m I EB Cornell University VB Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003266347 PRACTICAL PHYSIOLOGICAL CHEMISTRY. .1st Edition - 1904. 2nd Edition 1908. 3rd Edition 1913. 4th Edition 1914. 5th Edition - - 1919. {Completely revised and enlarged. 6th Edition 1920. Practical Physiological Chemistry. SYDNEY W. COLE, M.A. Trinity College, Cambridge. University Lecturer in Medical Chemistry, Cambridge. Lecturer in Pathological Chemistry, Charing Cross Hospital Medical School. With an INTRODUCTION by F. G. HOPKINS, M.B., D.Sc, F.R.C.P., F.R.S., Professor of Biochemistry ; Fellow and PrEelector of Trinity College ; Hon, Fellow of Emmanuel College, Cambridge. SIXTH EDITION. BALTIMORE, U.S.A. : WILLIAMS & WILKINS COMPANY CAMBRIDGE, ENGLAND • W. HEFFER & SONS LTD. 1920 Printed in Great Britain INTRODUCTION. My colleague's book, in its earlier editions, received so hearty and so general a welcome that any personal words of recommendation seem now uncalled for. In this edition, however, it emerges as a work of a somewhat different order. Like all good books which deal with a progressive subject, it has felt the growth impulse, and, notwithstanding the exceptional nature of the times, material for growth has during the last few years accumulated in abundance. If this edition has largely out- grown its predecessors the increase is not only desirable, but, in my opinion, necessary. Progress in any science caUs continuously for new methods, and for extension and improvement in technique. In the growth of any branch of knowledge there are, indeed, periods when the development of technique becomes the most pressing of needs, and its success the best measure of progress. Biochemistry has been successfully passing through such a period. Its methods have been greatly multiphed, extended and improved. We are now beginning to reap the reward in the accumulation of accurate quantitative data. One need peculiar to biochemistry, that of following changes in living tissues without terminating the Ufe of the animal, or harming the human subject, has now been largely met, at least so far as studies of the blood are concerned. This is due to the success of micro-methods of analysis. So significant are some of the results which can be obtained that we may hope to see the methods become as general in connexion with medical diagnosis as the use of the stethoscope or the electrocardiogram. It may be good for the advanced student, possessed of leisure, to determine for himself the exact conditions necessary for success in the use of a given method. For two classes, however, it is highly desirable that success should be reached as immediately as possible : for the elementary student, in order that his faith may not be weakened, and for the research worker not specially versed in chemical technique, who, with limited time, wishes to apply a method to medical or biological problems. Each of these will be VI. INTRODUCTION. the better for descriptions which secure against failure. Such are the descriptions found in this book. Indeed, the chief satisfaction I derive from being allowed to write this foreword arises from the opportunity it gives me of bearing witness to the fact that the author has always a first-hand acquaintance with his subject matter. In connexion with the newer, and less famiUar, tests and methods the directions are not copied from elsewhere, not even (when they are due to others) from the descriptions of their originators. They have been written at the laboratory bench, step by step with the successful accomplishment of the process they describe. When success has seemed doubtful, or too difficult of attainment, the method has found, no place in the book. Older methods have often been modified in detail, as a result of long experience of their use in practical classes. On the other hand not a few of the processes described are original. It is indeed a pleasant duty to emphasise the fact that the book is used as a mediimi for the pubhcation of a considerable amount of patient research work requiring abUities of a special order. It is to be trusted that this work wiU receive the same degree of recognition that it undoubtedly would if it were pubhshed through the more ordinary chaimels of the scientific periodicals. Some of the sections are intended for purposes wider than that of class instruction alone. In the chapter on the preparation of the amino-acids for instance, Mr. Cole has drawn on the collective experience of many workers in my laboratory. The descriptions are unique in their wealth of detail, and I feel confident that the preparation of these compounds by the methods described can be undertaken with every prospect of success by all workers. A supply of pure amino-acids is so important for the prosecution of many lines of research that the inclusion of the chapter will be welcomed by many who have been disappointed at the results of their previous attempts. Of my own Imowledge I can testify to the success that has