 
 21 . 
 
 
 
 1-13 
 
 4-84 
 
 49 . 
 
 
 2-64 
 
 11-55 
 
 22 . 
 
 
 
 1-18 
 
 5-16 
 
 50 . 
 
 
 2-69 
 
 11-76 
 
 23 . 
 
 
 
 1-24 
 
 5'42 
 
 51 . 
 
 
 2-74 
 
 11-98 
 
 24 . 
 
 
 
 1-29 
 
 564 
 
 52 . 
 
 
 2-8 
 
 12-25 
 
 25 . 
 
 
 
 1-34 
 
 5-86 
 
 53 . 
 
 
 2-85 
 
 12-46 
 
 26 . 
 
 
 
 1-40 
 
 6-12 
 
 54 . 
 
 
 2-91 
 
 12-73 
 
 27 . 
 
 
 
 1.45 
 
 6-34 
 
 55 . 
 
 
 2-96 
 
 12-95 
 
 28 . 
 
 
 
 1-50 
 
 6-56 
 
 56 . 
 
 
 3-01 
 
 13-16 
 
150 ERRORS IN UREA DETERMINATIONS. 
 
 It must be borne in mind that the foregoing factors 
 
 and table are intended for use with non-saccharine 
 
 urine. As already stated, the evolution of the nitro- 
 
 'I gen of urea is more complete in the presence of sugar 
 
 ( than in its absence, and hence the results yielded 
 
 | when diabetic urine is treated with hypobromite 
 
 ! should be multiplied by the factor 0'93 to obtain 
 
 more correct results. An alternative plan is to 
 
 add some simple syrup or honey (from 3 to 5 
 
 c.c.) to the urine in the sample-tube. Under these 
 
 circumstances, 40 c.c. of nitrogen (against 37 '1 
 
 c.c. in the absence of sugar) are yielded by O'l 
 
 gramme of urea. Hence if 5 c.c. of urine be the 
 
 volume employed, each c.c. of gas evolved represents 
 
 - 05 per cent, of urea in the sample. Thus, if the 
 
 nitrogen measure 26'4 c.c, the sample contains 26"4 
 
 x 0'05 = 1*32 per cent, of urea. 
 
 The author's experience of the hypobromite method 
 of estimating urea, under a great variety of conditions, 
 and with the utmost care to obtain accurate results, 
 leads him to the conclusion that the process is by no 
 means so accurate, nor even so constant in its indica- 
 tions, as is commonly supposed. But it is undoubt- 
 edly a method of great practical utility, and one which 
 is capable of doing excellent service in the hands of 
 intelligent operators. Its readiness of application, and 
 the great rapidity with which fair approximations can 
 be obtained, specially adapt it to the requirements of 
 the physician, who might employ it with advantage 
 far more frequently than is the case at present. 
 
 Influence of Varying Conditions on the Excre- 
 tion of Urea. 
 
 The amount of urea excreted varies considerably 
 with the diet, being increased by nitrogenous foods. 
 The weight of urea excreted per diem by an adult 
 man on mixed diet ranges from 25 to 40 grammes, 
 
EXCRETION OF UREA. 151 
 
 the average being about 33 grammes ( = 500 grains). 
 On a diet poor in proteids the excretion of urea may 
 fall to 15 to 20 grammes, while on a flesh diet the 
 daily output may rise to 100 grammes. The propor- 
 tion of urea in human urine averages about 2 per cent., 
 but dog's urine is stated to contain 10 per eent. 
 
 A large excretion of urea, if long continued, points to 
 increased tissue-metabolism or to surplus nitrogenous 
 ingesta, but a temporary increase may be simply due 
 to increased urination. Similarly, diminished excre- 
 tion of urea may be due to diminished metabolism or 
 to retention of urea in the system (as in uraemia). 
 
 A great number of observations have been recorded 
 of the influence of drugs, diseases, and other conditions 
 on the proportion of urea excreted. The results have 
 been classified byW. D. Halliburton {Chemical 
 Physiology and Pathology, 1891) as follow : — 
 
 An increased excretion of urea occurs: — 
 
 1. After administration of dilute sulphuric acid, 
 potassium chloride, ammonium salts (especially with 
 food), small doses of phosphorus, arsenic, antimony, 
 morphine, codeine, or large doses of quinine. 
 
 2. After poisoning by phosphorus or arsenic. 
 
 3. From application of cold to the skin ; after hot 
 baths ; from increase of oxygen inhaled ; from exces- 
 sive muscular work. 
 
 4. In diseases, as at the commencement of acute 
 febrile diseases, up to the acme of the fever; during the 
 paroxysms of intermittent fever (ague); in diabetes. 
 
 A decreased excretion of urea occurs : — 
 
 1. After administration of small doses of quinine. 
 
 2. During the sinking of the fever in acute febrile 
 diseases ; in most chronic and debilitating diseases 
 (anaemia, syphilis, phthisis, dropsical affections, &c.) ; 
 towards the fatal termination of most diseases (5 to 6 
 grammes daily) ; in uraemia (when the excretion may 
 
152 CREATININE AND CREATINE. 
 
 entirely cease); in diabetic coma ; and in all degenera- 
 tive changes of the liver, especially in acute yellow 
 atrophy. 
 
 Creatinine. Methyl-glycocyamidine. 
 
 C 4 H,N 3 0;or,NH:C{S>- CI M. 
 
 Creatinine is an anhydride of creatine, 
 C 4 H 9 N 3 2 , and is produced from the latter body with 
 great facility. 1 Creatinine occurs constantly in normal 
 human urine, the amount varying, according to V o i t, 
 from 0'5 to 4 - 9 grammes per diem, according to the 
 quantity of proteids eaten. The proportion is not 
 diminished by fasting, but is said to be increased in 
 typhus, intermittent fever, pneumonia and tetanus, 
 and diminished in convalescence from acute diseases, 
 anaemia, chlorosis, paralysis and phthisis. Creatinine 
 has been found in sweat and the muscles of fishes, and 
 Gr. S. Johnson has isolated creatinine, or a modifica- 
 tion of it, from the flesh of a healthy cow. 
 
 1 Creatine ; Methyl-glycocyamine ; or Methyl-guanidine-acetic acid. 
 C 4 H 9 N 3 2 ;o r ,NH:c{^).CH,COOH| > 
 
 Creatine is a constant constituent of muscle-substance, to the extent of - 2 
 to 0"3 per cent, of its weight, and is most conveniently prepared from Liebig's 
 
 extract of meat. Creatine has also been 
 found in nerve-tissue, and probably occurs in 
 minute traces in urine and other animal fluids. 
 But its isolation from these does not prove its 
 pre-existence therein, since it is very easily 
 formed by the dehydration of creatinine, into 
 which body, on the other hand, creatine is 
 very readily changed. Thus, on evaporating 
 a solution of creatine with the calculated 
 amount of dilute sulphuric acid, it yields a 
 residue of creatinine sulphate. A similar 
 Fig. 13.— Creatine (af terFrey). change occurs very readily on boiling creatine 
 with dilute hydrochloric acid, and the result- 
 ant creatinine can be readily recognised by conversion into the zinc salt. 
 
 Creatine crystallises in transparent rhombic prisms (fig. 13) containing water. 
 It is sparingly soluble in cold water to form a slightly bitter solution, neutral 
 to litmus. It reduces alkaline picric acid solution gradually in the cold, but 
 immediately on boiling. 
 
ISOLATION OF CREATININE. 
 
 153 
 
 Kg. 14. — Creatinine (after Frey). 
 
 Creatinine may be isolated from human urine by 
 L i e b i g's process, which consists in exactly neutral- 
 ising the liquid with milk 
 of lime, and adding calcium 
 chloride as long as calcium 
 phosphate continues to be 
 precipitated. The filtrate, 
 which should be neutral or 
 very faintly acid, is evapo- 
 rated to a small bulk, and 
 the crystals of common salt, 
 &c, removed. Thirty-two 
 parts of the mother-liquor 
 are treated with one part 
 of zinc chloride in very concentrated solution, and 
 the whole left for several days. The creatinine-zinc 
 chloride which separates in nodules is washed with 
 a little cold water, and then with alcohol. It is 
 then boiled with recently precipitated lead hydroxide, 
 the filtrate evaporated, and the residue digested with 
 absolute alcohol, which dissolves the creatinine leav- 
 ing any creatine insoluble. 
 
 G. Stilling fleet Johnson prepares creat- 
 inine by a modification of Maly's process. 1 He treats 
 a large volume of urine with 5 per cent, of its volume 
 of a saturated aqueous solution of sodium acetate and 
 25 per cent, of saturated mercuric chloride solution. 
 The precipitate which forms is filtered off immediately, 
 and the filtrate left at rest for forty-eight hours. The 
 creatinine separates in microscopic spherical masses of 
 the mercuric chloride compound, which is filtered off, 
 washed with cold water, and decomposed by sulphur- 
 
 1 Maly (Ann. Ohem. Pharm., clix. 279) recommends a preliminary con- 
 centration of the urine by heat, and precipitation by basic acetate of lead 
 before adding mercuric chloride. Johnson's modification is a marked improve- 
 ment on this, as it gives a purer product and wholly avoids the use of heat, 
 which is an essential condition if it be desired to obtain unchanged creatinine. 
 
154 
 
 CHARACTERS OF CREATININE. 
 
 etted hydrogen. The filtered liquid is decolorised 
 with purified animal charcoal, and concentrated by 
 spontaneous evaporation over sulphuric acid, when 
 creatinine hydrochloride separates in brownish-yellow 
 prisms. The crystals are redissolved in 15 parts of 
 cold water, and the solution treated with an excess of 
 lead hydroxide, prepared by precipitating a solution 
 of the acetate with ammonia. The mixture is well 
 stirred for twenty minutes and then filtered. The 
 filtrate is free from lead and chlorine, and on evapora- 
 tion over sulphuric acid in a vacuum yields free 
 creatinine in the form of efflorescent crystals containing 
 C 4 H 7 N 3 + 2H 2 0. If this creatinine be dissolved in 
 hot water the crystals obtained on spontaneous evapo- 
 ration are anhydrous tables, which, according to G. S. 
 Johnson, are not chemically identical with the efflor- 
 escent form, but reconvertible into it by modifying 
 the process of solution. The following table shows 
 the results obtained by Johnson by the recrystallisa- 
 tion of "artificial" urinary creatinine (Proc. Royal 
 Soc, xliii. 526), reproduced from creatine which had 
 been prepared by boiling urinary creatinine with 
 water : — 
 
 Nature of Crystals. 
 
 Solvent. 
 
 Evaporation. 
 
 Product. 
 
 Effloresced Ka . . 
 
 Water at 60° C. 
 
 In vacuo. 
 
 Tabular K0. 
 
 Tabular creatin- 
 
 
 
 
 ine a . . . . 
 
 Water at 60° C. 
 
 In vacuo. 
 
 Efflorescent Kj3. 
 
 Effloresced Ka . . 
 
 Water at 100° C. 
 
 In vacuo. 
 
 Tabular Ka (anhy- 
 
 Tabular Kj8 (anhy- 
 
 
 
 drous). 
 
 drous) . . . 
 
 Cold water. 
 
 In vacuo. 
 
 Efflorescent Ka. 
 
 Efflorescent K@ . 
 
 Cold water. 
 
 In vacuo. 
 
 Efflorescent K . 
 
 Creatinine as ordinarily obtained crystallises in 
 oblique rhombic prisms and stellate forms (fig. 14). 
 It dissolves in about 12 parts of cold water, and is 
 also soluble in alcohol, but almost insoluble in ether. 
 
 The aqueous solution of creatinine is, according 
 to some observers, neutral, but according to others 
 
COMPOUNDS OF CREATININE. 155 
 
 alkaline. 1 The solution readily undergoes change with 
 formation of creatine, especially if ammonia, oxide 
 of lead, or other base be present. By prolonged boil- 
 ing with caustic alkali, creatinine is completely decom- 
 posed. 
 
 By boiling with baryta- water, creatinine is hydro- 
 lysed to ammonia and methyl-hy dantoin, 
 C<*H) { ggWgj 1 + Ha0 = NHa + co | NjgEW^ J . 
 
 Boiled with water and mercuric oxide, it gives methyl- 
 guanidine. 
 
 Creatinine yields a series of crystallisable salts. 
 The hydrochloride, BHC1, crystallises in prisms from 
 alcohol or in laminae from water. It unites with zinc 
 chloride to form the double salt ZnCl 2 ,2BHCl. This 
 is very soluble in water and alcohol, and must not be 
 mistaken for the compound ZnCl 2 ,2C 4 H 7 N 3 0, which 
 is one of the most characteristic salts of creatinine. 
 Creatinine-zinc chloride is obtained by mixing con- 
 centrated aqueous or alcoholic solutions of zinc chloride 
 and creatinine, or by adding sodium acetate to the 
 solution of the double hydrochloride. It forms oblique 
 rhombic prisms or small needles, which have a ten- 
 dency to form rosettes or warty concretions. The 
 crystals are soluble in about 54 parts of cold or 27 of 
 boiling water. They are insoluble in absolute alcohol, 
 and require 9217 parts of alcohol of 98 per cent., or 
 5734 of alcohol of 87 per cent. 
 
 Mercuric chloride gives a white, curdy precipitate 
 in strong solutions of creatinine, but the separation 
 is not perfect unless sodium acetate be added, or 
 mercuric acetate substituted for mercuric chloride. On 
 allowing such a mixture to stand at the ordinary 
 temperature, the compound is gradually deposited in 
 
 1 A strongly alkaline sample leaves an alkaline ash on ignition, proving the 
 presence of mineral impurity. S a 1 k o w s k i finds creatinine quite free from 
 alkaline reaction, but it liberates ammonia from ammoniacal salts on boiling. 
 
156 REACTIONS OF CREATININE. 
 
 microscopic spherules. This reaction is applied by G. 
 S. Johnson to the isolation of creatinine from urine 
 (Proc. Royal Soc, 1888, xliii. 507). The compound 
 is almost insoluble in cold water, and is decomposed 
 with partial reduction of the mercury by hot water. 
 It is readily soluble in dilute hydrochloric acid, but is 
 nearly insoluble in acetic acid. Johnson attributes 
 to the spherical mercury compound the formula 
 C 16 H 2g N 12 Hg 7 Cl 10 , and suggests the following constitu- 
 tion :— 4(C 4 H 6 Hg"N 3 0,HCl),3HgCl 2 + 2H 2 0. Experi- 
 ments on specimens of the spherical salt prepared 
 in the author's laboratory, both from urine and from 
 pure creatinine prepared by Johnson's process (page 
 154), do not fully confirm this formula. The propor- 
 tion of chlorine, in particular, varies materially, a fact 
 which points to the presence of HgO, and possibly of 
 Hg 2 Cl 2 , in some preparations. 
 
 From a concentrated solution of creatinine, silver 
 nitrate precipitates crystals of the compound, 
 C 4 H 7 N 3 0,AgN0 8 . Mercuric nitrate does not precipi- 
 tate a dilute solution of creatinine till excess of sodium 
 carbonate is added, when B 2 Hg(N0 3 ) 2 ,HgO is thrown 
 down as a crystalline precipitate. 
 
 Creatinine possesses marked reducing properties. 
 The mercury of the spherical salt above described is 
 reduced, even in the cold, partly to the mercurous 
 state and partly to metal on adding caustic alkali. 
 Contact with boiling water produces a similar change. 
 
 Creatinine reduces Fehling's solution on boiling, the 
 blue liquid changing to yellow, but no cuprous oxide 
 separates. Creatinine appears also to prevent the 
 separation of a precipitate when glufiose is present, and 
 hence exerts an interfering action on the application 
 of Fehling's solution to the detection of dextrose in 
 urine (compare page 65). Pavy's solution is reduced 
 by creatinine without precipitation, and the reagent 
 
REACTIONS OF CREATININE. 157 
 
 may be used for its determination. According to G. 
 S. Johnson, the reducing power of creatinine 
 obtained direct from urine is greater than that of the 
 base prepared from creatine (compare page 154). Thus 
 he finds 12 grammes of tabular creatinine-a from 
 urine to have a cupric oxide reducing power equiva- 
 lent to 12 grammes of glucose ; that is two molecules 
 of this creatinine equal one of glucose in reducing 
 power ; against 1\ molecules required of creatinine 
 prepared from creatine. 
 
 Phosphomolybdic and phosphotungstic acids produce 
 micro-crystalline precipitates in solutions of creatinine 
 acidulated with nitric or hydrochloric acid. By treating 
 the precipitates with baryta, free creatinine is obtained. 
 
 If a concentrated solution of picric acid be added to 
 normal human urine a small crystalline sediment is 
 gradually formed. On separating this, and treating it 
 with hot water, uric acid remains, while the greater 
 part dissolves. The soluble portion is a double picrate 
 of potassium and creatinine, which forms lemon-yellow 
 needles or thin prisms, readily soluble in hot water, 
 sparingly in cold alcohol, and almost insoluble in 
 ether. With dog's urine the precipitate produced by 
 picric acid contains little or no uric acid, and the 
 kynurenic acid present is not precipitated. 
 
 When a solution of picric acid is added to a solu- 
 tion of creatinine not more dilute than 1 in 3000, on 
 adding a drop of dilute caustic alkali a deep red colour 
 is produced, which is intensified by boiling the liquid. 
 By this reaction the presence of creatinine can be 
 recognised in the urine of man, dog, and rabbit. 
 Acetone gives a similar but less intense colour. 
 Glucose gives a similar reaction on heating. It is 
 evident that the behaviour of creatinine with picric 
 acid gravely affects the value of that reagent as a test 
 for small quantities of sugar in urine. 
 
158 EE ACTIONS OF CREATININE. 
 
 T. Weyl (Berichte, 1878, page 228) has pointed 
 out that if a few drops of very dilute solution of sodium 
 nitroprusside be added to a solution of creatinine, and 
 dilute caustic soda then added drop by drop, a fine 
 ruby-red colour will be produced, which in a few 
 minutes changes to an intense straw-yellow. If the 
 liquid be now acidulated with acetic acid and warmed, 
 it turns greenish and prussian blue separates. Guar- 
 eschi recommends that 10 per cent, solutions of 
 nitroprusside and caustic soda should be used. K r u- 
 g e n b e r g states that the reaction is best obtained 
 by first adding caustic soda, and then a few drops of a 
 concentrated solution of the nitroprusside. S a Ik o w- 
 s k i confirms this. The reaction is very delicate, and 
 can be obtained with a solution containing 0'03 per 
 cent, of pure creatinine, or with urine containing 0"066 
 per cent. In applying the test to urine the absence of 
 acetone should be insured by distilling off a portion, 
 since that body gives a ruby-red colour with Weyl's 
 test, though no blue colour can be obtained on acidu- 
 lating, acetic acid merely restoring the yellow colour 
 to red. According to Guareschi, a red colour is 
 also yielded by hydantoin, methyl-hydantoin, and 
 other compounds containing the group N.CH 2 .CO.N. 
 Creatine gives no reaction with Weyl's test unless the 
 liquid be first boiled with a dilute acid, so as to convert 
 it into creatinine. In this manner, Weyl demonstrated 
 the presence of creatine in milk (Berichte, xi. 2175). 
 