attended the preparation of histidine and tryptophane, for example, by junior laboratory attendants following the descriptions here published. In my experience the teaching of practical biochemistry to students of ph3'siology presents a difficulty less felt in the practical teaching of the other branches of the science. A successful histo- INTRODUCTION. Vll. logical preparation, or the neat accomplishment of a graphic record, yields immediate satisfaction to a student biologically inclined. He is dealing directly with the animal, and the result seems an end in itself. It is otherwise with tests and estimations carried out as mere exercises. The interest of a quantitative result, which may be great when it is obtained during an actual study of metabolism, seems remote to the student who works without any such stimulus. Yet in large chemical classes it is almost impossible to provide closer touch with the animal, and interest cannot always be secured by maintaining an exact correspondence in the sequence of lectures and classes. It is desirable therefore that, even in a book with aims that are avowedly practical, there should be some judicious reference to theory and to the actual significance of results. In the present work this end seems to be reached without undue consumption of space. The book in its present form, while very fully covering the ground required by the medical student, can be profitably used by aU who seek for accurate and fuU descriptions of biochemical methods, whether for use in medical diagnosis or in biological researches. The earlier editions of the book have been used by the students at the Agricultural Laboratory here, and in its present form it would appear to be highly suited to the needs of those engaged in the study of animal nutrition. F. GOWLAND HOPKINS. BIOCHEMICAL DEPARTMENT, CAMBRIDGE. FROM THE PREFACE TO THE FIFTH EDITION I have added a considerable amount of new material, the most important being a chapter on the properties of solutions in which particular attention is paid to the hydrogen-ion concentration and to the colloidal state : a chapter on the preparation and properties of certain of the amino-acids : on the preparation and hydrolysis of nucleic acid : sections on the as5niimetric carbon atom and on the theory of the polarimeter : on the action of intestinal bacteria on proteins : on autolysis : on the action of oxidase systems and many new quantitative methods related to enzyme action and blood, urinary and gastric analyses. A good deal of this material has not been pubUshed previously, but the methods have stood the test of routine class work at Cambridge, and it is trusted that they will not faU when tried elsewhere. With the exception of the exercises on the preparation of the amino-acids and on the hydrolysis of nucleic acid, the book represents the course in Practical Physiological Chemistry for Medical Students at Cambridge. It may be objected that the course is overburdened with analjrtical exercises. They are inserted for two reasons that seem important to my Chief, Prof. F. G. Hopkins, and to myself. In the first place they have considerable educational value : one is enabled to train the student to make accurate observations and pay attention to the effect of variations in conditions on results. Secondly, it is hoped that the student, having acquired the technique necessary to determine the course of the metabohc changes in the normal individual, will be encouraged to extend his observations to those occurring in disease. Progress in medical science is largely dependent on the statistical method. I am convinced that a con- siderable body of trained medical men making accurate anal3rses of the abundant clinical material that must inevitably come their way wiU advance our knowledge much more rapidly than a few isolated speciahsts, who are apt to confine their attention to subjects in advanced disease. It is the border-land between health and iU- health that particularly requires exhaustive investigation, and for its exploration a whole army is required. We can safely rely on the Medical Research Committee for the Staff work necessary for the proper co-ordination of the results. PREFACE TO THE SIXTH EDITION. The present edition has been carefully revised and several new methods introduced. The most important of these is my micro- method for the determination of blood sugar, which my Cambridge students have used for nearly a year with very consistent results. The- method is a modification of McLean's method which in my hands and in class work proved most unsatisfactory. It is a sub- stitute for Bang's micro-method, which, though reliable in the hands of the expert, has been found too difficult for the average student. For the estimation of blood chlorides, Van Slyke's excellent method is described in preference to Bang's micro-method. The. Soya bean method for the estimation of the urea of blood also finds a place, owing to its importance in the diagnosis of renal disease. I am very grateful to my many correspondents for criticisms and suggestions, which enable me to render the book helpful to as wide a class of worker as possible. Chemicals and apparatus of pre-war standard are still difficult to obtain, but Messrs. Baird and Tatlock, of London, are making a praiseworthy effort to maintain a stock of all the essential apparatus and reagents required for the various exercises described. I have to thank them for the loan of the blocks of several new figures. SYDNEY W. COLE. Biochemical Laboratory, Cambridge, October, 1920. CONTENTS. CHAPTER I. The Pkoperties of Solutions A. Colloids and Crystalloids B. Diffusion and Dialysis C. Osmotic Pressure D. Freezing Point E. The Electrical Properties of Colloids F. The Precipitation of Colloids G. The Concentration of Hydrogen-Ions H. Ampholytes or Amphoteric Electrolytes PAGE I I 2 3 5 9 lO 30 CHAPTER II. The Proteins . . A. Definition B. Classification . . C. General Reactions D. Colour Reactions E. The Heat Coagulation of Albumins and Globuhns F. The Properties of Albumins and Globulins G. The Chemistry of Egg-White , H. The Metaproteins I. The Albumoses or Proteoses and Peptones J. The Gluco-Proteins . . K. The Reactions of certain Albuminoids 33 33 33 35 38 42 45 49 51 52 57 58 CHAPTER III. The Nucleoproteins, Nucleins and Nucleic Acids 60 CONTENTS. CHAPTER IV. Tnp Preparation and Properties of Certain Amino- ACIDS XI PAGE 67 CHAPTER V. The Carbohydrates . . 100 A. The Monosaccharides . . . . . . . . 100 B. The Disaccharides . . . . . . . . . . 113 C. The Polysaccharides . . . . . . . . . . 117 D. The Quantitative Estimation of the Carbohydrates 125 E. The Theory and Use of the Polarimeter . . . . 143 F. Optical Activity and the As3mimetric Carbon Atom 147 CHAPTER VI. The Fats, Oils and Lipines . . 153 CHAPTER VII. The Chemistry of Some Foods A. Milk C. Cheese D. Potatoes E. Flour F. Bread G. Meat (Muscle) . . CHAPTER A mi. 166 166 172 173 173 174 175 The Composition of the Digestive Juices and the Action of Certain Enzymes A. Saliva . . B. Ptyalin C. Gastric Juice . . D. Pepsin . . 183 186 186 194 201 2XU. ^B CONTENTS. CHAPTER VIII. — continued. PAGE E. Rennin and the Clotting of Milk 207 F. Trj'psin 210 G. Erepsin 218 H. Amylopsin 220 I. Maltase, Lactase and Sucrase 221 J- Bacterial Decomposition in the Intestine. . 222 K. Autolysis 228 L. Oxidases, Peroxidases and Tyrosinase 230 CHAPTER IX. The Coagulation of the Blood . . 234 CHAPTER X. The Red Blood Corpuscles and the Blood Pigments 238 A. The Taking of Blood. . . . . . . . . . 238 B. Haemoglobin and its Derivatives . . . . . . 240 C. The Spectroscopic Examination of the Blood Pigments . . . . . . . . . . . . 243 D. Blood Constituents and their Analysis . . . . 249 CHAPTER XI. The Constituents of Bile . . 265 CHAPTER XII. Urine and its Chief Constituents. . A. The Average Composition . . B. The Physical Chemistry of the Urine C. The Pigments of Urine D. The Inorganic Constituents . . E. Urea 270 270 271 278 280 286 CONTENTS. XIU. CHAPTER XII. — continued. F. Uric Acid • • • G. Purine Bases, other than Uric Acid . . H. Creatinine and Creatine I. Ammonia J- Hippuric Acid K. Certain Constituents of Abnormal Urines L. Urinary Sediments CHAPTER XIII. The Quantitative Analysis of Urine . . A. Total Nitrogen B. Ammonia C. Ammonia and Amino-Acids. D. Urea E. Creatinine and Creatine F. Uric Acid G. Glucose H. The Acetone Bodies . . I. Chlorides J. Phosphates K. Sulphates L. Albumin M. Diastase CHAPTER > [IV. The Detection of Substances of Physiological Interest A. Fluids . . B. Solids PAGE 292 298 298 301 301 302 319 321 321 329 333 334 338 343 346 349 355 357 358 361 362 365 365 374 APPENDIX. Chart for Spectroscopic Absorption Bands Chart for Recording Urinary Analysis Weights and Measures . . 376 378 379 XIV. CONTENTS APPENDIX — contin ued. PAGE Tension of Aqueous Vapour . ■ 380 Atomic Weights . 380 Specific Gravity Tables . 381 Boiling Points . 383 Standard Acids and Alkalies . ■ 384 Pipettes and Burettes . . • 385 Fume- Absorber • 387 Colorimeters . 388 Micro-balance • 392 Preparation of Reagents • 393 Index 396 Logarithm Tables Back cover ILLUSTRATIONS AND FIGURES. FIG. 1. Beckmann's freezing point apparatus . . 2. Beckmann's thermometer 3. Arrangement of tubes in Comparator . . 4. Cole and Onslow's Comparator . . 5. Dreyer's dropping pipette 6. Paraf&ned bottle for storing standard alkali 7. Reflux condenser . . 8. Distillation in vacuo 9. Connexions of vacuum pump ... 10. Saturation with dry hydrochloric acid gas 11. Buchner funnel and filtering flask 12. Removal of hydrogen sulphide by air current 13. Distillation in vacuo with Claisen flask. . 13a. Apparatus for Benedict's method 14. Micro-burette . . 15. Apparatus for maintaining a standard heating power 16. Filtering apparatus for reduced copper. . 17. Curve of copper values for glucose 18. Curve of copper values for lactose 19. Crystal of calc spar . . . . 20. Refraction in a Nicol's prism 21. Arrangement of a simple polarimeter . . 