 The determination of creatinine in urine is usually 
 based on its isolation as cre'atinine-zinc chloride, which 
 process is preferred by Neubauer. P. Grocco 
 (Chem. Centr., 1887, page 17) has described the follow- 
 ing modification of this method of isolating creatinine 
 from urine. The urine of twenty-four hours, kept 
 acid by acetic acid, and preserved in ice if possible, is 
 treated with milk of lime till only faintly acid, calcium 
 
DETERMINATION OP CREATININE. 159 
 
 chloride added and the liquid evaporated, the reaction 
 being maintained neutral or faintly acid by cautious 
 addition of acetic acid. The residue is extracted with 
 alcohol containing a little sodium acetate, and the 
 filtered liquid treated with alcoholic zinc chloride and 
 a little acetic acid, avoiding excess. If necessary, the 
 alcoholic extract is decolorised with animal charcoal 
 before adding the zinc reagent, and it should be 
 cooled with ice. The purity of the precipitated creat- 
 inine-zinc chloride should be proved by a microscopic 
 examination, with a high power, to make certain of its 
 freedom from sodium chloride. It should be completely 
 soluble in hot water. (Compare page 155.) 
 
 E. Salkowski operates in a very similar manner, 
 but uses a smaller quantity of urine. He directs that 
 240 c.c. should be rendered alkaline by the cautious 
 addition of milk of lime, and precipitated by calcium 
 chloride. The volume is made up to 300 c.c, and the 
 liquid filtered after ten minutes. 250 c.c. of the 
 filtrate, representing 200 of urine, which must be feebly 
 alkaline, is evaporated to about 20 c.c, and an equal 
 measure of absolute alcohol added. This is subse- 
 quently diluted to 100 c.c. with alcohol, allowed to 
 stand twenty-four hours, and filtered. To 80 c.c. of 
 the filtrate zinc chloride is added, and the creatinine- 
 zinc chloride collected after twenty-four hours. 
 
 Instead of weighing the compound of creatinine 
 with zinc chloride, the contained creatinine may be 
 deduced from the amount of ammonia produced on de- 
 composing it with boiling concentrated sulphuric acid. 
 For this purpose the precipitate should be dissolved in 
 the minimum quantity of sulphuric acid, previously 
 diluted with an equal measure of water, and the 
 solution treated as described on page 128. 
 
 A method of isolating creatinine from urine, which, 
 in the opinion of the author, is preferable to the zinc 
 
160 DETERMINATION OF CREATININE. 
 
 process, is that based on its precipitation as the 
 spherical mercuric compound (described on page 156), 
 which, according to G. S. Johnson, contains about 
 20 per cent, of creatinine. Instead of weighing the 
 precipitate, it may be washed with cold water, and 
 decomposed by treatment with strong sulphuric acid 
 as in Kjeldahl's process 1 described on page 127, the 
 creatinine being deduced from the amount of ammonia 
 obtained. This plan, in the opinion of the author, 
 is preferable to weighing the mercury salt. 
 
 Attempts to determine creatinine by treating the 
 zinc or mercury compound with alkaline hypobromite 
 solution, as employed for the estimation of urea, have 
 failed to give the author satisfactory results, the pro- 
 portion of the total nitrogen evolved on treatment 
 with the hypobromite being apparently dependent on 
 conditions not yet mastered. Hence the method, 
 which at first appeared very promising, cannot at 
 present be recommended. 
 
 Uric Acid. Lithic Acid. C 6 H 4 N 4 3 . 
 
 Uric acid is one of the most constant and character- 
 istic products of the metabolism of the animal organ- 
 ism. In its formation the nucleiin of the cell-nuclei is 
 specially concerned. It exists normally as urate of 
 sodium in human urine, the proportion present being 
 greatly increased in cases of gout and rheumatism, 
 when the urine contains large quantities of acid urate 
 of sodium. 2 Traces of uric acid exist normally in the 
 
 1 Kjeldahl's original method must be employed, as the modified process 
 described on page 128 is not applicable in presence of mercury compounds. 
 
 2 The amount of uric acid commonly excreted by an adult is usually stated 
 to be about 8 grains (0"5 gramme) in the twenty-four hours, but the results of 
 the more modern methods of determination lead to the conclusion that the 
 diurnal quantity eliminated under normal conditions is from 20 to 30 grains 
 (1"3 to 2 - grammes). The deposition of urates from urine on cooling does not 
 prove their presence in excessive amount. 
 
 During recent years, Haig and other observers have shown that uric acid 
 
OCCURRENCE OF URIC ACID. 161 
 
 brain, lungs, liver, and spleen, and uric acid is also 
 found in the saliva, gastric juice, sweat, &c. The 
 merest trace exists normally in blood, but in cases of 
 albuminuria, and especially of gout, the proportion 
 becomes very appreciable. The so-called " chalk- 
 stones," and other gouty concretions, commonly consist 
 of the sparingly soluble acid sodium urate, while the 
 buff-coloured sediment which frequently separates from 
 human urine usually consists of the quadri-urate of 
 sodium or ammonium. Acid urate of ammonium con- 
 stitutes the greater part of the urinary excrement of 
 birds (" guano "), while that of serpents and other 
 terrestrial reptiles contains it in a still purer form. 
 On the other hand, uric acid is nearly absent from the 
 urine of herbivorous mammals, being replaced therein 
 by hippuric acid. 
 
 The synthesis- of uric acid has been effected 1 by 
 Horbaczeweski by heating glycocine with ten 
 times its weight of urea to about 230° C. : — 
 C 2 H 6 N0 2 +' 3CH 4 N 2 = C 6 H 4 N 4 3 + 2H 2 + 3H 3 N. 
 
 Glycocine. Urea. Uric acid. Water. Ammonia. 
 
 has, in all probability, a much wider bearing in pathology than had previously 
 been supposed. Its connection with gout, rheumatism, and stone has long 
 been recognised, though its relation to these complaints has been much mis- 
 understood. Its relation to other affections (e.g., according to Haig as 
 a casual factor in migraine) is still more obscure. 
 
 1 Another interesting synthesis of uric acid has been effected by Behrend 
 and Ro osen (Serichte, xxi. 999) by the reaction of etho-acetie ether and 
 urea. From this synthesis the following constitutional formula, first proposed 
 bv M e d i c u s, has been assigned to uric acid : — 
 
 {NH— CO 
 I 
 C— NH} 
 ii ko. 
 NH— C-NHj 
 This formula shows that uric acid contains the residues of two molecules of 
 urea, and explains the fact that the decompositions of uric acid almost in- 
 variably yield either a molecule of urea or some derivative of urea, together 
 with a second body which can by further treatment be converted into urea. 
 Many of the decomposition-products of uric acid can indeed be prepared 
 directly from urea. In view of the close relationship existing between urea 
 and uric acid, it is not surprising that the foods which, in the mammal, cause 
 an increased excretion of urea, in "birds are converted into uric acid. 
 
 L 
 
162 XANTHINE DEBIVATIVES. 
 
 Conversely, when uric acid is heated under pressure to 
 ] 70° with hydriodic acid, it yields glycocine, ammonia, 
 and carbon dioxide. 
 
 Uric acid differs by an atom of oxygen from 
 xanthine, a feeble base of wide occurrence in both 
 the animal and vegetable kingdoms, and the physio- 
 logical and pathological relations of which are but 
 little understood. 1 
 
 1 Xanthine, C 6 H 4 N 4 2 , is the typical member of a series of feebly basic 
 bodies closely related to uric acid and to each other. Some of these com- 
 pounds {e.g., xanthine, hypoxanthine, guanine, carnine) occur in small 
 quantity in normal urine and animal organs and tissues, and are normal 
 products of the degradation of proteids. Other members of the group {e.g., 
 caffeine, theobromine, theophylline, and xanthine itself) occur in plants. 
 
 Hypoxanthine (C 6 H 4 N 4 0), xanthine (C 5 H 4 N 4 2 ), and uric acid (CJSJKfi^ 
 all occur in normal urine. They differ from each other only by an atom ofi 
 oxygen, bat, notwithstanding this close relationship, they do not seem to be 
 convertible, as has been alleged. Heteroxanthine (C 6 H 6 N 4 2 ) and para- 
 xanthine (C 7 H 8 N 4 2 ) also occur in normal urine, and the former is said to be 
 present in larger amount in the urine of anaemic persons. 
 
 The vegetable bases theobromine and theophylline are dimethyl- 
 xanthines, C 6 H 2 (CH 3 ) 2 !N 4 02, while caffeine has the constitution of a 
 trimethyl-xanthine, C 5 H(CH 3 ) 3 ]Sr 4 2 . 
 
 Guanine, C 5 H 5 N 5 0, a body abundant in Peruvian guano, has the constitu- 
 tion of an imido-xanthine; while adenine, C 5 H 5 N 5 (originally isolated 
 from the pancreas),, bears the same relationship to hypoxanthine. 
 
 Xanthine was originally discovered (M a r-c e t, 1819) in a urinary calculus. 
 It has been found in guano, and is a normal constituent of urine, especially 
 during the use of sulphur-baths, and is present in minute quantities in 
 various parts of the system. It has also been found in tea, lupines, malt- 
 seedlings, yeast, &c, and has been produced synthetically. 
 
 In their chemical and physical characters, the xanthine bases present a 
 close resemblance to uric acid. They have but a feeble affinity for acids, and 
 their salts are mostly decomposed by water. Some of them (including 
 xanthine itself) exercise an acid function in addition, and unite with bases. 
 They are mostly very slightly soluble in cold water, and, except caffeine and 
 theobromine, insoluble in alcohol, ether, or chloroform. They all yield white 
 precipitates with phosphomolybdic acid, mercuric chloride, and ammoniacal 
 lead acetate ; and guanine and adenine are very perfectly precipitated by 
 picric acid. 
 
 A general reaction of the xanthine bases is their precipitation from am- 
 moniacal solutions by ammonio-nitrate of silver, as a gelatinous compound of 
 the base with argentic oxide. On treating the precipitate with dilute nitric 
 acid, crystalline compounds of the bases with silver nitrate are obtained, the 
 xanthine compound containing C 6 H 4 N 4 2 ,AgN0 3 . The different solubility of 
 these compounds in water and nitric acid affords a means of distinguishing 
 and separating the xanthine bases from each other. 
 
 The xanthine derivatives (except caffeine and theobromine) are precipitated 
 
XANTHINE DERIVATIVES. 163 
 
 As commonly obtained, uric acid is said to be anhy- 
 drous, 1 and forms small crystalline scales which are very 
 apt to appropriate colouring matter. The microscopic 
 
 by oupric acetate, especially on heating. The precipitates are the cuprous salts 
 of the bodies, that yielded by xanthine containing Cu^CjH^N^. They 
 may be produced by treating the neutral solution of the body with a mixture 
 of cupric sulphate and sodium sulphite or thiosulpha.te, or by mixing the 
 ammoniacal solution with Fehling's solution, heating to boiling, and gradu- 
 ally adding a solution of dextrose. Instead of cuprous oxide separating 
 in the free state, it combines with the xanthine-derivative to form a white 
 insoluble compound. Hence it is evident that the presence of xanthine and 
 its allies, including uric acid, will prevent the detection of sugar in urine by 
 Fehling's test to an extent dependent on the amount of the interfering body 
 present. The fact is of considerable practical importance when small quanti- 
 ties of sugar are to be sought for, and therefore, in such case, Fehling's test 
 should be applied in the modified manner described on page 62. If, instead 
 of using dextrose as a reducing agent, the mixture of a xanthine base with 
 Fehling's solution be treated with hydroxylamine hydrochloride, reduction of 
 the copper to the cuprous state will occur in the strongly alkaline solution 
 and at the ordinary temperature. P. B alke {Jour. Pract. Chem., [2], xlvii. 
 537) employs the foregoing reaction for the determination of the xanthine 
 derivatives, and for their isolation from flesh, malt, &c. 
 
 The proportion of xanthine derivatives (other than uric, acid) ordinarily 
 present in urine is extremely small, but there is reason to believe that, under 
 circumstances not fully understood, their amount is much increased and may 
 then be of pathological importance. For the extraction of xanthine from 
 mine, a large quantity (5 to 10 gallons) should be treated by instalments of 
 about 1 quart at a time with neutral lead acetate in powder, as long as a pre- 
 cipitate is produced. The liquid is filtered and sodium sulphate added as 
 long as lead sulphate is thrown down, the liquid poured off or filtered from 
 the precipitate, copper sulphate added, and the liquid boiled. The precipitate, 
 which contains the xanthine derivatives as cuprous salts, is filtered off, 
 washed, dissolved in dilute nitric acid, and excess of ammonia added, followed 
 by silver nitrate. The precipitate, consisting of the argentic oxide compounds 
 of xanthine, &c, is separated, suspended in hot ammoniacal water, and decom- 
 posed by sulphuretted hydrogen, the resultant silver sulphide filtered off, and 
 the filtrate concentrated till the xanthine crystallises out. If, instead of de- 
 composing the silver precipitate with sulphuretted hydrogen, it be boiled 
 with nitric acid of 1*10 specific gravity, the silver nitrate compounds of 
 hypoxanthine and adenine will crystallise out immediately on cooling, while 
 those of xanthine, paraxanthine, and heteroxanthine will remain in solution, 
 and may be recovered as the silver-oxide compounds by rendering the filtrate 
 ammoniacal. 
 
 1 When slowly deposited from dilute solutions, uric acid sometimes separates 
 in large crystals containing C 5 H 4 N 4 3 + 2H 2 0. It is very probable that 
 some of the ordinary forms of uric acid are hydrated. Thus Dr. James Edmunds 
 finds that all crystals of uric acid, obtained by addition of hydrochloric acid to 
 cold filtered urine, effloresce and break up on heating to 200° F. or on exposure 
 in a desiccator at 60° F. over sulphuric acid for twenty-four hours. Prepara- 
 tions made by Dr. Edmunds show this in a marked manner. 
 
164 CHARACTERS OF URIC ACID. 
 
 appearance of uric acid is very variable, dumb-bell, 
 whetstone, and lozenge-like forms being among the 
 most characteristic (fig. 16, page 167). When precipi- 
 tated from urine by adding hydrochloric acid, it com- 
 monly forms small, transparent, rhombic tablets, with 
 a few elliptical and oblong plates ; but the forms 
 assumed greatly depend on the nature and amount of 
 the pigments and other co-existing substances. 
 
 When quite pure, uric acid forms a white crystal- 
 line powder without taste or smell, and of a specific 
 gravity ranging from l - 855 to l - 893. On heating, it 
 decomposes without melting, giving off hydrocyanic 
 acid and carbon dioxide, yielding a sublimate contain- 
 ing cyanuric acid, ammonium cyanate and urea, and 
 leaving a carbonaceous residue. 
 
 Uric acid is nearly insoluble in water, requiring 
 15,000 parts of cold or 1800 of boiling water for its 
 solution. Blarez and Deniges (Compt. rend., 
 civ., 1847) find that 100 grammes of water at 0° C. 
 dissolve 2 - milligrammes of uric acid ; at 10°, 37 ; 
 at 20°, 6*0 ; and at 100°, 62 - 5 milligrammes of uric 
 acid. Uric acid dissolves in glycerin, but in alcohol 
 and ether it is quite insoluble. Uric acid is soluble 
 in solutions of the borates, phosphates, carbonates, 
 acetates, and lactates of potassium and sodium, but 
 not in solutions of the corresponding ammonium salts. 
 
 In strong sulphuric acid uric acid dissolves to form 
 a crystallisable sulphate, which is decomposed by 
 water, the uric acid being precipitated unchanged. 
 When strongly heated with concentrated sulphuric 
 acid, uric acid is broken up, the nitrogen being ulti- 
 mately wholly converted into ammonia (see page 127). 
 
 Hydrochloric acid has neither solvent nor chemical 
 action on uric acid. By the action of oxidising agents 
 on uric acid a number of compounds of great theoreti- 
 cal interest are obtainable. These form two dis- 
 
PROPERTIES OP URIC ACID. 165 
 
 tinct series. The first, represented by alloxan, 
 C 4 H 3 N 2 4 , are produced by acid oxidising agents, such 
 as nitric acid. The second, of which allantoin, 
 C 4 H 6 N 4 3 , is the type, result from the oxidation of 
 uric acid in alkaline or neutral solution. 
 
 Hot dilute nitric acid converts uric acid into 
 alloxantin, C 8 H 4 N 4 7 , a body which is also pro- 
 duced by the action of reducing agents on alloxan. 
 Chlorine and bromine convert uric acid at ordinary 
 temperatures into urea and alloxan. On heat- 
 ing, parabanic and oxalic acids are also 
 produced. Hypobromites and hypochlorites cause the 
 evolution of a portion of the nitrogen of uric acid 
 in a gaseous state, but the reaction does not appear to 
 be sufficiently constant to serve as a method of deter- 
 mining uric acid. 
 
 Uric acid suspended in pure water remains un- 
 changed for a long time, but the addition of a very 
 small quantity of decomposed urine causes its rapid 
 and complete decomposition in hot weather, with 
 formation of ammonium carbonate and other 
 bodies. 
 
 Detection and Determination of Uric Acid. 
 
 Uric acid is commonly separated in the free state 
 by adding excess of hydrochloric acid to its solution. 
 When separated from urine in this manner, it forms a 
 coloured deposit which adheres to the sides of the 
 glass. 1 The best mode of operating is described in 
 the sequel. 
 
 When isolated, uric acid is readily identified by its 
 microscopic appearance, though the forms it assumes 
 are very numerous: When deposited from urine or 
 other impure solutions, dumb-bell, whetstone, and 
 lozenge-like forms are among the most common and 
 
 1 A drop of fresh urine, mixed with hydrochloric acid, may be observed 
 under the microscope to deposit uric acid crystals in the course of a few minutes. 
 
166 
 
 DETECTION OF URIC ACID. 
 
 Ik o ■ - 
 
 characteristic (fig. 16, 6 and c). A. E. Garrod has 
 shown that the pigments of urine are especially con- 
 cerned in modifying the forms assumed by the uric 
 
 acid, and that the presence 
 of excess of one particular 
 pigment will produce a cor- 
 responding definite varia- 
 tion in the form of the 
 crystals. Dr J. Edmunds 
 has independently found 
 that the forms assumed by 
 uric acid greatly depend on 
 the nature and amount of 
 the co-existing substances. 
 When precipitated from a 
 solution of a pure urate by 
 addition of hydrochloric acid, 
 uric acid generally forms 
 minute transparent rhombic 
 plates (fig. 16, a). Large 
 crystals are obtainable much 
 more readily from urine or 
 other impure solutions than 
 from pure urates. 
 
 A highly characteristic 
 and delicate reaction is that 
 known as the " murexide 
 test," which is based on the 
 
 Fig. 15.- Crystals of Ukic Acid, behaviour of uric acid on 
 oxidation. If uric acid, a urate, or even urine be 
 treated with a few drops of strong nitric acid, and 
 the liquid evaporated to dryness in porcelain at 100°, 
 a yellowish or red residue will be obtained, which 
 owes its colour to the formation of alloxantin, 
 C 8 H 6 N 4 8 . On inverting the capsule over another 
 containing ammonia, or otherwise subjecting the 
 
REACTIONS OF URIC ACID. 167 
 
 residue to ammoniacal vapours, it acquires a magni- 
 ficent purple colour, owing to the formation of mur- 
 exide or ammonium purpurate, NH 4 .C 8 H 4 N 6 6 . 
 On now adding caustic soda, ^^^^^^ /-^ 
 the purple becomes changed to ^sS^* 55 * 1 < > <=> 
 blue, the colour disappearing on ^-^ Z^7 t? 
 warming. Somewhat analogous ^-^ ^ 
 
 reactions are given by caffeine, 
 theobromine, guanine, and xan- 
 thine, but the differences do not 
 allow of their confusion with 
 uric acid. 
 
 The nitric acid prescribed in 
 the above test may be advan- 
 tageously replaced by bromine- Fig . i 6 .— Crystals of Ubic 
 water, or the material to be acid.— «, From decomposi- 
 
 •. •, , i ■ , i tion of urates ; i, from 
 
 tested may be evaporated with a human uriue . „, dumb . be n 
 
 few drops of strong hydrochloric forms. 
 
 acid and a minute crystal of potassium chlorate. 
 
 If uric acid be dissolved in a solution of sodium 
 carbonate and a drop of the liquid placed on filter- 
 paper previously moistened with silver nitrate, a 
 yellow, brown or black spot will be produced, owing 
 to the fact that silver carbonate is reduced by uric 
 acid even at the ordinary temperature. 
 
 On adding a little Fehling's solution to a solution 
 of uric acid in caustic soda, a greyish precipitate is 
 formed, said to consist of cuprous urate ; but with 
 excess of the reagent, and on application of heat, red 
 cuprous oxide separates. 
 
 Uric acid does not reduce a hot, alkaline solution of 
 picric acid, which fact distinguishes it from creatinine, 
 glucose, and other normal and occasional constituents 
 of urine which do react with Fehling's solution. 
 
 For the determination of uric acid in urine a 
 common practice is to add hydrochloric acid to the 
 
168 DETERMINATION OP URIC ACID. 
 
 previously concentrated sample, allow the liquid to 
 stand in the cold for a few hours, and filter from the 
 precipitated uric acid, which is subsequently washed 
 with cold water and rectified spirit, dried, and weighed. 
 But under the most favourable circumstances, and 
 however carefully the process be conducted, the separa- 
 tion of the uric acid is incomplete, and the results 
 consequently below the truth. 
 
 A preferable method of isolating the uric acid from 
 urine is that based on the insolubility of the acid 
 ammonium urate, C 6 H 3 (NH 4 )N 4 3 , in a solution of am- 
 monium chloride or sulphate. In the original process, 
 which is due to A. P. Fokker {Jour. Chem. Soc, 
 xxviii. 1293; also Salkowski, Fresenius' Zeits- 
 chrifb, xvi. 371), only a limited amount of ammonium 
 sulphate was used, and hence a considerable correction 
 was necessary for the solubility of the acid urate ; but 
 F. G. Hopkins ( Chem. News, lxvi. 106) has pointed 
 out that by saturating the liquid with ammonium 
 chloride no such correction is required, and the time 
 necessary for complete precipitation is much reduced. 
 Hopkins prescribes the following procedure : — 
 
 To 100 c.c. (or 4 fluid ounces) of urine, finely 
 powdered ammonium chloride is added in excess, 1 about 
 30 grammes (1 oz.) being necessary. When a 
 small quantity of the salt remains undissolved, even 
 after brisk stirring at intervals of a few minutes, satura- 
 tion is sufficiently complete, even if complete solution 
 occurs when the liquid recovers from the depression 
 of temperature caused by the solution of the ammonium 
 chloride. The solution is allowed to stand for two 
 hours with occasional stirring, and is then passed 
 
 1 When it is intended subsequently to decompose the precipitate by hydro- 
 chloric acid and weigh the liberated uric acid, it is essential that the ammonium 
 chloride used should dissolve to an absolutely clear solution in water, since 
 the quantity employed is very large relatively, and any insoluble matter would 
 seriously vitiate the estimation of uric acid. 
 
PEECIPITATION OF URIC ACID. 169 
 
 through a thin filter and washed twice with a saturated 
 solution of ammonium chloride. When time is an 
 object, the urine may be made alkaline with ammonia 
 after saturation with ammonium chloride. The phos- 
 phates which are thus precipitated with the ammonium 
 urate occasion no inconvenience, while precipitation is 
 complete in ten minutes. 
 
 When a normal acid urine is saturated with pure 
 ammonium chloride, the precipitate of acid ammonium 
 urate, after being washed with the cold, saturated solu- 
 tion of ammonium chloride, yields a mere trace of ash 
 on ignition, showing that no mineral salts are carried 
 down. Of the ordinary constituents of urine, uric 
 acid, xanthine, and certain pigments appear to be the 
 only bodies precipitated. Xanthine is thrown down 
 still more completely from ammoniacal solutions, but 
 it is left in solution when the ammonium urate is 
 subsequently decomposed by hydrochloric acid. Hypo- 
 xanthine and creatinine are not precipitated by 
 ammonium chloride. Certain pigments are thrown 
 down, so that the precipitate is always more or less 
 coloured. Hsemato-porphorin, in particular, is very 
 perfectly precipitated, but remains in solution when 
 the urate is subsequently decomposed by acid. 
 
 The acid ammonium urate, isolated in the foregoing 
 manner, admits of several alternative treatments, as 
 follow : — 
 
 1. When it is desired to determine the uric acid by 
 weight, the precipitate is rinsed off the filter with a 
 jet of hot water, and the liquid heated just to boiling 
 with excess of dilute hydrochloric acid. The liquid is 
 thoroughly cooled and allowed to stand for two hours. 
 It is then filtered on to a smooth filter, and the crystals 
 of uric acid washed twice with cold water, then with 
 alcohol, till the washings are no longer acid, dried at 
 100° C. ( = 212° F.), and weighed. To the weight of 
 
170 ACID AMMONIUM URATE. 
 
 uric acid thus obtained 0"001 gramme should be added 
 for every 15 c.c. ( = ^ oz.) of mother-liquor, the bulk 
 of which need never exceed 30 c.c. ; but no correction 
 need be made for the insignificant trace of uric acid 
 dissolved by the aqueous and alcoholic washings. The 
 uric acid thus isolated is usually only slightly coloured 
 and is practically pure. When derived from highly pig- 
 mented urines, the uric acid may retain so much colour- 
 ing matter as to suggest the presence of an amount of 
 impurity sufficient to vitiate the result. In such case, 
 after washing the precipitate of acid ammonium urate 
 off the filter, rectified spirit equal in bulk to the water 
 present should be added, and, after adding hydro- 
 chloric acid, the beaker should be covered and heated 
 for some time on the water-bath. 
 
 Instead of weighing the uric acid isolated in the 
 foregoing manner, it may, if preferred, be dissolved in 
 a little hot solution of sodium carbonate, and the 
 liquid treated by process 3 or 4. 
 
 2. The precipitate of acid ammonium urate having a 
 perfectly definite composition, it may be titrated with 
 standard alkali and an indicator giving no reaction 
 with uric acid. Such an indicator exists in methyl- 
 orange. The precipitate from 200 c.c. of urine is 
 treated with a known measure, e.g., 20 c.c, of deci- 
 normal hydrochloric or sulphuric acid, the liquid boiled 
 for some minutes, cooled, diluted to 200 c.c, a few 
 drops of a 1 per cent, aqueous solution of methyl- 
 orange added, and ^-normal caustic soda ( = 2*0 
 grammes of NaHO per litre) dropped in from a burette 
 until the orange colour of the acid liquid becomes 
 yellow, which change indicates the point of neutrality. 
 The difference between the volume of acid employed 
 and that of the alkali required to neutralise it repre- 
 sents the ammonia of the precipitate, uric acid having 
 no action on methyl-orange. Each centimetre of ~ 
 
TITRATION OF URIC ACID. 171 
 
 solution of soda shows the presence of 0"0084 gramme 
 of uric acid. 
 
 3. An alternative plan, when the acid urate of am- 
 monium is not very strongly coloured, is to rinse the pre- 
 cipitate off the filter with hot water, cool the solution, 
 and dilute it with distilled water to, 100 c.c. Twenty 
 c.c. of pure concentrated sulphuric acid is then added, 
 so as to acidify the liquid and raise its temperature to 
 about 60° C. ( = 140° F.), and then a standard solution 
 of potassium permanganate is run in, till the liquid 
 acquires a pink tint surviving agitation and lasting some 
 seconds. Further decolorisation may occur on standing, 
 but this should be disregarded. Each centimetre of 2*0 
 normal permanganate ( = T578 gramme of KMn0 4 
 per litre) decolorised represents 0"00375 gramme of 
 uric acid. 1 F. G. Hopkins strongly recommends this 
 process {Jour. Pathology and Bacteriology, June 1893). 
 When it is intended to titrate the ammonium urate with 
 standard permanganate in the above manner, it is very 
 desirable to wash the precipitate with a saturated 
 solution of ammonium sulphate, instead of ammonium 
 chloride, since the latter salt somewhat affects the 
 accuracy of the titration. 
 
 The same method of titration by permanganate may 
 be applied to the uric acid isolated in process 1, after 
 simply dissolving it in a little hot solution of sodium 
 carbonate. 
 
 4. Bay rac (Compt. rend., ex. 352) determines uric 
 acid by evaporating 50 c.c. of the urine to dryness at 
 100° C, treating the residue with 10 c.c. of dilute 
 hydrochloric acid (l : 5), and washing with alcohol to 
 
 1 This factor is due to F. G. H o p k i n s, as the result of experiment. As 
 it corresponds to no simple reaction, the process has been investigated in the 
 author's laboratory by A. R. Tankard. The first results on pure uric acid 
 were not very constant, ranging from 96 to 104 per cent, of the truth, but on 
 taking as the end-reaction the point at which the permanganate ceased to be 
 instantly decolorised much closer figures were obtained. When the titration 
 was conducted at a boiling heat, instead of at 60° C, the results were higher. 
 
172 DETERMINATION OF TJKIC ACID. 
 
 remove urea and creatinine. The residue is dissolved 
 on the water-bath in 20 drops of caustic soda solution, 
 and heated to 90° or 100° C. with 15 c.c. of a concen- 
 trated solution of sodium hypobromite solution in 
 the apparatus for estimating urea shown in fig. 1], 
 page 145. 22'38 c.c. of nitrogen measured at 0° C. and 
 760 mm. barometric pressure, or 23 "5 5 c.c. at the 
 ordinary temperature and pressure, are said to repre- 
 sent 0'084 gramme of uric acid. 
 
 Experiments made in the author's laboratory to 
 test the possibility of estimating uric acid by measur- 
 ing the amount of nitrogen evolved on treatment with 
 alkaline hypobromite have not yielded very encourag- 
 ing results, the reaction being subject to variations 
 from causes not yet understood. 
 
 5. Uric acid can be determined by heating it with 
 concentrated sulphuric acid, and determining the am- 
 monia by treatment with hypobromite or distillation 
 with alkali. The method can be applied to a uric acid 
 precipitate. (See page 127 et seq.) 
 
 F. B. Haycraft has described a method of deter- 
 mining uric acid based on its precipitation as silver 
 urate, on adding a solution of am monio- nitrate of silver. 
 The process has been modified by Salkowski and 
 others. It is somewhat complex, and the results are 
 not more accurate than those yielded by methods 
 already detailed. 
 
 F. W. Pavy (Trans. Royal Med. and Ghirurg. 
 Soc, London) has proposed to determine uric acid 
 volumetrically by an ammoniacal cupric solution, in 
 the manner so successfully employed for glucose (page 
 67) ; but the reducing power of uric acid on Pavy's 
 solution is found in practice to vary so greatly with 
 the method of conducting the titration and other 
 working conditions as to render the results of very 
 little value. 
 
QUADRI -URATES. 173 
 
 Urates. 
 
 Uric acid is a feeble acid which is usually stated to 
 possess a dibasic function. But it was shown by 
 Bence Jones (Jour. Chem. Soc, xv. 201), and has 
 been confirmed more recently by Sir Win. Roberts 
 (Croonian Lectures, 1892), that a third series of urates 
 exist and have great physiological significance. The 
 salts of the formula M 2 C 6 H 2 N 4 3 , commonly called 
 neutral or normal urates, dissolve readily in water, 
 and are exclusively laboratory-products, not being 
 met with in the animal system under either healthy 
 or pathological conditions. The acid urates, or 
 " bi-urates," of the formula MHC 5 H 2 N 4 3 are very 
 sparingly soluble, and exist in the urine only after it 
 has undergone ammoniacal fermentation. They are 
 known pathologically as components of gouty con- 
 cretions in the tissues, but it is questionable if they 
 ever exist physiologically in the blood or tissues. The 
 third class, or " quadri-urates," have the composition 
 MHC 6 H 2 N 4 3J H 2 C 5 H 2 NA. They are more soluble 
 than the bi-urates, and are specially the physiological 
 combinations of uric acid. They exist normally in 
 the urine, and probably also in the blood, and con- 
 stitute the whole of the urinary excretion of birds 
 and serpents. Roberts considers that all the morbid 
 phenomena due to uric acid probably arise from 
 secondary changes in the quadri-urates. 
 
 Quadri-urates, MHUr,H 2 Ur, usually present them- 
 selves as amorphous powders, but the spheres of birds' 
 and serpents' urine are distinctly crystalline, and dis- 
 play a black cross when examined by polarised light. 
 These forms are permanent in the air if kept perfectly 
 dry, but readily assume a gelatinous character, and 
 then appear under the microscope as large translucent 
 globules. The quadri-urates are difficult to obtain pure. 
 When produced artificially, they are apt to be mixed 
 
174 QUADRI-URATES. 
 
 with free uric acid or bi-urates, and when prepared 
 from urine to be contaminated with pigments and traces 
 of extraneous saline matters. Roberts prepares potas- 
 sium quadri-urate by adding 2 grammes of uric acid 
 to a boiling solution of 9 grammes of potassium 
 acetate in 300 c.c. of water. The liquid is agitated 
 for about a minute, filtered hot, and cooled rapidly 
 in a stream of cold water. The voluminous pre- 
 cipitate which forms is filtered off, washed in succes- 
 sion with rectified spirit and absolute alcohol, and 
 dried at a temperature not exceeding 40° C. The 
 results of the analysis of the product obtained agree 
 well with the formula KHC 6 H 2 N 4 O s ,H 2 C 5 H 2 N 4 3 . 
 Other quadri-urates can be obtained by similar means, 
 but they are less stable than the potassium salt. 
 
 The quadri-urates are insoluble in alcohol, ether, 
 chloroform, glycerin, and volatile oils, and cannot be 
 dissolved without change in any simple menstruum. 
 When treated with hot water, they pass momentarily 
 into solution, but are almost immediately decomposed 
 into bi-urate and free uric acid. The same decompo- 
 sition is effected by neutral saline solutions, but in 
 this case, and notably with a solution of common salt, 
 the decomposition is greatly retarded. When treated 
 with solutions of alkaline carbonates, or disodium 
 hydrogen phosphate, the quadri-urates are converted 
 into bi-urates. In healthy urine of feeble acid reaction 
 the quadri-urates dissolve unchanged, but the solution 
 undergoes gradual but complete decomposition, with 
 ultimate separation of the whole of the uric acid in 
 a free state. This change is retarded in normal urine 
 by the salts and colouring matters present (urea has 
 no influence), occurring with greater facility the larger 
 the proportion of free acid there is present. 
 
 In studying the action of water on quadri-urates 
 Sir W. Roberts recommends that about 0"4 gramme of 
 
QUADRI-TJRATES. 175 
 
 the dried deposit should be stirred up with 1000 parts 
 ( = 400 c.c.) of distilled water, the mixture heated nearly 
 to the boiling point until solution is complete, and then 
 left at rest for forty-eight hours. The supernatant liquid 
 is then syphoned off and the remainder passed through 
 a weighed filter. The crystals of uric acid are washed 
 very sparingly with cold water, then more freely with 
 rectified spirit, dried, and weighed. 1 A correction of 
 0*0055 gramme per 100 c.c. of mother liquor is applied 
 to compensate for the solubility of the uric acid. The 
 decanted liquid, filtrate, and washings are next heated 
 nearly to boiling, strongly acidulated with hydrochloric 
 acid, and allowed to stand forty-eight hours as before. 
 By the foregoing method, Sir W. Roberts obtained 
 the following figures from two specimens of quadri-urate 
 prepared by the acetate of potassium method. 
 
 Sample A. Sample B. 
 
 Uric acid separated by water 
 
 (corrected for solubility), O080 gramme. (H64: gramme. 
 Uric acid dissolved as bi-urate, O077 „ 0*159 „ 
 
 These results sufficiently establish the existence of 
 the quadri-urate and the manner of its decomposition 
 by water. 
 
 The decomposition of sodium quadri-urate under the 
 influence of water can be conveniently observed by 
 filtering off the buff-coloured sediment deposited by 
 healthy urine, washing it thoroughly with cold recti- 
 fied spirit, and drying it at a blood-heat. When the 
 quadri-urate thus purified is mixed with a considerable 
 volume of water it is speedily disintegrated, a portion 
 passing into solution in combination with the bases, 
 and the remainder falling as an insoluble precipitate of 
 crystalline uric acid. The change is readily observed 
 under the microscope by intimately mixing a particle 
 
 1 It is evident that the modified methods of determining uric acid, described 
 on page 168 et seg., may with advantage be employed here. 
 
176 QUADRI-URATES. 
 
 of the purified deposit on a glass slide with a drop of 
 water and protecting the mixture with a covering-glass. 
 In the course of ten minutes ovoid crystals of uric acid 
 make their appearance, and grow and multiply till in 
 the course of half an hour the entire field is thickly 
 studded with crystals ; the process continuing, pro- 
 vided that water be added as required, until the amor- 
 phous substance is entirely replaced by crystals of uric 
 acid. 1 
 
 The quadri-urates readily assume a gelatinous form. 
 Thus, if a 5 per cent, solution of ordinary sodium phos- 
 phate be heated to boiling with excess of uric acid, and 
 the liquid filtered hot, the filtrate sets to a jelly on 
 cooling. This jelly, after being pressed between blot- 
 ting-paper to free it from mother liquor, exhibits the 
 characteristic behaviour of a quadri -urate, being rapidly 
 decomposed by water with copious formation of 
 crystals of uric acid. On keeping in a moist condi- 
 tion, gelatinous sodium quadri-urate gradually passes 
 into a crystalline condition, and then appears under the 
 microscope in radiating spheres, exactly similar to the 
 spheres so common in serpents' and birds' urine. 
 