22. Plan of a three-field polarimeter 23. Appearance of field of polarimeter 24. Model of carbon atom 25. Two superposable models 26. Model of an asymmetric carbon atom . . 27. Plane of symmetry of model 28. Meig's method of fat extraction . . 29. Automatic measuring apparatus. . PAGE 8 8 20 21 23 25 72 73 74 75 76 90 99 128 130 136 137 138 140 143 144 144 145 145 148 149 149 150 170 191 XVI. ILLUSTRATIONS AND FIGURES. FIG. 30. Zeiss' direct-vision spectroscope 31. Apparatus for constant heating . . 32. Method of drawing blood 33. Curve for Cole's blood sugar method 34. Apparatus for Micro-Kjeldahl 35. Titration in CO^- free atmosphere 36. Urinometer 37. Comparator for large tubes 38. Gauge for calibrating tubes 39. Hall's two-way tube for burettes 40. Kjeldahl apparatus: direct boiling 41. Kjeldahl apparatus : steam distillation 42. Kjeldahl apparatus : alcohol distillation 43. Apparatus for ammonia estimation (Folin) 44. Apparatus for ammonia and urea (Van Slyto 45. Flask for ammonia and urea . . 46. Apparatus for urea by hypobromite method 47. Filtering flask and pump connexion 48. Gooch crucible and apparatus . . 49. Apparatus for estimation of acetone 50. Esbach's albuminometer 51. Ostwald pipette 52. Method of reading burette 53. Apparatus for reading burette 54. Fohn's fume-absorber 55. Duboscq Colorimeter 56. Path of rays in Duboscq Colorimeter 57. Kober's Colorimeter 58. Torsion balance PAGE 243 255 255 facing 258 262 262 272 276 276 322 325 326 328 330 331 332 337 345 351 353 361 385 386 386 387 388 389 390 392 CHAPTER I. THE PROPERTIES OF SOLUTIONS. A. Colloids and Crystalloids. The condition of a substance in " solution " is one that differs considerably with different substances ; moreover, it may differ with the same substance, depending on the method of preparing the solution.. All "solutions" can be regarded as suspensions of particles in the "solvent." The size and nature of the particles cause variations in the physical properties of the solution. There are four classes of so-called "solutions," which are not, however, very sharply differentiated from one another. They are CRV^T/iT T HTD'^ [ELECTROLYTES. u/t: J- o i ^j^j^uiu::, "y^QN-ELECTROL YTES. COLLOIDS [EMULSOIDS. ^ "^ [SUSPENSOIDS. Probably the essential difference between colloids and crystalloids is the size of the particles suspended in the fluid. In the crystalloids these particles are sniall, con- sisting of ions or single, relatively small molecules. In the colloids the particles are large, either because the molecules themselves are large, or because they^tend to aggregate and form relatively large masses. The difference between emulsoids and suspensoids is probably that suspensoids are two-phase liquids in which the " solvent " (external phase) does not combine with the " solute " (internal phase), that is to say, the solute is in real suspension in the so-called solvent. In emulsoids, on the 2 THE PROPERTIES OF SOLUTIONS. [CH. I. other hand, we have two-phase hquids, each, phase con- taining both components in different concentrations. The solute is able to combine to a certain extent with' the solvent. They are intermediate between suspensions and true solu- tions. In certain cases there is evidence to show that the solute may be partially ionised. Solutions of native pro- teins, starch, dextrins, etc., are emulsoids, whereas the denaturised proteins behave more like suspensoids. Sorensen's view as to the main differences between the two types of colloids is as follows : — ■ " The suspensoids show a viscosity differing but Uttle from that of the pure external phase. There is generally a well-marked difference in electrical charge between the two phases. Only comparatively small concentrations of electrolytes are required to bring about coagulation, which is in most cases irreversible. "The emulsoids show a great viscosity and power of foam formation; the system commonly does not show any marked difference in electrical charge between the two phases. Great concentrations of electrolytes are commonly necessary to bring about coagulation, which is in most cases reversible." B. Diffusion and Dialysis. The difference between crystalloids and colloids that has been most emphasised is the disparity in the rate of diffusion of the two substances. If a solution of sodium chloride or glucose be separated from distilled water by means of a film of collodion, parchment paper, or gold beater's skin, the dissolved substance is found to pass through the membrane, the process being known as diffusion. If a colloidal solution be tested in the same way, it will be found to pass through either very slowly or not at all. In other words, the colloids are relatively indiffusible. This is sometimes employed as a convenient method of separating crystalloids from colloids, and is known as dialysis. It should be noted, however, that abrupt transitions are not common in nature, and that all emul- soids do diffuse through such membranes, though extremely slowly as compared to crystalloids. I. Preparation of collodion sacs for dialysis. A convenient size is made by use of a large boiling tube (200 x 15 mm.). Into a clean, dry tube pour about 10 cc. of the collodion solution described CH. I.] DIALYSIS. 