 If the white mortar-like substance which constitutes 
 the urinary excretion of birds and serpents be examined 
 in its fresh and uncontaminated state, and not after 
 contact with water or bacterial fermentation, it will be 
 found to behave in an exactly similar manner to arti- 
 ficial quadri-urates. Under the microscope it appears 
 as minute spheres, which exhibit a radiated structure 
 and display a black cross with polarised light. On 
 adding a drop of water, the spheres are seen gradually 
 to melt away, with formation of hexagonal tablets of 
 uric acid. 
 
 Bi-urates have the general formula MHC 5 H 2 N 4 3 , 
 
 1 Crystals of the sodium bi-urate simultaneously formed are never observed, 
 since this salt is liberated in the gelatinous form. 
 
ACID URATE OF SODIUM. 177 
 
 or MHUr. They result from the action of water on 
 the quadri-urates, and exist in the body under various 
 pathological conditions. The sodium salt, which is the 
 most important and characteristic member of the series, 
 possesses the following properties : — 
 
 Acid Sodium Urate, or Sodium Bi-urate, contains 
 2(NaHC 5 H 2 N 4 3 ) + H 2 0. It generally fqrms a crystalline 
 powder, which, under the microscope, 
 appears in needles (often crossed), 
 rosettes, stellate, and hedgehog-like 
 forms. It requires about 1200 parts 
 of cold or 120 of boiling water for 
 solution. Sodium bi-urate is readily 
 obtained by passing carbon di-oxide 
 through a solution of uric acid in 
 caustic soda, or by boiling uric acid Sodium Urate. 
 with sodium carbonate, phosphate, or ( After Frfj y)- 
 acetate, or with borax. The buff or brick-red sediment 
 often thrown down by urine is commonly stated to 
 consist of sodium bi-urate, but Roberts has shown 
 that it consists essentially of sodium quadri-urate 
 (page 175). 
 
 The solubility of acid urate of sodium in water im- 
 pregnated with salt and other substances has an im- 
 portant bearing on the cause and cure of gout, and has 
 been investigated by Sir Wm. Roberts, who gives the 
 figures on next page, which represent the parts by 
 weight of sodium urate dissolved at 100° F. ( = 37'8° C.) 
 by 1000 parts of the solution of the strengths indicated. 
 The amount of sodium bi-urate dissolved by 1000 parts 
 of distilled water at 100° F. was found to be 1 '0. From 
 the following results it appears that the solvent action 
 of the various salts depends on the nature of the metal, 
 and has no reference to its form of combination. Salts 
 having an alkaline reaction to litmus, like the car- 
 bonates and phosphates, behave exactly similarly to 
 
 M 
 
178 
 
 SOLUBILITIES OF ACID SODIUM URATE. 
 
 those of neutral reaction, such as the chlorides and 
 sulphates. Salts of potassium exert no appreciable 
 influence on the solubility of sodium bi-urate in water. 
 Salts of sodium decrease the solubility, the influence 
 being greater the larger the proportion of salt present. 
 Salts of ammonium, calcium, and magnesium behave 
 similarly to, but less powerfully than, salts of sodium. 
 
 
 o-i 
 
 2 
 
 0-3 
 
 0-5 
 
 07 
 
 10 
 
 Percentage of Salt in Solvent. 
 
 per 
 cent. 
 
 per 
 cent. 
 
 per 
 cent. 
 
 per 
 cent. 
 
 per 
 cent. 
 
 per 
 cent. 
 
 Sodium bicarbonate, 
 
 0-50 
 
 0-34 
 
 0-20 
 
 0-13 
 
 0-09 
 
 0-08 
 
 Sodium chloride, 
 
 
 0-45 
 
 0-30 
 
 0-16 
 
 o-io 
 
 0-08 
 
 0-05 
 
 Sodium phosphate (crystallised; 
 Sodium sulphate (crystallised), 
 Sodium salicylate, 
 Potassium bicarbonate, 
 
 t 
 
 
 0-70 
 0-55 
 0-65 
 0-96 
 
 1-00 
 
 0-86 
 1-00 
 
 0-32 
 0-24 
 0-25 
 0-97 
 
 1-02 
 
 0-98 
 
 Potassium chloride, . 
 
 
 
 0-96 
 
 
 1-01 
 
 1-10 
 
 
 
 Potassium phosphate, 
 Ammonium chloride, 
 
 
 
 1-01 
 0-85 
 
 V 50 
 
 0-42 
 
 1-00 
 0-35 
 
 
 
 Calcium chloride, 
 
 
 
 
 
 
 0-27 
 
 
 
 Calcium sulphate, 
 
 
 
 ; 65 
 
 0-44 
 
 
 
 
 
 Magnesium chloride, 
 
 
 
 0-85 
 
 
 
 ; 68 
 
 
 
 Magnesium sulphate (crystallised), 
 
 
 0-90 
 
 
 
 
 
 
 Crystalline sodium bi-urate is ten times as soluble 
 in boiling water as in cold, but a saturated hot solution 
 does not deposit the excess of salt immediately on 
 cooling. The bi-urate remains in complete solution 
 for a considerable time, and is not entirely deposited 
 for some days. Roberts has shown that this behaviour 
 is not merely due to supersaturation of the liquid, but 
 is owing to the formation of a gelatinous modification 
 of the bi-urate of greater solubility than the crystalline 
 form. Thus if a saturated solution of sodium bi-urate 
 in boiling water be prepared, and when cold mixed with 
 an equal measure of a 20 per cent, solution of common 
 salt, a voluminous gelatinous precipitate will be thrown 
 down. Saturated solutions or solid crystals of other 
 salts {e.g., sodium phosphate or acetate, potassium 
 chloride, phosphate, acetate, &c.) may be substituted 
 for the common salt. The precipitate, if filtered off, 
 
ACID URATE OP LITHIUM. 179 
 
 allowed to drain, and cautiously washed with cold 
 water, consists of sodium bi-urate in a state of approxi- 
 mate purity. It dissolves at 100° F. in blood-serum, 
 or in a liquid containing 0*5 gramme of sodium 
 chloride and 0"2 of sodium carbonate per 100 c.c. (which 
 represents the saline ingredients of serum), sufficiently 
 freely to cause a considerable separation of uric acid 
 after acidulating with acetic acid ; whereas crystalline 
 sodium bi-urate is taken up by water at 100° F. so 
 slightly that no deposition of uric acid is obtainable on 
 acidulating the liquid. 
 
 The gelatinous form of sodium bi-urate gradually 
 changes into the crystalline variety, and the gradual 
 deposition of the salt from its solution in water, blood- 
 serum, or imitation-serum is evidently due to the same 
 change of condition. 
 
 Acid Potassium Urate, or Potassium Bi-urate, 
 KHUr, is said to be sometimes formed as a urinary 
 deposit in cases of fever. It is amorphous, and more 
 soluble than the corresponding sodium salt, requiring 
 for solution only 800 parts of cold or 70 to 80 parts 
 of boiling water. 
 
 Acid Lithium Urate or Lithium Bi-urate, LiHUr, 
 forms crystalline grains, soluble in 370 parts of cold or 
 39 of boiling water. Lipowitz states that if equal 
 parts of uric acid and lithium carbonate be treated 
 with 90 parts of water at blood-heat, a clear solution 
 is obtained, while at 100° C. four times the amount 
 of uric acid can be dissolved without increasing the 
 weight of lithium carbonate. Seeing that lithium 
 carbonate itself requires about 200 parts of water 
 for solution, its solvent action on uric acid is remark- 
 able, and is of much interest in connection with the 
 extensive application of lithium salts in the treatment 
 of gout. On the other hand, it is stated by L. 
 Siebold (Year-book Pharm., 1889, page 413), as 
 
180 LITHIUM URATE. 
 
 the result of direct experiment, that the relative 
 solvent action of solutions of lithium, sodium, and 
 potassium carbonates on a given weight of uric acid, 
 under equal conditions of dilution and at a temperature 
 of 37° C. (blood-heat), is strictly proportional to the 
 ratio of the molecular weight of these solvents. Hence 
 lithium carbonate has the advantage that 74 parts 
 are chemically equivalent to 106 of the sodium salt 
 or 138 of potassium carbonate ; but there the advan- 
 tage ceases. Urinary sediments are similarly dissolved 
 by these carbonates with equal facility if molecular 
 proportions are used, and equivalent weights of the 
 citrates of lithium, sodium, and potassium produce 
 equal alkalinity in the urine of the" person taking 
 them. Siebold further states that lithium chloride 
 and sulphate have no solvent action on uric acid and 
 acid urates, and that natural mineral waters contain- 
 ing these salts have none beyond that exercised by 
 basic constituents simultaneously present, and by the 
 water. 
 
 Acid Ammonium Urate, (NH 4 )HUr, is soluble in 
 about 1500 parts of cold water, and quite insoluble 
 in saturated solutions of ammonium chloride and sul- 
 phate (compare page 168). The urinary excrement 
 of serpents is commonly stated to consist almost 
 wholly of a mixture of acid urate of ammonium 
 with free uric acid. This is often true of the altered 
 product, but Sir W. Roberts has shown that, in a fresh 
 and undecomposed state, serpents' urine consists sub- 
 stantially of quadri-urates, which undergo decompo- 
 sition into a mixture of bi-urates and free uric acid by 
 contact with water (page 176). Guano, the excrement 
 of various aquatic birds, consists chiefly of oxalate and 
 acid urate of ammonium in admixture with phosphates. 
 Guanine, C 5 H 5 N 5 0, a base forming crystallisable 
 salts with acids, is also a constituent of guano, and 
 
METALLIC URATES. 
 
 181 
 
 replaces uric acid in the urine of spiders and other 
 invertebrate animals. 
 
 Neutral or Normal Urates of the light metals do 
 not exist naturally, but they may be obtained by dis- 
 solving uric acid in the theoretical amount of alkali. 
 The normal urates of lithium and ammonium are un- 
 known. Neutral Potassium Urate, K 2 C 6 H 2 N 4 03, forms 
 small crystals having an alkaline reaction and caustic 
 taste. It dissolves, with partial decomposition into 
 the acid salt, in about 36 parts of cold water, forming 
 a liquid of soapy taste which froths strongly when 
 shaken. Normal Sodium Urate, Na 3 Ur + H 2 0, forms 
 hard nodules which closely resemble the potassium 
 salt, but are less soluble in water. 
 
 On passing carbon dioxide through a solution of 
 the normal urate of potassium or sodium the corre- 
 sponding acid urate is precipitated. The same 
 decomposition occurs by prolonged boiling of the 
 solution. 
 
 The following table, due to Ralph {Practical 
 Treatise on Diseases of the Kidneys, 1885), shows 
 the characters of the urates of the light metals : — 
 
 Urate. 
 
 Solubility in 
 Cold Water. 
 
 Character of Deposit. 
 
 Potassium; acirl, . 
 
 ,, normal, 
 Sodium ; acid, 
 
 ,, normal, 
 Lithium ; acid, . 
 Calcium ; acid, . 
 
 ,, normal, 
 Ammonium ; acid, 
 
 1-800 
 
 1-44 
 
 1-1200 
 
 1-77 
 
 1-60 
 
 1-600 
 
 1-1500 
 
 1-1600 
 
 Amorphous. 
 
 Amorphous, or in fine needles. 
 
 Amorphous ; rarely crystalline. 
 
 Nodular masses. 
 
 Amorphous, or in fine needles. 
 
 Amorphous, or in fine needles. 
 
 Fine granules. 
 
 Amorphous, or spiked globular masses. 
 
 The urates of lead, copper,, mercury, and silver are 
 quite insoluble in water. Hence solutions of these 
 metals are used for determining uric acid or for sepa- 
 rating urates from urine. 
 
182 URATES IN THE SYSTEM. 
 
 The behaviour of the urates with water and saline 
 solutions has an important bearing on the cause and 
 treatment of gout. It is probable that in media con- 
 taining alkaline carbonates — such as the serum of 
 the blood, and its derivatives lymph and synovia — 
 uric acid passes into solution in the first instance as 
 quadri-urate, and it may be inferred that it circulates 
 in the blood and is voided in the urine in the same 
 form. In perfect health, the elimination of the quadri- 
 urate proceeds with sufficient speed and completeness 
 to prevent any undue detention or any accumulation 
 of it in the blood. But in gouty subjects, either from 
 defective action of the kidneys or from excessive intro- 
 duction of uric acid into the circulation, the quadri- 
 urate lingers unduly in the blood and accumulates 
 therein. The detained quadri-urate, circulating in a 
 medium rich in carbonate of sodium, gradually takes 
 up an additional atom of base, and is thereby converted 
 into bi-urate, which at first exists in the hydrated or 
 gelatinous condition, but with lapse of time and 
 accumulation passes into the insoluble crystalline 
 condition, and then symptoms of gout manifest them- 
 selves. 
 
 A. Haig (Med. Chirurg. Trans., lxxi. 125, 283) 
 has shown that administration of acids diminishes the 
 relative amount of uric acid excreted, while that of 
 ; alkalies increases it. Thus the normal proportion of 
 uric acid to urea is 1 : 35, but after a few doses of 
 citric acid the relation was 1 : 41, and after similar 
 doses of potassium citrate 1 : 28. In these cases 
 there was not only a relative but also an absolute 
 diminution and increase in the uric acid excreted. 
 Salicylic acid forms an important exception to the 
 general behaviour of acids, for while it increases 
 urinary activity it does not in any way diminish the 
 excretion of uric acid. Moreover, acids given while 
 
ACTION OF SALICYLATES. 183 
 
 salicylates are present in the circulation have no 
 longer the power of diminishing the excretion of uric 
 acid, nor is excessive excretion of uric acid under 
 salicylates accompanied by any headache. Both uric 
 and salicyluric acids are present in the urine passed 
 under the influence of salicylates, probably owing to 
 the salicylate acting on the uric acid in the blood, but 
 not on that secreted by the kidney itself. Benzoates 
 do not act in the same way as salicylates, probably 
 because the hippuric acid formed from them is less 
 soluble than salicyluric acid. The value of salicylates 
 in uric acid diseases is largely due to their power of 
 preventing acids from causing retention of uric acid. 
 Thus salicylates prevent gout, the peculiar headache 
 due to uric acid and frequent after breakfast, and also 
 epilepsy, which is probably due to uric acid acting on 
 the nerve-centres. 
 
 Hippuric Acid. Benzoyl-amidoacetic Acid. Ben- 
 zoyl-glycocine. C 9 H 9 N0 3 ; or, C 6 H 3 .CO.NH.CH 2 .COOH. 
 
 Hippuric acid derives its interest less from its 
 pathological importance than as affording a typical 
 example of the so-called " conjugated bodies " of 
 which the synthesis is readily effected within the 
 living organism. Thus if benzoic acid be taken inter- 
 nally, it appears in the urine as hippuric acid, and 
 hippuric acid may be obtained artificially by heating 
 benzoic anhydride with amido-acetic acid (glycocine), 
 or the zinc salt of the latter with benzoyl chloride : — 
 
 C 7 H 5 O.C1 + C 2 H 2 (NH 2 )O.OH = C r H s O.O.C 2 H 2 (NH 2 )0 + HC1 
 
 Benzoyl Chloride. Glycocine. Hippuric Acid. 
 
 Benzoic aldehyde, toluene, cinnamic acid, quinic 
 acid and phenyl-propionic acid when ingested, are 
 also excreted as hippuric acid. Substituted benzoic 
 acids appear in the urine as substituted hippuric 
 acids. Salicylic acid, which has the constitution of 
 
184 HIPPURIC ACID. 
 
 ortho-hydroxybenzoic acid, C 6 H 4 (OH).COOH, is con- 
 verted in the system into hydroxy-hippuric or 
 salicyluric acid, C 9 H 8 (OH)N0 3 , which may be 
 detected in the urine by the bluish-violet coloration 
 produced on adding dilute ferric chloride. 
 
 The quantity of hippuric acid excreted in normal 
 human urine is stated to range from 5 to 60 grains 
 ( = 0'3 to 3'8 grammes) in twenty-four hours, but an 
 increase results from a vegetable diet. This has been 
 particularly noticed after eating plums, pears and 
 cranberries, and the cuticular parts of many plants 
 act similarly. In the urine of diabetic patients, hip- 
 puric acid is frequently present in much increased 
 proportion, as also in jaundice and other liver-com- 
 plaints, and it is abundant in the acid urine of persons 
 suffering from all kinds of fevers. 
 
 Hippuric acid replaces uric acid in the urine of herbi- 
 vorous animals, which are stated to contain it to the 
 extent of about 2 per cent. j 1 its origin being doubtless 
 in bodies of the aromatic series existent in the food. 
 Hippuric acid is also found in the excrement of the 
 lower animals, except that of birds, which contains 
 the allied substance ornithuric acid, having the 
 constitution of a dibenzoyl-diamidovaleric 
 acid : 2 — 
 
 C 6 H 5 .CO.NH ) . pt tt nnnti 
 C 6 H 6 .CO.NH J • l4tl7 ' lUUh ' 
 
 1 Hippuric acid may be conveniently prepared from fresh cows' or horses' 
 urine, which often contains sufficient to yield a precipitate on mere addition of 
 excess of hydrochloric acid. If not, the urine should be boiled with milk of 
 lime, and the filtrate neutralised, concentrated, and treated with excess of 
 hydrochloric acid ; or the neutralised filtrate may be precipitated with ferric 
 chloride, and the washed precipitate decomposed by hydrochloric acid. The 
 hippuric acid is freed from colouring matter by recrystallising it from chlorine- 
 water, or by treating it with bleaching powder and hydrochloric acid. 
 
 2 On boiling ornithuric acid with hydrochloric acid, it almost immediately 
 parts with one benzoyl group and yields benzoyl -ornithine, which on 
 further boiling splits up into benzoic acid and diamido- valeric acid or orni- 
 thine, (NH 2 ) 2 C 4 H 7 .COOH, a base of strong alkaline reaction and of caustic 
 taste. 
 
PROPERTIES OF HIPPUKIC ACID. 185 
 
 Hippuric acid crystallises in milk-white rhombic 
 prisms ending in two or four facets, the crystals being 
 often grouped in clumps (fig. 18). As liberated by 
 adding hydrochloric acid to 
 cows' urine or one of its 
 salts, hippuric acid is apt to 
 form ill-defined crystals. 
 
 Hippuric acid has a 
 slightly bitter taste, free 
 from acidity. It melts at 
 187-5° C, and above 240° 
 decomposes, with an odour 
 of hay or fresh urine, 
 and formation of h y d r o - „. 
 
 J . Fig. 18. — Hippuric Acid (after 
 
 Cyanic and benzoic Frey). a, a, Prisms ; b, Crystals 
 acids and benzonitril formed by slow evaporation. 
 
 (phenyl cyanide), C 6 H 5 .CN, a dark resinous or coaly 
 mass being left. 
 