3 below. Pour this back into the stock, revolving the tube with the mouth downwards so that an even film is left adherent to the walls of the tube. Add another portion of collodion solution and repeat as before. Allow the film to dry, so that it does not stick to the finger. When this point is reached fill the tube with cold water. Cut round the rim of the tube with a knife, pour off the water, and carefully detach the membrane from the side of the tube. Allow water to run between the sac and the glass. By means of a glass rod with a spatulate end, and by traction and twisting, the sac can usually be removed from the glass tube. Fill the sac with water. It should be perfectly transparent. A cork, bored with a large hole, can be tied into the upper end, and by means of this it can be sus- pended in a jar of distilled water. The secret of success is to fill the tube with water at a particular moment, determined by trial on each specimen of collodion. If the water be added too soon the sac is opaque and feeble. If it be added too late, it is somewhat difficult to remove the film from the glass without damage. Sacs prepared in this way are very much better than those made of parchment paper. They should be kept wet, as on drying they become porous. Note. — Preparation of Collodion Solution, To 3 gm. of gun cotton (pyroxylin) add 75 cc. of dry ether and allow to stand for 10 or 15 minutes in a flask closed with a cork. 25 cc. of absolute ethyl alcohol are then added, and the pyroxylin dissolves to a mobile fluid, which does not require filtration. It should be allowed to stand until all bubbles have disappeared. 2. Dialysis. Mix 2 per cent, starch paste (see Ex. 135) with about one-tenth of its volume of saturated ammonium sulphate solution. Place the mixture in a collodion sac and suspend this in water contained in a tall jar. Care must be taken to avoid spiUing any of the mixture into the jar. Examine the " dialysate " (the fluid in the jar) for starch by the iodine test (Ex. 136) and for am- monium sulphate by means of barium chloride at the end of half an hour. The starch test will probably be negative, whilst the sulphate test will be positive. The dialysate should also be examined for starch after 2 to 7 days. C. Osmotic Pressure. Certain membranes can be prepared which allow of the passage of water molecules, but do not allow dissolved substances to pass through them. Such membranes are 4 THE PROPERTIES OF SOLUTIONS. [CH. I. called " semi-permeable." If a dissolved substance, like glucose, be separated from water by means of a semi- permeable membrane, the sugar solution is diluted by water passing through the membrane. This process, the passage of water through a membrane into a solution, is known as " osmosis," and is to be carefully distinguished from "diffusion," the passage of a dissolved substance through a membrane. If the sugar solution be contained in a vessel connected to a manometer, and arrangements are made to keep the volume of the solution constant, it is found that the water passing into the vessel causes a rise of pressure. The final pressure reached is known as " the osmotic pressure " of the solution. It is not necessary here to enter into the theories of osmotic pressure, but it is important to note that the osmotic pressure of a solution depends on the number of particles in a given volume, no matter whether these particles be ions, molecules, or aggregates of molecules. Thus the osmotic pressure of a dilute solution of sodium chloride is nearly double that of an equimolecular solution of glucose, because in dilute solution the sodium chloride is almost completely dissociated into its constituent ions. From these considerations it follows that the osmotic pressure of a colloidal solution is extremely low in com- parison with that of a crystalloid of the same percentage, for the number of particles in a given volume of the colloidal solution is very small compared with that in the crystalloid solution. For non-electrolytes it has been shewn by Van 't Hoflf that Boyle's law for gases can be appUed to solutions, if we substitute osmotic pressure for gas pressure. It has also been found that the Law of Charles is obeyed, namely, that at constant volume the pressure varies as the absolute temperature. It follows that in dilute solute solution V.P. = R.T. ; where V = Volume ; P = Osmotic Pressure ; T = Temperature (absolute), and R = a Constant. Moreover, it has been shewn by Van 't Hoff that R in the case of osmotic pressure has the same value as in the case of gases, that is, the solution exerts the same osmotic pressure as the pressure that the dissolved substance would exert if it were gasified at the same temperature and confined in the same volume as that of the solution. It follows that one gramme-molecule of a non-electrolyte will exert an osmotic pressure at o° C. of 760 mm. of mercury when the volume of the solution is 22-4 litres. CH. I.] OSMOTIC PRESSURE. 