 Hippuric acid requires about 600 parts of ice-cold 
 water for solution, but dissolves tolerably readily in hot 
 water. It is also soluble in alcohol, especially when hot. 
 The aqueous and alcoholic solutions have an acid re- 
 action. Hippuric acid is but slightly soluble in cold 
 ether, but dissolves in acetic ether, and readily in boil- 
 ing amylic alcohol. In chloroform, benzene, petroleum 
 spirit, and carbon disulphide it is practically insoluble. 
 
 When boiled for a time (half an hour) with dilute 
 nitric, hydrochloric, or oxalic acid (or more rapidly if 
 strong hydrochloric acid be used), hippuric acid under- 
 goes hydrolysis, the liquid on cooling depositing 
 benzoic acid, while a salt of glycocine 
 (amido-acetie acid) remains in solution : — C 9 H 3 N0 3 -|- 
 H 2 = C 7 H 6 02-t-C2H s N0 2 . A similar reaction takes 
 place spontaneously in urine containing hippuric acid, 
 under the influence of ferments. Hence only perfectly 
 fresh urine will yield hippuric acid. If the urine be 
 
186 DETERMINATION OF HIPPURIC ACID. 
 
 alkaline, as is usually the case with that of her- 
 bivorous mammals, the glycocine first produced splits 
 up into ammonia and acetic acid : — C 2 H 5 N02+H 2 = 
 
 C 2 HA+NH,. 
 
 If hippuric acid be evaporated to dryness with con- 
 centrated nitric acid, and the residue heated, an odour 
 of nitrobenzene is evolved. 
 
 Hippuric acid decomposes carbonates, and dissolves 
 zinc with evolution of hydrogen. Its salts are mostly 
 soluble and crystallisable. The hippurates of silver, 
 lead, and copper are sparingly soluble. 
 
 When ferric chloride is added to a solution of a 
 hippurate, a cream-coloured precipitate of ferric 
 hippurate is thrown down, which contains more 
 or less basic salt, according to the greater or less 
 dilution of the solution. The precipitate is almost 
 insoluble in pure water, but dissolves in free hippuric 
 acid, in excess of ferric chloride, and in alcohol. The 
 reaction with ferric salts may be employed for the 
 determination of hippuric acid in urine. For this 
 purpose, the urine is acidulated with nitric acid, heated 
 to boiling to remove carbon dioxide, neutralised with 
 calcium carbonate, treated with excess of lead nitrate, 
 and then diluted to a known volume and filtered. An 
 aliquot part of the filtrate is then heated and titrated 
 with a solution of neutral ferric nitrate which has 
 been standardised with pure hippuric acid. The re- 
 action is at an end when a drop of the clear liquid 
 gives a blue coloration with potassium ferrocyanide. 
 The distinction between this point and the previous 
 formation of the white ferrocyanide of lead is very 
 sharp. 
 
 On treating ferric hippurate or the solution of a 
 soluble hippurate with excess of hydrochloric acid, the 
 hippuric acid separates sooner or later in long crystal- 
 line needles, Hippuric acid is distinguished from 
 
DETECTION OF HIPPDRIC ACID. 
 
 187 
 
 Fig. 19.— Benzoic Acid. 
 
 benzoic and salicylic acids by its crystalline form (figs. 
 18, 19, pages 185, 187), by charring when heated with 
 strong sulphuric acid, by giving off ammonia on ignition 
 with soda-lime, and by not being dissolved on agitating 
 its solution with chloroform or petroleum-spirit. 
 
 For the detection of hip- 
 puric acid in the urine of 
 herbivora, the fresh liquid 
 should be treated with milk 
 of lime, filtered, the filtrate 
 concentrated to a syrup as 
 rapidly as possible, and excess 
 of hydrochloric acid added, 
 when hippuric acid crystallises 
 out on standing. If the urine be at all stale, benzoic 
 acid will be obtained instead, and may be distin- 
 guished from hippuric acid as just described. 
 
 For the detection of traces of hippuric acid in 
 human urine, the perfectly fresh liquid should be 
 evaporated nearly to dryness at 100° C, the residue 
 mixed with powdered barium sulphate, a little 
 hydrochloric acid added, and the whole exhausted 
 with rectified spirit. The alcoholic solution is care- 
 fully neutralised with soda, and the liquid evapo- 
 rated. The residue is mixed with a little oxalic acid, 
 and again evaporated to dryness on the water-bath. 
 The residue is exhausted with a mixture of equal 
 measures of alcohol and ether, the solution distilled 
 to a small bulk, boiled with milk of lime, filtered, 
 concentrated, and acidulated with hydrochloric acid. 
 Immediately, or on standing, according to the quantity 
 present, crystalline needles of hippuric acid separate, 
 and may be filtered off and purified by washing, first 
 with diluted hydrochloric acid and then with a little 
 ether. 
 
 Another method is to precipitate the urine (1000 to 
 
188 ISOLATION OF HIPPURIC ACID. 
 
 1200 c.c.) with a slight excess of strong baryta-water. 
 The filtered liquid is treated with dilute sulphuric 
 acid till exactly neutral to litmus, decanted or filtered 
 from the precipitated barium sulphate, and evaporated 
 to a syrup on the water-bath. The residue, which 
 should be exactly neutral, is treated while still hot 
 with 150 to 200 c.c. of absolute alcohol and thoroughly 
 agitated. Barium succinate, sodium chloride, and 
 other compounds are thus precipitated. The liquid 
 is decanted, the alcohol evaporated, and the syrupy 
 residue treated while still hot with hydrochloric 
 acid. The liberated hippuric acid is extracted by 
 repeated agitations with ether (100 to 150 c.c), 
 the separated ether distilled off, the residue diluted 
 with water, and heated to boiling with a little milk 
 of lime. The liquid is filtered, concentrated, and 
 treated with excess of hydrochloric acid, when hippuric 
 acid separates in fine crystals, which can be obtained 
 colourless by treatment with purified animal charcoal. 
 
COLOURING MATTERS OF URINE. 
 
 The colour of urine is liable to modification under the 
 influence of a variety of pathological conditions. The 
 following resume" is based on that of Krukenberg : — 
 
 Nearly colourless or pale yellow urine may be due 
 either to greater dilution or to diminution of the 
 normal pigments. It occurs specially in anaemia, 
 chlorosis, diabetes, granular kidney, &c. 
 
 Yellowish and milky urine may be due to the 
 presence of floating globules of fat, as in chyluria ; or 
 to suspended pus-corpuscles, as in pyelitis or other 
 purulent disease of the urinary tract. 
 
 Orange-coloured urine is excreted after the inges- 
 tion of certain drugs, such as chrysophanic acid, 
 rhubarb, santonin, &C 1 
 
 Brownish-yellow to red-brown urine, which becomes 
 blood-red on addition of an alkali, is likewise indica- 
 tive of excreted drugs, such as rhubarb, senna, 
 chelidonium, &c. Port-wine coloured urine is some- 
 times excreted after sulphonal has been taken. 
 
 Dark yellow to brown-red urine, which easily deposits 
 a sediment, occurs in certain febrile diseases, and owes 
 its colour to an increase of the normal pigments or to 
 the occurrence of pathological colouring matters. 
 
 Red or reddish urine may be due to pigmentary 
 
 1 If the urine be treated with caustic alkali and then shaken with amylic 
 alcohol, the colouring matter of santonin is dissolved out, and becomes yellow 
 on exposure to air. The colouring matter of rhubarb (chrysophanic acid) is not 
 sensibly dissolved by amylic alcohol. 
 
190 COLOURING OF URINE. 
 
 matters (e.g., logwood, madder, bilberries, coal-tar 
 dyes) in the food, or to the presence of unchanged 
 haemoglobin, occurring in haemoglobinuria or in urinary 
 haemorrhage. A brown colour may also be due to 
 haemorrhage into the kidneys or to methaemoglobinuria. 
 
 Brown or brownish-black urine is excreted in cases 
 of methaemoglobinuria and haemorrhage, and is especi- 
 ally indicative of melanitic sarcoma. It is also highly 
 characteristic of poisoning by carbolic acid, in which 
 case hydrochinone and catechol (pyrocatechin) appear 
 in the urine. 1 
 
 Yellowish-green, green, or greenish-brown urine is 
 indicative of the presence of bile-pigments, and of 
 course it occurs in jaundice and other affections of 
 the liver. 
 
 Dirty green or blue urine, sometimes showing a 
 dark blue scum with a blue deposit, indicates the 
 presence of excess of indigogens. It frequently 
 occurs in cholera and typhus, 2 especially when the 
 urine is putrefying. 
 
 • The chemistry of urochromes is still in a very con- 
 fused state, notwithstanding the great attention the 
 subject has received. An exhaustive recapitulation of 
 
 1 Melanitic urine, on addition of ferric chloride, yields a brown turbidity or 
 black precipitate soluble in excess. On adding a dilute solution of sodium 
 nitroprusside, followed by caustic alkali, they frequently give a pink or red 
 coloration, and on adding an acid Prussian blue is precipitated. 
 
 2 The urine of healthy persons gives a negative reaction with the "diazo 
 test," while, on the other hand, the urine of patients suffering from typhus 
 and certain other fevers exhibits the reaction in a marked manner. The re- 
 action is also produced in cases of acute tuberculosis, but not by the urine in 
 intestinal catarrh, which can thus be differentiated from typhus fever. (See 
 Rutimeyer, Lancet, ii., 1890, page 413.) To apply the diazo test, 1 gramme 
 of sulphanilic acid is dissolved in 10 c.c. of pure hydrochloric acid, and the 
 solution diluted with water to 200 c.c. Fifty c.c. of this reagent is mixed with 
 5 c.c. of a solution of 1 gramme of potassium or sodium nitrite (NaN0 2 ) in 
 200 c.c. of water, and 50 c.c. of the sample of urine added. The mixture is 
 made strongly alkaline by ammonia, and the whole well shaken. A blight 
 red colour is produced by the pathological urines above mentioned, and on 
 standing for twenty-four hours a deposit forms, the upper part of which is 
 green or black. Healthy urine gives no such reaction. 
 
UROBILIN. 191 
 
 the existing knowledge on the subject is beyond the 
 scope of this work, but the following is a brief 
 outline of the leading facts known respecting the 
 principal colouring matters of normal and pathological 
 urine. 
 
 Urobilin. 
 
 The best known and most definite of the colouring 
 matters of normal urine is urobilin, to which the 
 formula C 32 H 40 N 4 O 7 or 2C 16 H 18 N 2 3 +H 2 is ascribed. 
 It is a yellowish-brown amorphous substance, almost 
 insoluble in pure water but soluble in presence of 
 small quantities of free acids or neutral salts, slightly 
 soluble in ether and benzene, and readily so in alcohol 
 and chloroform. The neutral alcoholic solution is 
 reddish-yellow, and when concentrated exhibits a 
 green fluorescence. On addition of acid, the fluor- 
 escence is destroyed, and the dilute solution acquires a 
 rose tint. Alkaline solutions of urobilin are yellowish 
 or yellowish-green, according to their concentration, 
 and show a fluorescence which is much more marked 
 after addition of a solution of zinc chloride, the liquid 
 then appearing rose-coloured by transmitted and green 
 by reflected light. Neutral and alkaline solutions of 
 urobilin in alcohol, when examined in the spectroscope, 
 exhibit an absorption-band between the Fraunhofev- 
 lines b and F. The absorption is much increased by the 
 addition of zinc chloride. Normal urobilin is regarded 
 by MacMunn as an oxidation-product of effete hsematin 
 and bile-pigments and not as a reduction-product. It 
 appears to be identical with choletelin, a body resulting 
 from the treatment of hsematin in acid solution with 
 hydrogen peroxide, and said by Maly to have the 
 formula C 16 H 18 N 2 6 . 
 
 The proportion of urobilin in fresh normal urine is 
 often extremely small, but appears to increase on 
 
192 ISOLATION OF UROBILIN. 
 
 exposure of the urine to the air. This behaviour is 
 probably due to the presence in the urine of a 
 chromogen or mother-substance, which is converted 
 into urobilin by oxidation. 
 
 For the extraction and estimation of urobilin, A. 
 Studensky (Chem. Centr., 1893, ii. 668) recom- 
 mends that 20 c.c. of the urine should be treated 
 with 2 c.c. of a saturated solution of cupric sulphate, 
 the liquid saturated with crystallised ammonium sul- 
 phate, 10 c.c. of chloroform added, and the mixture 
 shaken for some minutes. The urobilin dissolves in 
 the chloroform with copper-red colour, while bilirubin 
 and its allies are not extracted in presence of copper 
 sulphate. A portion of the chl oroformic layer is tapped 
 off, and its colour compared with that of a standard 
 solution of urobilin, prepared in a similar manner from 
 a large quantity of urine. The chloroformic solution 
 thus obtained is evaporated to dryness, the residue 
 washed with ether, and weighed. Standard solutions 
 of urobilin are prepared by dissolving known weights 
 of this residue in chloroform, and the solution yielded 
 by the sample under examination is compared with 
 them. The standard solutions remain unchanged for 
 some months, if preserved in the dark in closed vessels, 
 and covered with a layer of saturated solution of am- 
 monium sulphate. 
 
 Pathological Urobilin is, according to MacMunn, 
 a substance distinct from normal urobilin, though 
 obtainable in the same manner and soluble in the 
 same menstrua as that body. The absorption -band at 
 F, which characterises the spectrum of the urobilin 
 series, is much broader and darker in the case of the 
 pathological than in that of the normal colouring 
 matter, and other characteristic differences are recog- 
 nised by MacMunn, who regards the former as less 
 oxidised than the normal product and indistinguish- 
 
URINARY PIGMENTS. 193 
 
 able from and probably identical with stercobilin, 
 the colouring matter of faeces. 1 
 
 Uroerythrin is the colouring matter of certain 
 bright pink urinary deposits, and possibly of the highly 
 coloured urine excreted in rheumatism. It may be 
 extracted by boiling pink urates with alcohol. The 
 solution exhibits two ill-defined absorption-bands be- 
 tween- D and F, and the residue left on evaporation is 
 turned green by caustic alkalies. 
 
 Uromelanin is a dark-coloured pigment produced 
 by boiling urine with hydrochloric acid. According 
 to L. von U d r a n s k y, it has its origin in the carbo- 
 hydrates of the urine (Zeitsch. physiol. Chem., xi. 
 537 ; xii. 33). 
 
 Uro-eosein is the name given to an extremely 
 unstable pigment observed by N e n c k i and S i e b e r 
 in diabetic urine which became bright pink on adding 
 hydrochloric acid. It was extracted by amylic 
 alcohol, and showed a characteristic absorption-band 
 between D and E. 
 
 Bile Pigments in Urine. 
 
 The colouring matters characteristic of bile are 
 not present in normal urine, but in certain diseases 
 (jaundice, &c.) they exist in very appreciable amount. 
 Such urine exhibits a yellowish-green, green, greenish- 
 brown, or almost black colour. The most characteristic 
 colouring matter of bile is bilirubin, C 16 H 18 N 2 03, while 
 biliverdin, biliprasin, bilifuscin, bilicyanin, &c, are 
 
 ' J MaoMunn points out that, " if this be so, then the presence of pathological 
 urobilin in urine is, to a certain extent, an indication of the absorption of 
 fecal matters from the intestine, and with them of poisonous alkaloidal 
 bodies — ptomaines, which have escaped the destructive action of the liver. . . . 
 By such an assumption we can explain many of the symptoms which accompany 
 its presence in urine. . . It would appear that, in those cases where patho- 
 logical urobilin occurs in urine, there is either some vaso-motor disturbance 
 of the hepatic circulation, or some mechanical interference with it, as in 
 passive congestion." 
 
 N 
 
194 DETECTION OF BILE-PIGMENTS. 
 
 products of its oxidation. In bilious urine of saffron- 
 yellow colour bilirubin predominates, while biliverdin 
 and other oxidation-products are present in greenish 
 urine. Bilious urine gives a yellow froth on agitation, 
 and stains linen and filter-paper yellow. 1 Bilirubin may 
 be extracted from its acidulated solutions by agitation 
 with chloroform. It dissolves in caustic alkalies, and 
 on exposing the solution to the air it is converted 
 into biliverdin, C 16 H 18 N 2 4 ; subsequently into 
 biliprasin, C 16 H 22 N 2 6 ; and, according to Maly, 
 ultimately into choletilin, C 16 H 18 N 2 6 . 
 
 A variety of tests have been proposed for the 
 detection of bile-pigments in urine, but the following 
 are the most delicate and answer every purpose :— 
 
 Gmeliris Test consists in treating the urine with 
 strong nitrie acid and observing the change of colour 
 produced. The reaction is best observed by allowing 
 some of the urine to run gently onto the surface of 
 some fuming nitric acid contained in a test-tube. If 
 bile-pigments be present, a green ring will become 
 apparent at the point of contact, while below this will 
 appear violet, red, and yellow zones, in the order 
 named. The green colour is alone characteristic of 
 bilious urine, since indigogens give rise to blue and 
 red colorations. 
 
 Various modifications of Gmelin's reaction have 
 been proposed, but they possess no advantage over 
 the above mode of applying the test. 
 
 Rosin's Test for bile-pigments consists in allowing 
 very dilute iodine solution or bromine -water to flow 
 on to the surface of the urine from a pipette. A 
 grass-green ring is produced at the junction of the 
 two strata. 
 
 1 If the dyed filter-paper be treated with a drop of nitric acid, the margin of 
 the spot will become violet or deep bine, while the centre gradually changes 
 to emerald-green. 
 
DETECTION OF BILE-PIGMENTS. 195 
 
 For the detection of traces of bile-pigments, often 
 of great clinical and physiological importance, 1 the 
 urine should be treated with a moderate excess 
 of lime-water, and the excess of lime precipitated as 
 carbonate, by passing carbon dioxide gas or adding 
 seltzer-water, till the liquid no longer exhibits an 
 alkaline reaction to litmus (or preferably to phenol- 
 phthalein). The precipitate is collected on a filter, 
 and treated with fuming nitric acid, when the green 
 and other colours already described will become 
 evident if bile be present. Or the precipitate may 
 be boiled with alcohol acidulated with sulphuric acid, 
 when the supernatant liquid will acquire a grass-green 
 colour, a white deposit of calcium sulphate being 
 simultaneously formed. 
 
 Another reliable test for bile-pigments in urine is 
 to treat 30 c.c. (or 1 oz.) of the sample with about 
 one-third of its bulk of a 20 per cent, solution of zinc 
 acetate, 2 after previously neutralising most of the free 
 acid by sodium carbonate. The voluminous precipi- 
 tate is filtered off, washed, and treated with a little 
 ammonia. In presence of bile-pigments the ammo- 
 niacal liquid is usually fluorescent, and either at once 
 or on standing shows the absorption-spectrum of 
 bilicyanin, characterised by bands on each side of 
 the D line, and a third between b and F. 
 
 i In two cases of highly icteric urine, after the occurrence of ammoniacal 
 fermentation Salkowski could detect no bilirubin by Omelin's test, and 
 extraction yielded no unchanged biliary pigment. He suggests that this de- 
 composition of bilirubin -without the formation of any characteristic products 
 was probably the result of the activity of bacteria, and may explain other 
 cases of jaundice, in which the urine, though dark coloured, gave no evidence 
 of the presence of bile-pigments. 
 