5 If w grams of a substance be dissolved in V cc. of solvent at t° C. and the osmotic pressure produced be P mm. of mercury, the volume at o° C. and at 760 mm. mercury will be V X 273 X P (273 + t) X 760 ~ »• Now Vo contains zti grams of substance, so 22400 cc. contains 22400 X w Since this weight of substance produces a pressure of 760 mm. at 0° C. when in a volume of 22-4 litres it follows that it is the molecular weight of the substance. Instead of the formula V.P. = R.T., we must use the following for electro- lytes, V.P. = > ~ — I R.T., where i is the percentage of the substance 100 ionised. 3. Chemical garden. To a dilute aqueous solution of potassium ferrocyanide add a particle of solid ferric chloride. A film of Prussian blue is formed round the solid. This membrane is semi- permeable, and allows water to pass in to dissolve the ferric chloride. The osmotic pressure of this solution being greater than that of the solution outside, Water passes into the cell, which expands, and may assume remarkable forms. 4. A drop of a fairly strong solution of potassium ferrocyanide is added to a dilute solution of copper sulphate. A semipermeable cell of copper ferrocyanide is thus formed around the drop. The osmotic pressure of the potassium ferrocyanide being greater than that of the copper sulphate, pure water passes from the sulphate into the cell. This results in a concentration of copper sulphate immediately around the cell, and blue striae can be seen descending owing to the greater density of the strong copper sulphate solution thus formed. D. Freezing Point. The freezing point of a solution of a substance is always lower than that of the solvent. The depression of the freezing point (A) depends on the number of particles in a given volume of the solution. We have already seen that the osmotic pressure of a solution also varies with the number of particles in a given volume of the solution. It therefore follows that A varies with the osmotic pressure. 6 THE PROPERTIES OF SOLUTIONS. [CH. I. Consequently the osmotic pressure of a solution is most con- veniently estimated by a determination of its freezing point. The depression of the freezing point (A) for a given concentration of a substance varies with the solvent employed, the relationship for non-electrolytes being — X M = C, where w is the weight of a substance of mole- w cular weight M dissolved in lOO grams of the solvent and C is the "coefficient of depression" for the particular solvent. If s grms. of solvent are taken instead of lOO, it follows that since A is proportional to the concentration ^_ X - X M = C. lOO w The value of C for water is i8-6° C. : for acetic acid it is 39° C. Van 't Hoff has shewn that the value of C can be 2 T^ calculated from the formula C = -, where T is the lOO L absolute temperature of the freezing point, and L is the latent heat of fusion of the solvent. Thus for water T = 273° L = 80 So C = 1^^^^'= i8-6=. 100 X 80 With non-electrolytes, therefore, the gramme-molecule in 1,000 gm. of water causes a depression (A) of 1-86° C. So that — ^ = molecular concentration. 1-86 For electrolytes = concentrations of (ions -f- molecules), so that if a substance be ionised to the extent of i per cent, the molecular concentration is A X 100 1-86 X {2.1 + 100— i) ' CH. I.J FREEZING POINT. 7 The quantitative relationship between osmotic pres- sure and A for aqueous solutions can be readily calculated as follows. The gramme-molecule in 22-4 litres gives an osmotic pressure of 760 mm. of mercury at 0° C. The gramme-molecule in i litre gives a A of i • 86° C. So the gramme-molecule in 22-4 litres gives a A of 1-86 o o r- = 0-083 c. 22-4 So a A of 0-083 C. corresponds to an osmotic pressure of 760 mm. So a A of o-ooi C. corresponds to an osmotic pressure of 9-1 mm. Thus a 5 per cent, solution of glucose (Mol.wt. = 180) has a A of 1-86 X -^ = 0-517° C, 180 and an osmotic pressure of 51-7 x 9-1 = 470 mm. Hg. The A of Blood is about 0-55" C, corresponding to an osmotic pressure of about 500 mm. Hg. Owing to the relatively small number of particles in a given volume of a colloidal solution, it follows that the freezing point of such solutions is only very slightly lower than that of .distilled water. Since it is very difficult to remove the last traces of electrolytes by dialysis, it is not easy to obtain reliable figures for the osmotic pressure of the colloids. Sorensen has recently investigated the problem and has been successful in overcoming the technical diffi- culties. He states that the osmotic pressure of crystalline egg-albumin indicates a molecular weight of 34,000. 