 2 Zinc acetate may be readily extemporised by treating lead acetate with 
 zinc sulphate in slight excess, and filtering from the precipitated lead sul- 
 phate. Or sodium acetate may be added to a solution of zinc sulphate or 
 chloride, the sodium sulphate or chloride formed simultaneously with the zinc 
 acetate being disregarded. 
 
19G BLOOD IN URINE. 
 
 Blood Pigments in Urine. 
 
 Blood occurs in urine in certain diseases. In 
 Hematuria blood is present, and blood-corpuscles may 
 be recognised by examining the deposit formed on 
 standing under a high microscopic power. In Hsemo- 
 globinuria, the urine contains the red colouring 
 matter of the blood, but no actual blood-corpuscles 
 unless blood also be present. 
 
 The micro-spectroscope affords the most satisfactory 
 means of detecting blood-pigments in urine. In its 
 absence, the following tests may be applied : — 
 
 Heller's Test consists in making the urine strongly 
 alkaline with caustic soda or potash, and heating the 
 liquid to boiling. In presence of blood a bottle- 
 green coloration is produced, and a brownish-red 
 precipitate, consisting of earthy phosphates coloured 
 by blood, is thrown down. 
 
 Hsemin Test. — The deposit which separates from the 
 urine on standing is evaporated to dryness at a gentle 
 heat with a small fragment of common salt. The resi- 
 due is treated with two or three drops of glacial acetic 
 acid, and heated on a glass microscope-slide. On 
 cooling, if blood-pigments were present, microscopic 
 crystals of hsemin as reddish-brown rhomboidal plates 
 will be observed. 
 
 According to C. Rosenthal (Chem. Centr., 1886, 
 page 251), Heller's test for haemoglobin, based on the 
 red coloration produced by warming with aqueous 
 soda, fails when the proportion of blood is less than 1 
 part per 1000 of the urine. S t r u v e's test, consisting 
 in the isolation of haemin from the precipitate produced 
 by tannin, is uncertain in its results, but the presence 
 of more than a minute trace of iron in the ash result- 
 ing from the ignition of this precipitate affords 
 satisfactory evidence of the presence of haemoglobin in 
 the urine. 
 
BLOOD PIGMENTS. 197 
 
 UroH/Ematoporphorin is regarded by M a c M u n n 
 as solely a reduction-product of haematin, which has 
 been produced in the organism by reduction of effete 
 haemoglobin or effete histo-haematin. It can be pre- 
 pared artificially by the action of sodium-amalgam, 
 zinc and dilute acid, and other reducing agents on 
 hsematin. In acid solutions, the spectrum exhibits a 
 narrow absorption-band almost coincident with the D 
 line, and another darker band between D and E ; be- 
 sides a feeble shading between these two, and a band at 
 F closely resembling that of urobilin. If the alcoholic 
 solution of the isolated colouring matter be treated with 
 ammonia, the liquid shows a five-banded spectrum 
 closely resembling that of hsematoporphorin. 
 
 H^ematoporphyrin is a colouring matter produced 
 by the action of strong sulphuric acid on hsematin 
 or haemin. According to Nencki and Sieber's first 
 accounts it contains Cgat^N^, but in a more recent 
 research (Monatsh., ix. 115) they regard this body as 
 either a mixture or an anhydride of true h ae m a t o- 
 porphyrin, which they prepare by acting on 
 haemin with a saturated solution of hydrobromic acid 
 gas in glacial acetic acid. To this body they ascribe 
 the formula C 16 H 18 N 2 3; identical with that of anhy- 
 drous bilirubin, which body haeniatoporphyrin re- 
 sembles in many of its properties. When introduced 
 into the system, haematoporphyrin is partly expelled 
 in the urine, but the greater portion is retained, and 
 probably utilised in the formation of haemoglobin. 
 
 Haematoporphyrin is very frequently present in the 
 dark-coloured urine excreted after the administration 
 of sulphonal. 0. Hammer sten (Jour. Chem. 
 Soc, Ixii. 649 and 1136) examined four samples of 
 urine from patients to whom sulphonal had been 
 administered, and found hEematoporphyrin in each 
 case, but adds that more observations will be 
 
198 HSEMATOPORPHYRIN. 
 
 needed before it can be positively stated that 
 sulphonal is the cause of the appearance of haema- 
 toporphyrin in the excretion. 1 For the detection 
 of hsematoporphyrin, Hammersten precipitates the 
 urine with barium acetate and filters. The filtrate 
 is precipitated alternately with barium acetate and 
 sodium carbonate until a small filtered portion gives a 
 white precipitate with these reagents. The hsemato- 
 porphyrin is carried down in the precipitate. Both 
 precipitates are washed well and extracted with acidi- 
 fied alcohol. The acid alcoholic solution is diluted with 
 several times its measure of water, and shaken with 
 chloroform, which extracts most of the colouring 
 matter. The chloroformic layer is rapidly tapped off 
 from the upper stratum, washed well with water, and 
 evaporated in shallow basins in the dark. According 
 to Hammersten, the brown residue left after evapora- 
 tion is soluble with splendid purple colour in chloro- 
 form, insoluble in cold water and in very dilute acids, 
 sparingly soluble in cold alcohol, but soluble in hot 
 alcohol, from which it crystallises in needles resembling 
 those of the hsematoporphyrin hydrochloride of Nencki 
 and Sieber. On spectroscopic examination, the absorp- 
 tion-bands were found to be slightly nearer to the 
 red end of the spectrum than those of the hsemato- 
 porphyrin obtained by Nencki and Sieber. In only 
 one case out of the four did the substance appear to 
 be absolutely identical with their product. In 
 another case the cbromogen of a similar colouring 
 matter was met with. A solution of hsematoporphyrin 
 in ammonia and zinc chloride gives four absorption- 
 bands. The two lying between C and D and between 
 b and F disappear within twenty- four hours, the former 
 first. The other two bands are permanent. 
 
 1 Hammersten's suggestion has been fully confirmed by Salkowski and others. 
 
POTASSIUM INDOXYL-SULPHATE. 199 
 
 Urinary Indigogens. 
 
 Normal urine contains traces of the potassium salt of 
 indoxyl-sulphuric acid, CgHgN.SOJL 1 TmVj 
 body is derived primarily from indole, C 8 H 7 N, / 
 which by oxidation yields indoxyl, C 8 H 5 (NH).OH. | 
 By reaction with the elements of sulphuric acid this is 
 converted into an ethereal salt, the potassium com- 
 pound of which is the substance in question. 
 
 Potassium Indoxyl-sulphate, C 8 H 6 N.S0 4 K, has 
 received the unfortunate name of " urinary indican," 
 from a supposed identity with plant-indican, the 
 glucoside from which indigo is obtained. The only 
 similarity between the two bodies is that both yield 
 indigo-blue as one of the products of their decom- 
 position. 2 
 
 Potassium indoxyl-sulphate crystallises from hot 
 alcohol in colourless lustrous tables, readily soluble in 
 water but only sparingly in cold alcohol. When boiled 
 with dilute acid it is decomposed into indoxyl and acid 
 potassium sulphate, but is not attacked by alkalies. 
 When the crystals are heated, indigotin (indigo-blue) 
 sublimes, and the same substance is found quanti- 
 tatively when the acidulated solution is warmed with 
 ferric chloride. 
 
 For the. detection of indoxyl-sulphuric acid in urine, 
 Jaffe (PJluger's Archiv., iii. 448) first separates 
 any albumin by boiling the liquid, and treats the 
 filtrate with an equal measure of hydrochloric acid. 
 A dilute solution of bleaching powder is then cautiously 
 added, until the blue colour no longer increases. On 
 
 1 Indoxyl-sulphuric acid is described by some writers as indoxyl- 
 sulphonic acid. The latter name would be applicable to a body of the 
 constitution C 8 H 4 (S0 3 H)(NH).0H. This would be isomeric with indoxyl- 
 sulphuric acid, and would not exhibit the readiness of the latter in hydro- 
 lysing into sulphuric acid and indoxyl (page 6). 
 
 2 Decomposing urine occasionally forms a bluish-red pellicle, and ultimately 
 deposits microscopic crystals of indigo-blue. A calculus of the same nature 
 has been described. 
 
200 URINARY IND1UOGENS. 
 
 agitating with chloroform the colouring matter is taken 
 up and can be obtained on evaporation. Jaffe's method 
 is not suitable for the detection of traces of indigogen, 
 as the colouring matter is destroyed by the least excess 
 of the oxidising agent. Hence MacMunn boils the 
 urine with an equal measure of hydrochloric acid and 
 a few drops of nitric acid, cools, and agitates with 
 chloroform. The chloroform is generally coloured 
 violet, and, when examined in the spectroscope, shows 
 two broad absorption-bands, one on either side of the 
 D line. The less refrangible is due to indigo-blue 
 and the more refrangible to indigo-red ; though it is 
 doubtful if the latter colouring matter is identical 
 with the indirubin which occurs in commercial indigo. 1 
 A. C. Mehu (Jour. Pharm., [5], vii. 122) adds to 
 the urine about 0"5 c.c. of strong sulphuric acid to 1 
 litre of the sample, and then saturates the liquid with 
 powdered ammonium sulphate, whereby any indigotin 
 or indirubin is precipitated. 2 On treating the pre- 
 cipitate in the cold with proof-spirit the indirubin 
 will be dissolved, while the insoluble indigotin is 
 purified by washing with water, followed by spon- 
 taneous drying. Mehu proposes a colorimetric process 
 for the estimation of indigotin, which he dissolves in 
 
 1 For the detection of indirubin, 0. Rosenbach (Jour. Chem. Soc, 
 lviii. 1032) adds nitric acid to the boiling urine, cools, adds a large excess 
 of ammonia, and agitates with ether, which will acquire a purple colour if 
 indirubin be present. For its isolation, Rosenbach treats the fresh urine with 
 lead acetate, heats the filtered liquid to boiling, and adds nitric acid, drop by 
 drop, until a purple colour is produced, carefully avoiding excess of acid. 
 The liquid is then cooled and treated with ammonia till alkaline. The 
 precipitate is filtered off, washed in succession with ammonia, dilute hydro- 
 chloric acid, and water, and then dissolved in boiling alcohol. The solution 
 deposits indigo-blue on cooling. It is filtered and the filtrate treated with 
 alcoholic lead acetate, again filtered, and most of the alcohol boiled off. On 
 diluting the residual liquid with water, impure indirubin is precipitated as a 
 brown powder, which, after washing with water, may be purified by crystallisa- 
 tion from chloroform or ether. 
 
 2 Apparently, Menu's method is intended to apply to ready-formed indigotin 
 and indirubin, but in Michailoff s process it appears to be the indigogens which 
 are precipitated by ammonium sulphate. 
 
INDOXYL-SULPHURIC A(JID. 201 
 
 hot carbolic acid, to which sufficient glycerin or 
 absolute alcohol has been added to prevent crystal- 
 lisation on cooling. The colour of a solution of 
 indigo-blue of known strength, prepared in this 
 manner, is compared with that of the urinary 
 pigment. 
 
 W. Michailoff (Jour. Ch&m. Soc., liv. 880) also 
 saturates the acidified urine with finely powdered 
 ammonium sulphate, and then extracts the urobilin by 
 repeated agitations with ethyl acetate (acetic ether). 
 The aqueous layer is next mixed with an equal measure 
 of fuming hydrochloric acid, chloroform added, and then 
 cautiously treated with dilute bromine- water, agitating 
 well between each addition. By presenting the indigo 
 with the solvent when in the nascent state its extraction 
 is said to be very readily and perfectly effected. 1 
 
 Indoxyl-sulphuric acid occurs in very small quanti- | 
 ties in normal human urine, Jaf f e finding from j 
 "004 to 0"019 gramme in 1500 c.c. of the excretion. 
 Horse's urine contains twenty-three times as much. 2 
 The proportion in human urine is much increased 
 in certain diseases, such as cholera, typhus, periton- 
 itis, dysentery, and Addison's disease. In obstructive 
 diseases of the small intestine the increase is enor- 
 mous. The presence of a large amount of indigogens 
 in the urine generally implies that abundant albumin- 
 ous putrefaction is in progress in some part of the 
 system, these putrefactive products being absorbed 
 and eliminated by the kidneys in the forms of 
 iudoxyl-sulphuric acid and its analogue 
 
 1 All the oxidising agents mentioned in the test are liable to destroy the 
 indigo-blue if used in excess. A preferable plan is to employ ferric chloride in 
 presence of hydrochloric acid. 
 
 From 25 litres of normal dogs' urine, J. Hoppe-Seyler (Jour. Chmn. 
 Soc, xlvi. 1058) isolated 1 gramme of crystallised potassium indoxyl-sulphate 
 and 0'5 gramme of potassium phenyl-sulphate. Neither orthocinnamic acid, 
 orthoamidocinnamic acid, or orthonitrobenzaldehyde, alone or with acetone, 
 produced any increase in the quantity of indigogens excreted. 
 
202 INDOXYL-GLYCURONIC ACID. 
 
 skatoxyl-sulphuric acid, C 8 H 6 C(H 3 )N.S0 4 H. 
 The latter body is also found in sweat, and is said 
 to be somewhat more abundant in human urine than 
 the indoxyl-compound. When decomposed by hydro- 
 chloric acid or an oxidising agent, it gives a colour- 
 ing matter usually reddish, but which may possess a 
 marked purple tint. 
 
 Traces of compounds of indoxyl and skatoxyl with 
 glycuronic acid (page 37) not improbably exist 
 in normal urine, and their proportions appear to be 
 greatly increased under certain conditions. 
 
APPENDIX. 
 
 Weights and Measures. 
 
 English Weights. 
 
 1 grain, gr. 
 
 1 ounce, oz. = 43 7 '5 grains. 
 
 1 pound, lb. = 16 ounces = 7000 grains. 
 
 English Measures op Capacity. 
 
 1 minim (min.) =0*91146 grain-measure. 
 
 1 fluid drachm (fl. drm). = 60 minims. 
 1 fluid ounce (fl. oz.) = 8 fluid drachms. 
 1 pint = 20 fluid ounces. 
 
 1 gallon = 8 pints. 
 
 The term fluid grain is not official, but is sometimes used to 
 denote the volume occupied by 1 grain weight of distilled water. 
 
 Metric System of Weights and Measures. 
 
 The basis of the metric system is the length of one ten-millionth 
 of a quadrant of a meridian of the earth's surface, passing through 
 Paris. This length is called a metre, and is equal to 39"37079 
 inches, or very nearly the length of a pendulum vibrating seconds. 
 
 The sub-divisions of the metre are the decimetre ( = 3 - 937 inches), 
 the centimetre (0 - 3937 inch), and the millimetre ( = 0'03937 inch). 
 A gramme is the weight of water which occupies, at 4° C, a 
 cube measuring 1 centimetre in the side. 
 
 1 milligramme = j^j^ part of a gramme, or 0-001 grm. 
 1 kilogramme = 1000 grammes = the weight of 1000 c.c. or 
 1 litre of water at 4° C. 
 
204 
 
 WEIGHTS AND MEASURES. 
 
 Eelation op English Weights to Metric Weights. 
 
 0-0648 gramme or 64 - 8 milligrammes. 
 „ 129-58 
 „ 194-37 
 „ 259-16 
 „ 323-95 
 „ 388-74 
 ' „ 453-53 
 „ 518-32 
 „ 583 11 
 „ 648-0 
 437-5 grains = 28-3495 grammes. 
 1 pound = 7000 grains, = 453-5927 „ 
 
 1 grain 
 
 = 
 
 0-0648 
 
 o 
 
 
 = 
 
 0-1296 
 
 3 , 
 
 
 = 
 
 0-1944 
 
 4 , 
 
 
 = 
 
 0-2592 
 
 5 , 
 
 
 = 
 
 0-3239 
 
 6 , 
 
 
 = 
 
 0-3887 
 
 7 , 
 
 
 = 
 
 0-4535 
 
 8 , 
 
 
 = 
 
 0-5183 
 
 9 , 
 
 
 = 
 
 0-5831 
 
 10 , 
 
 
 = 
 
 0-648 
 
 1 OUl 
 
 ice 
 
 = 
 
 437-5 gra 
 
 Relation of 
 
 Metric Weight 
 
 s to Eng 
 
 1 milligramme = 0-01543 
 
 g 
 
 ain. 
 
 1 gramme 
 
 = 15-4323 
 
 
 ti 
 
 •> 
 
 - 1 j) 
 
 = 30-8647 
 
 
 >) 
 
 3 „ 
 
 = 46-2970 
 
 
 5J 
 
 * „ 
 
 = 61-7294 
 
 
 )) 
 
 5 „ 
 
 = 77-1617 
 
 
 )) 
 
 6 „ 
 
 = 92-5941 
 
 
 J» 
 
 7 „ 
 
 = 108-0264 
 
 
 5) 
 
 8 .. 
 
 = 123-4588 
 
 
 JJ 
 
 9 „ 
 
 = 138-8911 
 
 
 )) 
 
 10 „ 
 
 = 154-3234 
 
 
 >> 
 
 1 kilogramme = 15432-349 
 
 grains = 2 1 
 
 >s>. 3 oz. 1198 grains. 
 
 Relation op English Measures to Metric Measures. 
 
 1 minim = 0-05916 cubic centimetre. 
 
 1 fluid drachm = 3"5495 „ ,, 
 
 1 fluid ounce = 28-396 ,. „ 
 
 1 pint = 567-92 
 
 1 gallon 
 
 = 4543-36 
 
 or 4'5434 litres. 
 
WEIGHTS AND MEASURES. 
 
 205 
 
 Relation op Metric Measures to English Measures. 
 
 1 cubic centimetre = 16-9034 minims, 15-432 grain-measures, or 
 
 0-28172 fluid drachm. 
 1 litre = 1000 c.c. = 281 "72 fluid drachms or 35-2154 fluid ounces. 
 
 Relation of English Measures to English Weights. 
 
 1 minim is the measure of 0-91146 grain of water at 60° F. 
 
 1 grain-measure (" fluid-grain ") is the volume occupied by 1 grain 
 
 of water at 60° F. 
 1 fluid drachm is the measure of 54-6875 grains of water. 
 1 fluid ounce „ ,, 437-5 „ or 1 ounce of water. 
 
 1 pint „ „ 8750-0 ,, or 1 -25 pound of water. 
 
 1 gallon „ „ 70000-0 „ or 10 pounds „ 
 
 Conversions. 
 
 To convert grammes per 100 c.c. of liquid into grains per gallon, 
 multiply by 700. 
 
 To convert grammes per 100 r.e. of liquid to grains per fluid 
 ounce, multiply by 4-375. 
 
 To convert grammes per litre of liquid into grains per gallon, 
 m u 1 1 i p 1 y by 70. 
 
 To convert grains per gallon of liquid into grammes per litre, 
 divide by 70. 
 
 Relations of Thermometric Degrees. 
 
 To convert degrees Fahrenheit into degrees Centi- 
 grade, subtract 32, multiply the remainder by 5, and divide the 
 product by 9 ; or C = (F - 32) x 5 -=- 9. 
 
 To convert degrees Centigrade into degrees Fahren- 
 heit, multiply by 9, divide by 5 and add 32 ; or F. = f C + 32. 
 
 Tensions of Aqueous Vapour in Millimetres 
 of Mercury. 
 
 °C. mm. 
 
 °C. mm - 
 
 °C. 
 
 10 = 9-126 
 
 14 = 11-882 
 
 18 
 
 11 = 9-751 
 
 15 = 12-677 
 
 19 
 
 12 = 10-421 
 
 16 = 13-519 
 
 20 
 
 13 = 11-130 
 
 17 = 14-409 
 
 21 
 
 mm. 
 15-351 
 16-345 
 17-396 
 18-509 
 
 "C. 
 
 
 mm. 
 
 22 
 
 = 
 
 19-675 
 
 23 
 
 = 
 
 20-909 
 
 24 
 
 = 
 
 22-211 
 
 25 
 
 = 
 
 23-582 
 
206 
 
 NORMAL SOLUTIONS. 
 
 Symbols and 
 
 Element. 
 
 Symbol. 
 
 Barium, 
 
 Ba 
 
 Bromine, 
 
 Br 
 
 Calcium, 
 
 Ca 
 
 Carbon, 
 
 C 
 
 Chlorine, 
 
 CI 
 
 Copper, 
 
 Cu 
 
 Gold, 
 
 Au 
 
 Hydrogen, 
 
 H 
 
 Iodine, 
 
 I 
 
 Iron, 
 
 Fe 
 
 Lead, 
 
 Pb 
 
 Lithium, 
 
 Li 
 
 Combining Weights of Elements. 
 
 Element. 
 
 Symbol. 
 
 Combining 
 Weight. 
 
 Magnesium, 
 
 Mg 
 
 12 
 
 Manganese, 
 
 Mn 
 
 55 
 
 Mercury, 
 
 Hg 
 
 200 
 
 Nitrogen, 
 
 N 
 
 14 - 
 
 Oxygen, 
 
 
 
 16 
 
 Phosphorus, 
 
 P 
 
 31 
 
 Platinum, 
 
 Pt 
 
 194 
 
 Potassium, 
 
 K 
 
 39-1 
 
 Silver, 
 
 Ag 
 
 108 
 
 Sodium, 
 
 Na 
 
 23 
 
 Sulphur, 
 
 S 
 
 32 
 
 Zinc, 
 
 Zn 
 
 65 
 
 Combining 
 
 Weight. 
 
 137 
 
 80 
 
 40 
 
 12 
 
 35-5 
 
 63 
 
 196-5 
 
 1 
 
 127 
 
 56 
 
 207 
 
 7 
 
 The combining weights in the above table are in most cases the nearest 
 whole numbers, compared with oxygen as 16. The figures are sufficiently exact 
 for the purposes of this work, but make no pretence to rigidly accurate 
 expression. 
 
 Normal and Standard Solutions. 
 
 Anormal solution is one containing in 1000 cubic centi- 
 metres ( = 1 litre) such an amount of its active constituent as 
 will combine with, replace, or oxidise 1 gramme of hydrogen. 
 Hence normal (expressed ?) solutions of the following substances 
 have the strengths given on next page. 
 
 A normal solution may also be described as one containing 
 the hydrogen-equivalent in grammes of the essential substance, 
 with the addition of sufficient distilled water to make up the final 
 volume at 60° F. ( = 15-5° C.) to 1 litre ( = 1000 c.c). 
 
 Decinormal solutions are -^ of the strength of normal 
 solutions, and are expressed thus, 5. 
 
 Centinormal solutions are T ^ 7S of the strength of normal 
 solutions, and are expressed thus, iL 
 
STANDAKD SOLUTIONS. 207 
 
 Grammes 
 per Litre. 
 
 Normal caustic soda contains Na = 23-0 
 
 NaOH = 40-0 
 
 „ potash „ KOH = 56-1 
 
 „ sodium carbonate „ — \ — 3 = 5 3 
 
 „ hydrochloric acid „ HC1 = 36-5 
 
 ,, sulphuric acid „ 2 4 = 49-0 
 
 -j 
 
 „ oxalic acid „ IA9j^H 2 q = ^ 
 
 Decinormal silver nitrate „ ° 3 = 17-0 
 
 KMnO, „„„„ 
 
 ,, potassium permanganate „ -= — ^n = 3162 
 
 Liquor Potassm and Liquor Sodce, B.P., are approximately normal 
 solutions, the former containing 61 -8 grammes of KHO and the 
 latter 43 grammes of NaHO in 1 litre. 
 
 Other volumetric solutions of definite strength, but not falling 
 within the above classification, are often very convenient for special 
 purposes. These standard solutions are usually designed to measure 
 one particular substance, and are generally prepared so that each 
 cubic centimetre will react with - 001 or O010 gramme of the 
 substance to be measured. Thus, if 4*789 grammes of silver nitrate 
 be dissolved in distilled water, and the solution diluted with distilled 
 water to exactly 1 litre ( = 1000 c.c.) at 60° F., each 1 c.c. of the 
 solution will exactly precipitate - 001 gramme of chlorine. 
 
 These standard solutions are simply reciprocals of normal solu- 
 tions ; that is, they have the strength .of normal solutions divided 
 by the hydrogen-equivalent of the substance to be measured. 
 Thus, 1 c.c. of normal silver nitrate solution precipitates 35 - 5 milli- 
 grammes of chlorine ; but if diluted 35 - 5 times it will give a 
 standard solution 1 c.c. of which will precipitate 1 milligramme of 
 chlorine. Such solutions are of great utility in the practical work 
 of the laboratory. 
 
 A good example Of the application of such a standard solution 
 is afforded by the method employed for determining the proportion 
 of chlorides in urine. Direct precipitation of the sample by silver 
 nitrate is not applicable, since much organic matter is thrown down 
 together with the silver chloride. The simplest process generally 
 
208 DETERMINATION OF CHLORIDES. 
 
 applicable is to evaporate 20 c.c. of the urine to dryness in platinum 
 with 3 or 4 grammes of nitre (potassium nitrate) free from 
 chlorides. On gently heating the residue, the organic matter is 
 oxidised by the oxygen of the nitre, and on raising the temperature 
 to incipient redness complete combustion of the carbonaceous matter 
 results, and a perfectly white product is obtained. This, when 
 cold, is treated with hot water, the solution acidulated with nitric 
 acid, a little prepared chalk added, and the whole thoroughly agitated 
 till neutral to litmus. The liquid is then diluted to 100 c.c, and 
 passed through a dry filter. Fifty c.c. of the filtrate ( = 10 c.c. of 
 the original urine) should then be placed in a porcelain basin and two 
 drops of a saturated solution of neutral potassium chromate added. 
 A standard solution of silver nitrate containing 4 - 789 grammes of 
 pure AgN0 3 per litre is then gradually added, with constant 
 stirring, until the lemon-yellow colour of the contents of the basin 
 changes to reddish-yellow. This point indicates the conversion of 
 the whole of the chlorides present into white silver chloride, AgCl, 
 and the commencement of the formation of the red silver chromate 
 Ag 2 Cr0 4 . Every 1 c.c. of the silver solution used represents 0-001 , 
 gramme of chlorine in the 10 c.c. of urine employed ( = - 01 gramme 
 per 100 c.c). Hence if 37 c.c be required, the urine contains 0*37 
 per cent, of chlorine, which figure, multiplied by 4'375, equals T62 
 grains of chlorine per fluid ounce. 
 
 The chlorine contained in the urine is largely dependent on the 
 quantity of common salt taken with the food, but a portion of it 
 is derived from chlorides of potassium and sodium naturally present 
 in the food. The chlorine found can be calculated into its equivalent 
 of common salt by multiplying it by the factor 1 -648 (or, approxi- 
 mately, by dividing it by 0-6). Bromides and iodides, which are 
 not natural constituents of urine but appear after administration of 
 medicines containing them, react like chlorides with silver nitrate. 
 In cases of pneumonia, the chlorides almost entirely disappear 
 from the urine, while the sputum contains an excessive amount. 
 
 ERRATUM. 
 
 Page 90, line 2, after the word " be " insert the words "treated with caustic 
 soda and." 
 
INDEX. 
 
 Aceto-acetic acid, 97. 
 
 ether, 97. 
 
 Acetone, 91. 
 
 detection of, in urine, 92. 
 
 Acetone-carboxylic acid, 97. 
 Acetonuria, 90. 
 Acidity of urine, 10. 
 Adenine, 162. 
 Albumin, 101. 
 — acid-, 101, 103. 
 
 alkali-, 101, 103. 
 
 Almen's reagent for, 117. 
 
 coagulated, 102. 
 
 detection of, in urine, 105. 
 
 determination of, 116. 
 
 — - ferrocyanide test for, 108. 
 
 - — - heat test for, 105. 
 
 metaphosphoric acid test for, 
 
 113. 
 
 nitric acid test for, 107. 
 
 relative delicacy of tests for, 
 
 115. 
 separation of, from globulin, 
 
 118. 
 
 Spiegler's reagent for, 113. 
 
 Tanret's reagent for, 114. 
 
 Albuminates, 101. 
 Albuminous urine, 101. 
 Albuminuria, 102. 
 Albumoses, 101, 103, 120. 
 
 separation of, 121. 
 
 Alkaline picric acid as a test for 
 
 glucose, 53, 79. 
 Alkalinity of urine, 10. 
 Allantoin, 165. 
 Alloxan, 165. 
 Alloxantin, 165. 
 Ammoniacal cupric solution, 72. 
 
 . Pavy's, 67. 
 
 Animal gum, 31. 
 
 Antipeptones, 102. 
 Appearance of urine, 7. 
 Appendix, 203. 
 
 Basic lead acetate as a precipitant, 
 
 53. 
 Benzoic acid, 187. 
 
 Benzoyl chloride test for glucose, 90. 
 Bile-pigments in urine, 193. 
 Bismuth oxide, reduction by glucose, 
 
 51, 77. 
 Blood pigments in urine, 196. 
 Bbttger's test for glucose, 77. 
 Bright's disease, 102. 
 
 Caffeine, 162. 
 
 Cane-sugar in urine, 31. 
 
 Carbohydrates, 21. 
 
 of urine, 21. 
 
 Chlorides in urine, 3, 4, 207. 
 
 Colour of urine, 7. 
 
 Composition of urine, general, 1. 
 
 variation in, 5. 
 
 Constituents, urinary, 2. 
 
 action of oxidising agents 
 
 on, 53. 
 
 nitrogenous, 126. 
 
 precipitation of, 53. 
 
 Copper solutions, reaction of glucose 
 with, 56. 
 
 Creatine, 152. 
 
 Creatinine, 152. 
 
 determination of, 158. 
 
 isolation of, from urine, 153. 
 
 — • — reactions of, 156. 
 
 salts of, 155. 
 
 Crismer's safraniue test for glucose, 
 83. 
 
 Cupric acetate, action on urinary con- 
 stituents, 53. 
 
 O 
 
210 
 
 INDEX. 
 
 Cupric oxide, reduction by glucose, 
 50. 
 
 salts, reduction to cuprous, 57. 
 
 Cyano-cupric process, Gerrard's, 74. 
 
 Deuteko-albumose or deutero-pro- 
 
 teose, 123. 
 Dextrin, 81. 
 
 Dextro-glucose, see Dextrose. 
 Dextrose, 22 (see also Glucose). 
 
 isolation of, 42, 43. 
 
 1 optical activity of, 27, 43. 
 
 Diabetes Insipidus, 15. 
 Diabetes Mellitus, 14. 
 Diabetic urine, typical, 15. 
 
 sugar in, 16, 71. 
 
 Diacetic acid, 97. 
 ether, 97. 
 
 Egg-albumin, 101. 
 Esbaeh's albumin test, 111. 
 
 tube, improved, 112. 
 
 Ethereal salts in urine, 6. 
 Ethyl aceto-acetate, 97. 
 Euxanthic acid, 38. 
 Euxanthone, 38. 
 
 Fehling's solution, action on urinary 
 constituents of, 53. 
 
 action of, on glucose, 60. 
 
 — oxidising power of, 67. 
 
 preparation of, 59. 
 
 reduction of, 59. 
 
 • volumetric use of, 66. 
 
 Fehling's test, modified, 62. 
 Eerricyanides, reduction by glucose, 
 
 51. 
 Ferrocyanide test for albumin, 108. 
 Fletcher's automatic stopper, 75. 
 
 Gases in urine, 2. 
 
 Gerrard's cyano-cupric process, 76. 
 
 ureometer, 142. 
 
 Globulins, see Paraglobulin. 
 
 Gluconic acid, 27. 
 
 Glucose, bismuth test for, 77. 
 
 Fehling's test for, 58. 
 
 modified, 62. 
 
 fermentation of, 42, 48. 
 
 optical determination of, 66. 
 
 Glucose, reaction of, with benzoyl 
 chloride, 90. 
 
 copper solutions, 56. 
 
 mercuric compounds, 
 
 78. 
 
 organic colouring mat- 
 ters, 78. 
 
 phenyl-hydrazine, 85. 
 
 reducing action of, 43. 
 
 titration of, by Pavy's solution, 
 
 66. 
 
 varieties of, 21. 
 
 Glycerol-cupric solutions, 72. 
 
 Glycogen, 32. . 
 
 formation of, in the liver, 34. 
 
 Glycosuria, 14. 
 
 Glycosuric acid, 41. 
 
 Glycuroni5 acid, 37. 
 
 Gmelin's test for bile pigments, 194. 
 
 Gravity of urine, 11. 
 
 Grape sugar, 22. 
 
 Guanine, 162. 
 
 Gulonic acid, 40. 
 
 HffiMATOPOEPHORIN, 169, 197. 
 
 Hsemin test for blood pigments, 196. 
 
 Htemoglobin, 103. 
 
 Heller's test for blood pigments, 196. 
 
 Hemipeptones, 102. 
 
 Heteroxanthine, 162. 
 
 Hippuric acid, 183. 
 
 detection of, 187. 
 
 in urine, 184. 
 
 isolation of, 188. 
 
 Horse-flesh, detection of, 33. 
 Hydroxybutyric acid, 99. 
 Hypobromite solution, 141. 
 Hypoxanthine, 162. 
 
 Indian-yellow, 38. 
 Indigo-blue, reduction of, 51. 
 Indoxyl-glycuronic acid, 38, 202. 
 
 -sulphuric acid, 199, 201. 
 
 detection of, 199. 
 
 Inorganic compounds in urine, 1. 
 Inosite, 35. 
 
 Interfering substances in urine, 70. 
 Isolation of sugar from urine, 42, 43. 
 
 Knapp's mercuric cyanide solution, 
 53, 78. 
 
INDEX. 
 
 211 
 
 Lactose in urine, 29. 
 
 Lsevo-glucose, see Laevulose. 
 
 Lsevulose, 26, 28. 
 
 Lardaoein, 102. 
 
 Lead acetate, precipitation by, 53. 
 
 Liquor Potasstx, B.P., 207. 
 
 Maltose, 31. 
 
 Mercuric acetate, precipitation by, 53, 
 55. 
 
 chloride, precipitation by, 54. 
 
 compounds, reduction of, by 
 
 glucose, 51, 78. 
 Metaphosphoric acid as a test for 
 
 albumin, 113. 
 Methylene-blue, reduction of, by 
 
 glucose, 51, 81. 
 Milk-sugar, 29. 
 Mcmo-saccharids, 21. 
 Moore's test for glucose, 24. 
 Mucin, 103, 124. 
 
 . constitution of, 125. 
 
 separation from pus, 125. 
 
 Nessler's mercuric iodide solution, 
 
 53. 
 Nitric acid test for albumin, 107. 
 Nitrogen, total, in normal urine, 4. 
 
 determination of, 127. 
 
 Nitrometers, 144. 
 
 Normal urine, albumin in, 102. 
 
 analyses of, 4. 
 
 mucin in, 124. 
 
 sugar in, 17. 
 
 traces of sugar in, 17. 
 
 urea in, 133. 
 
 uric acid in, 160. 
 
 Nylander's bismuth solution, 53, 77. 
 
 Odotte of urine, 8. 
 Orthonitrophenyl-propiolic acid, 51. 
 Oxidising agents, action of, on urin- 
 ary constituents, 53. 
 
 Paraglobulin, 103, 111, 118. 
 
 ■ separation from albumin, 118. 
 
 Paraxanthine, 162. 
 Pavy's solution, 53. 
 
 oxidising value of, 67. 
 
 titration by, 66. 
 
 Peptones, 101, 102, 103, 122. 
 
 distinction from proteoses, 122. 
 
 tests for, 123. 
 
 • volumetric determination of, 
 
 124. 
 
 Phenyl-hydrazine hydrochloride, 86. 
 
 reaction of sugars with, 85. 
 
 Phenyl-osazones, 85. 
 
 Phloridzin diabetes, 35. 
 
 Picramic acid, 79. 
 
 Picric acid test for albumin, 110. 
 
 glucose, 51, 78. 
 
 Picro-saccharometer, Johnson's, 80. 
 
 Piuri or Purrie, 38. 
 
 Polarimeters, 50. 
 
 Polyuria, 15. 
 
 Potassio-mercuric cyanide, 78. 
 
 — — iodide as a test for albu- 
 min, 114. 
 
 Potassium indoxyl-sulphate, 199. 
 
 Preliminary examination of urine, 7. 
 
 Propeptoues, 103. 
 
 Proteids, classification of, 101. 
 
 insoluble, 102. 
 
 reactions of, 103. 
 
 tests for, 102. 
 
 urinary, 103. 
 
 Proteoses, 101, 103, 120. 
 
 separation of, 121. 
 
 Qttadri-urates, 173. 
 Reaction of urine, 9. 
 
 Saccharic acid, 24, 40. 
 Sacchse's mercuric solution, 53, 78. 
 Safranine test for glucose, 51, 83. 
 Salicyl-sulphonic acid, 112. 
 Sediments, urinary, 8. 
 Serum-albumin, 101, 103. 
 
 globulin, 101, 103. 
 
 Skatoxyl-glyeuronic acid, 38. 
 Spiegler's test for albumin, 113. 
 Sucrose in urine, 31. 
 Sugar in urine, 17, 42. 
 
 diabetic, see Glucose. 
 
 Braun's test for, 79. 
 
 Sulphates in urine, 6. 
 Syntonin, 101, 103. 
 
 Tanret's reagent for albumin, 114. 
 
212 
 
 INDEX. 
 