5. The determination of the freezing point by Beckmann's method. (Cryoscopy.) Take the freezing point of (a) distilled water; (b) M/5 NaCl (1-16 per cent.) ; (c) M/5 glucose (3-6 per cent.). THE PROPERTIES OF SOLUTIONS. [CH. I. Use tfie apparatus shown in fig. i. In the outer chamber (c) place a mixture of ice and water and solid sodium chloride, or a saturated solution of salt. ^IJl MMMIwnniiiiMi Ij' i^i Fig. I. Beckmann's freez- ing point apparatus. Fig. 2. Beckmann's Ther- mometer. In the tube A place enough distilled water to cover the bulb of the Beckmann thermometer D. This is graduated to i/iooth° C. and can be read by means of a magnifjdng glass to i/iooo° C. The thermometer must not touch the sides or bottom of the tube A. CH. I.] COLLOIDS. 9 The tube B serves as an air jacket to A. Stir the water regularly by means of the (platinum) stirrer E. The temperature falls, and then after a time rises sharply, and remains steady for a considerable time. The temperature to be read is the highest obtained at this rise. This is the freezing point (W) of distilled water. Now replace the water by the fluid, rinsing the tube out with it once or twice. Repeat the experiment and note the freezing point (F) as before, W - F = A- Notes. — i. It is of the utmost importance to take care to prevent too great a super-cooling of the fluid. This should never exceed i ° C. If it has exceeded this in a preliminary experiment, it must be repeated, and when the temperature has fallen 0-5° C. below the freezing point, a minute crystal of ice must be introduced through the side tube. These crystals are best prepared by taking, in a dry test-tube, some hollow glass beads (that have been care- fully dried), adding a small amount of the fluid, pouring off the excess, and immersing the tube in a freezing mixture. They should be introduced by means of a pair of cooled forceps. 2 The observed A is usually too great, owing to the super-cooUng. The simplest correction is A corrected = A observed x I i - -5- where C = the super-cooling in degrees. 3. To set the thermometer. Turn the thermometer upside down, and by gentle shaking mix the mercury in the upper portion with that in the capillary tube. Then place the thermometer in water at about 2° C. Give a slight knock, and thus break the mercury column. It is now ready for use. 4. When reading the thermometer during an experiment it should be tapped with a piece of indiarubber tubing. E, The electrical properties of colloids. Under certain conditions it is found that colloidal particles carry an electric charge. In some cases tbey exhibit electricial conductivity, due to the fact that the substance? are partially ionised. But even if they do not exhibit this phenomenon it is often found that they tend to move towards one of the poles when a strong ( 1 00 volts) con- stant current is sent through the solution. In some cases this movement (" kataphoresis ") is towards the anode, i.e. the particles carry a negative charge ; in other cases it is towards the kathode. It is important to note that the direction of the migration can be changed in many cases by varying the reaction of the fluid in which the colloid is suspended. Thus metaproteins, albumins, etc., carry a 10 THE PROPERTIES OF SOLUTIONS. [CH. I, positive charge in acid solution and a negative charge in alkahne solution. At some particular reaction they seem to be electrically neutral, i.e. kataphoresis cannot be observed. This reaction is known as " the iso-electric point " of the particular colloid. It is discussed in more detail on p. 31. F. The precipitation of colloids. Colloids, as we have seen, are two-phase solutions. One, the solid, phase contains a high concentration of the solute and a low concentration of the solvent : the other, the liquid, phase contains a low concentration of the solute in the solvent. By certain changes in the condi- tions the solid phase can be dehydrated, so that the solution may become opalescent. An increase of this effect may result in the formation of particles visible to the naked eye, or even a dense precipitate that can, in some cases, be removed completely by filtration. In some cases this precipitate can be " dissolved " or " dispersed " by revert- ing to the original conditions. In other cases the change is irreversible, the material having been " coagulated." It is impossible to discuss fully the various conditions that tend to cause aggregation {i.e. precipitation) on the one hand, or dispersion {i.e. solution) on the other, since they vary considerably with different colloids. But it is important to note that many cases can be explained fairly satisfactorily on the theory that the dispersed or dissolved condition of a colloid is due to the fact that it carries an electric charge, the removal of which causes precipitation. Some examples of this are given below : — {a) By colloids with an opposite electrical charge. If ferric chloride be thoroughly dialysed a colloidal suspension of ferric hydroxide is obtained, generally known as " dialysed iron." This carries a positive charge. If this be added to certain albumins which carry a negative charge the two colloids mutually precipitate one another. This gives us a valuable method for removing certain proteins from solution. (See Ex. 310, p. 350.) CH. I.] ISO-ELECTRIC POINT. 