 Taste of urine, 9, 
 Theobromine, 162. 
 Theophylline, 162. 
 Thermo-hydrometer, Fletcher's, 12. 
 Torula Ureas, 10. 
 Trichloracetic acid, 112. 
 Trammer's test for glucose, 58. 
 
 Urates, 173. 
 
 acid, 173, 177, 179, 180. 
 
 M-, 176. 
 
 characters of, 181. 
 
 normal, 173, 181. 
 
 quadri-, 173. 
 
 TJrea or carbamide, 133. 
 
 compound of, with NaCl, 137. 
 
 HgO, 138. 
 
 decreased excretion of, 151. 
 
 determination of, 138. 
 
 increased excretion of, 151. 
 
 nitrate, 135. 
 
 oxalate, 136. 
 
 phosphate, 137. 
 
 reactions of, 134. 
 
 Uric acid, 160. 
 
 amount of, in urine, 182. 
 
 characters of, 164. 
 
 detection of, 165. 
 
 — determination of, 167. 
 
 Urinary constituents, behaviour of, 
 with reagents, 53. 
 
 specific gravities of, 12. 
 
 indigogens, 199, 
 
 pigments, 193. 
 
 sediments, 8. 
 
 Urine, aceto-acetic acid in, 98. 
 
 acetone in, 96. 
 
 acidity of, 10. 
 
 albuminous, 101. 
 
 alkalinity of, 10. 
 
 appearance of, 7. 
 
 average volume of, 3. 
 
 carbohydrates of, 21. 
 
 chlorides in, 3, 4, 207. 
 
 clarification of, 54. 
 
 Urine, creatinine in, 152. 
 
 colour of, 7. 
 
 colouring matters of, 189. 
 
 composition of normal, 4. 
 
 constituents of, 2. 
 
 dextrose in, 23. 
 
 diabetic, characters of, 14. 
 
 ethereal salts in, 6. 
 
 gases in, 2. 
 
 general composition of, 1. 
 
 inorganic compounds in, 1. 
 
 lsevulose in, 28. 
 
 milk-sugar in, 29. 
 
 — — • mucin in, 124. 
 
 odour of, 8. 
 
 preliminary examination of, 
 
 reaction of, 9. 
 
 specific gravity of, 11. 
 
 sugar in, 17, 42. 
 
 sulphates in, 6. 
 
 taste of, 9. 
 
 total nitrogen in normal, 4. 
 
 total solids of, 1 1. 
 
 uric acid in, 160. 
 
 variation in composition 
 
 5. 
 Urobilin, 191. 
 
 isolation of, from urine, 192 
 
 Uro-chloralic acid, 38. 
 
 -glycuronic acid, 38. 
 
 Urohaematoporphorin, 197. 
 
 VoLtfME of urine, average, 3. 
 
 "Wedinski's glucose test, 90. 
 Wender's reagent for glucose, 53. 
 "White-indigo, formation of, 51. 
 
 Xanthine, 162, 169. 
 
 hetero-, 162. 
 
 hypo-, 162. 
 
 para-, 162. 
 
 Zinc salt of creatinine, 155, 159. 
 Zouchlos' albumin test, 110. 
 
 of, 
 
 NEILL AND COMPANY, PRINTERS, EDINBURGH. 
 
No. 3. 
 
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12 
 
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INDEX TO J. & A. CHURCHILL'S LIST. 
 
 Adams (W.) on Clubfoot, 9 
 
 on Contractions of the Fingers, &c, 9 
 
 ■■ on Curvature of the Spine, 9 
 
 Allen's Chemistry of Urine, 12 
 
 Commercial Organic Analysis, 53 
 
 Armatage*s Veterinary Pocket Remembrancer, 14 
 Barnes (R.) on Obstetric Operations, 3 
 
 on Diseases of Women, 3 
 
 Beale (L. S.) on Liver, 6 
 
 ■ Microscope in Medicine, 6 
 
 Slight Ailments, 6 
 
 Urinary and Renal Derangements, 12 
 
 Beale (P. T. B.) on Elementary Biology, 2 
 Beasley's Book of Prescriptions, 5 
 
 Druggists' General Receipt Book, 5 
 
 -. Pocket Formulary, 5 
 
 Bell on Sterility, 3 
 
 Bellamy's Surgical Anatomy, 1 
 
 Bentley and Trimen's Medicinal Plants, 5 
 
 Bentley's" Systematic Botany, 5 
 
 Berkart's Bronchial Asthma, 6 
 
 Bernard on Stammering, 7 
 
 Bernay's Notes on Analytical Chemistry, 1 
 
 Bigg's Short Manual of Orthopaedy, 9 
 
 Bloxam's Chemistry, 12 
 
 ■ Laboratory Teaching, 12 
 Bousfield's Photo-Micrography, 14 
 Bowlby's Injuries and Diseases of Nerves, 9 
 
 Surgical Pathology and Morbid Anatomy, 9 
 
 Brockbank on Gallstones, 8 
 
 Brodhurst's Anchylosis, 9 
 
 Curvatures, &c, of the Spine, g 
 
 Talipes Equino-Varus, 9 
 
 Bryant's Practice of Surgery, 8 
 
 Burckhardt's (E.) and Fenwick's (E. H.) Atlas of 
 
 Cystoscopy, 11 
 Burdett's Hospitals and Asylums of the World, 2 
 Butler- Smythe's Ovariotomies, 4 
 Butlin's Malignant Disease of the Larynx, 11 
 -— Operative Surgery of Malignant Disease, n 
 
 Sarcoma and Carcinoma, 11 
 
 Buzzard's Diseases of the Nervous System, 7 
 Peripheral Neuritis, 7 
 
 Simulation of Hysteria, 7 
 
 Cameron's Oils, Resins, and Varnishes, 14 
 
 Soaps and Candles, 14 
 
 Carpenter and Dallinger on the Microscope, 14 
 Carpenter's Human Physiology, 2 
 Charteris on Health Resorts, 8 
 
 Practice of Medicine, 6 
 
 Chauveau's Comparative Anatomy, 14 
 Chevers' Diseases of India, 5 
 Churchill's Face and Foot Deformities, 9 
 Clarke's Eyestrain, 10 
 
 Clouston's Lectures on Mental Diseases, 2 
 Clowes and Coleman's Quantitative Analysis, 13 
 
 ..— "Elementary Analysis, 13 
 
 Clowes' Practical Chemistry, 13 
 
 Cooky's Cyclopaedia of Practical Receipts, 13 
 
 Cooper on Syphilis, 12 
 
 Cooper and Edwards' Diseases of the Rectum, 12 
 
 Cripps' (H.) Cancer of the Rectum, 12 
 
 Diseases of the Rectum and Anus, 12 
 
 Air and Faeces in Urethra, 12 
 
 Cripps' (R. A.) Galenic Pharmacy, 4 
 Cuffs Lectures to Nurses, 4 
 Cullingworth's Manual of Nursing, 4 
 
 Short Manual for Monthly Nurses, 4 
 
 Dalby's Diseases and Injuries of the Ear, 10 
 —————Short Contributions, 10 
 
 Day on Diseases of Children, 4 
 
 on Headaches. 8 
 
 Domville's Manual for Nurses,_4 
 
 Doran's Gynaecological Operations, 3 
 
 Druitt's Surgeon's Vade-Mecum, 8 
 
 Duncan (A.), on Prevention of Disease in Tropics, 5 
 
 Duncan (J. M.), on Diseases of Women, 3 
 
 Dunglison's Dictionary of Medical Science, 12 
 
 Ellis's (E.) Diseases of Children, 4 
 
 Ellis's (T. S.) Human Foot, 9 
 
 Fagge's Principles and Practice of Medicine, 6 
 
 Fayrer's Climate and Fevers of India, 5 
 
 Natural History, &c, of Cholera, 5 
 
 Fenwick(E. H.), Electric Illumination of Bladder, n 
 
 Symptoms of Urinary Diseases, 11 
 
 Fenwick's (S.) Medical Diagnosis, 6 
 
 Obscure Diseases of the Abdomen, 9 
 
 Fenwick's (S.) Outlines of Medical Treatment, 6 
 
 The Saliva as a Test, 6 
 
 Fink's Operating for Cataract, 10 
 
 Flower's Diagrams of the Nerves, 1 
 
 Fowler's Dictionary of Practical Medicine, 6 
 
 Fox (Wilson), Atlas ofPathological Anatomy of Lungs, 6 
 
 Treatise on Diseases of the Lungs, 6 
 
 Frankland and Japp's Inorganic Chemistry, 13 
 Fraser's Operations on the Brain, 8 
 Fresenius' Qualitative Analysis, 13 
 
 Quantitative Analysis, 13 
 
 Galabin's Diseases of Women, 3 
 
 Manual of Midwifery, 3 
 
 Gardner's Bleaching, Dyeing, and Calico Printing, 14 
 
 — Brewing, Distilling, and Wine Manuf. 14 
 
 Gimlette on Myxcedema, 6 
 Glassington's Dental Materia Medica, 10 
 Godlee's Atlas of Human Anatomy, 1 
 Goodhart's Diseases of Children, 4 
 Gowers Diagnosis of Diseases of the Brain, 7 
 
 Manual of Diseases of Nervous System, 7 
 
 Clinical Lectures, 7 
 
 Medical Ophthalmoscopy, 7 
 
 Syphilis and the Nervous System, 7 
 
 Granville on Gout, 7 
 
 Green's Manual of Botany, 5 
 
 Groves' and Thorp's Chemical Technology, 14 
 
 Guy's Hospital Reports, 7 
 
 Habershon's Diseases of the Abdomen, 7 
 
 Haig's Uric Acid, 6 
 
 Haig-Brown's Elementary Physiology, 2 
 
 Harley on Diseases of the Liver, 7 
 
 Harris's (V. D.) Diseases of Chest, 6 
 
 Harrison's Urinary Organs, 11 
 
 Hartridge's Refraction of the Eye, 10 
 
 Ophthalmoscope, 10 
 
 Hawthorne's Galenical Preparations of B P. , 4 
 Heath's Certain Diseases of the Jaws, 8 
 
 Clinical Lectures on Surgical Subjects, 8 
 
 — ■ Injuries and Diseases of the Jaws, 8 
 
 Minor Surgery and Bandaging, 8 
 
 Operative Surgery, 8 
 
 , Practical Anatomy, 1 
 
 — Surgical Diagnosis, 8 
 
 Hellier's Notes on Gynaecological Nursing, 4 
 Higgens' Ophthalmic Out-patient Practice, 10 
 Hill on Cerebral Circulation, 2 
 Hillis' Leprosy in British Guiana, 10 
 Hirschfeld's Atlas of Central Nervous System, 2 
 Holden's Human Osteology, r 
 
 Landmarks, 1 
 
 Hooper's Physicians' Vade-Mecum, 5 
 
 Hovell's Diseases of the Ear, 10 
 
 Howden's Index Pathologicus, 2 
 
 Hyde's Diseases of the Skin, 10 
 
 Hyslop's Mental Physiology, 3 
 
 lmpey on Leprosy, 10 
 
 Jacobson's Male Organs of Generation, 12 
 
 Operations of Surgery, 8 
 
 Johnson's Asphyxia, 6 
 
 Medical Lectures and Essays, 6 
 
 ■ — Cholera Controversy, 6 
 
 Granular Kidney, 6 
 
 Journal of Mental Science. 3 
 
 Keyes' Genito-Urinary Organs and Syphilis, 12 
 Kohlrausch's Physical Measurements, 14 
 Lancereaux's Atlas of Pathological Anatomy, 2 
 Lane's Rheumatic Diseases, 7 
 
 Langdon-Down's Mental Affections of Childhood, 3 
 Lee's Microtomists' Vade Mecum, 14 
 Lescher's Recent Materia Medica, 4 
 Lewis (Bevan) on the Human Brain, 2 
 Liebreich's Atlas of Ophthalmoscopy, 10 
 MacMunn's Clinical Chemistry of Urine, 12 
 Macnamara's Diseases and Refraction of the Eye, 9 
 
 ; of Bones and Joints, 8 
 
 McNeill's Epidemics and Isolation Hospitals, 2 
 Malcolm's Physiology of Death, 9 
 Mapother's Papers on Dermatology, 10 
 Martin's Ambulance Lectures, 8 
 Maxwell's Terminologia Medica Polyglotta, 12 
 Maylard's Surgery of Alimentary Canal, 9 
 Mayne's Medical Vocabulary, 12 
 Mercier's Lunacy Law, 3 
 Microscopical Journal, 14 
 
 [Continued an the next page . 
 
 LONDON: 7, GREAT MARLBOROUGH STREET. 
 
Index to J. & A. Churchill's List — continued. 
 
 Mills and Rowan's Fuel and its Applications, 14 
 Moore's (N.) Pathological Anatomy of Diseases, 1 
 Moore's (Sir W. J.) Family Medicine for India, 5 
 
 Manual of the Diseases of India, s 
 
 — - — — Tropical Climates, 5 
 
 Morris's Human Anatomy, 1 
 Moullin's (Mansell) Surgery, 8 
 Nettleship's Diseases of the Eye, 9 
 Notter and Firth's Hygiene, 2 
 Ogle on Tympanites, 8 
 Oliver's Abdominal Tumours, 3 
 
 Diseases of Women, 3 
 
 Ophthalmic (Royal London) Hospital Reports, 9 
 
 Ophthalmological Society's Transactions, 9 
 
 "Qrmerod's Diseases of the Nervous System, 7 
 
 Owen's Materia Medica, 4 
 
 Parkes' (E.A.) Practical Hygiene, 2 
 
 Parkes' (L.C.) Elements of Health, 2 
 
 Pavy's Carbohydrates, 6 
 
 Pereira's Selecta e Prescriptis. 4 
 
 Phillips' Materia Medica and Therapeutics, 4 
 
 Pitt-Lewis's Insane and the Law, 3 
 
 Pollock's Histology of the Eye and Eyelids, 9 
 
 Proctor's Practical Pharmacy, 4 
 
 Purcell on Cancer, 11 
 
 Pye-Smith's Diseases of the Skin, n 
 
 Quinby's Notes on Dental Practice, 10 
 
 Ramsay's Elementary Systematic Chemistry, 13 
 
 '■ Inorganic Chemistry, 13 
 
 Reynolds' Diseases of Women, 3 
 Richardson's Mechanical Dentistry, 10 
 Roberts' (D. Lloyd) Practice of Midwifery, 3 
 Robinson's (Tom) Eczema, 1 1 
 
 Illustrations of Skin Diseases, n 
 
 Syphilis, 11 
 
 Ross's Aphasia, 7 
 
 — Diseases of the Nervous System, 7 
 
 Royle and Harley's Materia Medica, 5 
 St. Thomas's Hospital Reports, 7 
 Sansom's Valvular Disease of the Heart, 7 
 Schweinitz's (G. E. de) Diseases of Eye, 10 
 Shaw's Diseases of the Eye, q 
 Short Dictionary of Medical Terms, 12 
 Silk's Manual of Nitrous Oxide, 10 
 Smith's (E.) Clinical Studies, 4 
 
 Diseases in Children, 4 
 
 Wasting Diseases of Infants and Children, 4 
 
 Smith's (J. Greig) Abdominal Surgery, 8 
 
 Smith's (Priestley) Glaucoma, 10 
 
 Snow's Cancer and the Cancer Process, it 
 
 — ■ Palliative Treatment of Cancer, 11 
 
 Reappearance of Cancer, 11 
 
 Southall's Materia Medica, 5 
 
 Squire's (P.) Companion to the Pharmacopoeia, 4 
 Squire's (P.) London Hospitals Pharmacopoeias, 4 
 
 Methods and Formulae, 14 
 
 Starling's Elements of Human Physiology, 2 
 Sternberg's Bacteriology, 6 
 Stevenson and Murphy's Hygiene, 2 
 Sutton's (H. G.), Lectures on Pathology, 1 
 Sutton's (J. B.), General Pathology, 1 
 Sutton's (F.) Volumetric Analysis, 13 
 Swain's Surgical Emergencies, 8 
 Swayne's Obstetric Aphorisms, 3 
 Taylor's (A. S.) Medical Jurisprudence, 2 , 
 Taylor's (F.) Practice of Medicine, 6 
 Taylor's (J. C.)» Canary Islands, 8 
 Thin's Cancerous Affections, of the Skin, n 
 
 Pathology and Treatment of Ringworm, 11 
 
 Thomas's Diseases of Women, 3 
 Thompson's (Sir H.) Calculous Disease, 11 
 
 Diseases of the Prostate, n 
 
 Diseases of theUrinaryOrgans,iz 
 
 Lithotomy and Lithotrity, 11 
 
 Stricture of the Urethra, 11 
 
 Suprapubic Operation, 1 1 
 
 Surgery of theUrinaryOrgans.ti 
 
 Tumours of the Bladder, 11 
 
 Thome's Diseases of the Heart, 7 
 
 Tirard's Prescriber's Pharmacopoeia, 5 
 
 Tomes' (C. S.) Dental Anatomy, 10 
 
 Tomes' (J. and C. S.) Dental Surgery, 10 
 
 Tooth's Spinal Cord, 7 
 
 Treves and Lang's German-English Dictionary, 12 
 
 Tuke's Dictionary of Psychological Medicine, 3 
 
 Influence of the Mind upon the Body, 3 
 
 Tuson's Veterinary Pharmacopoeia, 14 
 
 Valentin and Hodgkinson's Qualitative Analysis, 13 
 
 Vintras on the Mineral Waters, &c, of France, 8 
 
 Wagner's Chemical Technology, 13 
 
 Walsham's Surgery : its Theory and Practice, 8 
 
 Waring's Indian Bazaar Medicines, 5 
 
 Practical Therapeutics, 5 
 
 Watts' Manual of Chemistry, 12 
 
 West'sfS.) How to Examine the Chest, 6 
 
 Westminster Hospital Report, 7 
 
 White's (Hale) Materia Medica, Pharmacy, &c, 4 
 
 Wilks' Diseases of the Nervous System, 7 
 
 Williams' Veterinary Medicine, 14 
 
 — . Surgery, 14 
 
 Wilson's (Sir E.) Anatomists' Vade-Mecum, 1 
 Wilson's (G.) Handbook of Hygiene, 2 
 Wolfe's Diseases and Injuries of the Eye, 9 
 Wynter and Wethered's Practical Pathology, x 
 Year-Book of Pharmacy, 5 
 Yeo's (G. F.) Manual of Physiology, 2 
 
 N.B.—f. fy A. ChurchilVs larger Catalogue of about 600 works on Anatomy, 
 Physiology, Hygiene, Midwifery, Materia Medica, Medicine, Surgery, Chemistry, 
 Botany, fyc. fyc, with a complete Index to their Subjects, for easy reference, 
 will be forwarded -post free on application. 
 
 America. — /. 8f A. Churchill being in constant communication with 
 various publishing houses in America are able to conduct negotiations 
 favottrable to English Authors. 
 
 LONDON: 7, GREAT MARLBOROUGH STREET,