11 (b) By changing the reaction of the fluid. In acid solution most colloids " adsorb " the positively charged and readily diffusible hydrogen ions and acquire a positive charge. In alkaline solutions they adsorb hydroxyl ions and become negative. Many proteins are therefore soluble both in acids and alkalies, but at some particular reaction of the fluid they adsorb equal numbers of H and OH ions, lose their charge, and are precipitated. The exact reaction at which this takes place varies with different colloids, and is the above-mentioned iso-electric point. Another way of explaining this phenomenon will be found on p. 31. 6. The determination of the iso-electric point of casein. Into a 50 cc. measuring flask place 0-3 gm. of pure casein (Hammersten's). Add about 25 cc. of distilled water, previously warmed to about 40 C. and exactly 5 cc. of N. sodium hydroxide. Agitate till the casein dissolves, taking care to prevent frothing. Rapidly add 5 cc. of N. acetic acid, mix, cool, and make up to 50 cc. with distilled water. A faintly opalescent solution of casein in o-i N. sodium acetate is thus obtained. Make up the following series of tubes, using clean dry test-tubes. Tube No. I 2 3 4 5 6 6 8 9 cc. Casein in o-i N. sod. acetate . . I I I I I I I I I cc. Distilled water 8-38 775 8-73 8-5 8 7 5 I 7-4 cc. o-oi N. acetic acid 0-62 1-25 cc. o-i N. acetic acid . . 0-25 0-.5 I 2 4 8 N. acetic acid 1-6 Place the casein solution in the tubes first, then the water, and mix. Now add the acetic acid to the first tube and shake immedi- ately. Then add the acid to the second tube and shake this, and so on. Examine the tubes at intervals and record observations as below. o = no change. + = opalescence. x = precipitate. 12 THE PROPERTIES OF SOLUTIONS. [CH. Tube No. I 2 3 4 5 6 7 8 9 On mixing + + + + + + + + + + After 10 mins. . . + +++ XXX X X + + + After 20 mins. . . + X XXX X X + + + The precipitation is greatest in tube 5. The concentration of hydrogen ions (see p. 19) can be calculated approximately from the following formula, no allowance being made for the acidity of the casein. ,„. _ K (acetic acid in mols. per litre) a (sodium acetate mols. per Mtre) (H) = Hydrogen ions in grams per litre. K = dissociation constant of acetic acid = 1-85 x 10-^. a = degree of dissociation of sodium acetate. 0-87 for o-oi N. 079 for o-i N. The theoretical basis for the formula is given on p. 19. Thus in tube 5, (H) = 1-85 X io-» x 10-2 "—^ -^ = 2-13 X 10-^ 0-87 X 10-2 Below are the (H) and Py (see p. 16) of the various tubes. Tube (H) Ph Tube (H) Ph I 1-32 X 10-* 5-88 6 4-26 X 10-^ 4-37 2 2-66 X 10-6 575 7 8-52 X 10-^ 4-07 3 5-32 X 10-6 5-27 8 170 X I0-* 3-77 4 I -06 X 10-^ 4-97 9 3-40 X 10-^ 3-47 5 2-13 X 10-^ 4-67 Still finer adjustments of the reaction can be obtained by suit- ably var5nng the concentration of acetic acid. The (H) can be calculated from the formula. CH. I. COLLOIDS. 13 (c) By the addition of neutral salts. If a suspensoid carries a negative charge it exerts an attraction for positively charged ions (kations). The adsorption of these by the colloid may cause a neutralisa- tion of the charge, and therefore precipitation. In such cases it is found that a bi- or tri-valent ion is very much more potent than a monovalent ion. Thus, if a colloid is negatively charged it may be readily precipitated by BaCla ; if it carries a positive charge it may be readily precipitated by Na2S04. The precipitation ofan em ulsoid by a large excess of neutral salt, such as by saturation with ammonium sulphate, is probably a different phenomenon. 7. The precipitating effect of various ions on colloids. Prepare a solution of casein in o-i N. sodium acetate as de- scribed in Ex. 6. To 2 cc. add 17-5 cc. of distilled water, and then 0-5 cc. of O-I N. acetic acid and mix quickly. A solution of casein is thus obtained, alkaline to the iso-electric point, and therefore carrpng a negative charge. Divide the solution into four equal parts and place them into four clean tubes labelled -i, -2, -3 and -4. To another 2 cc. of the original solution of casein add 10 cc. of distilled water and 8 cc. of o-i N. acetic acid, and mix quickly. An acid solution of casein is thus obtained. Divide into four parts and place them into four clean tubes labelled -)-i, +2, +3 and +4. To the tubes marked i add 3 drops of N. KCl (7-45 per cent.). To the tubes marked 2 add i drop of N. BaClg (i0'4 per cent.). To the tubes marked 3 add i drop of N. K2SO4 (8-7 per cent.). Mix the contents of each tube and place the set of 8 tubes in a water bath at about 35 C. Examine them after 15 minutes, record- ing the results as in the previous exercise. - + I + -I- + 2 X + 3 or -1- XX 4 14 THE PROPERTIES OF SOLUTIONS. [CH. I. It will be noted that the electro-negative colloid is most readily precipitated by BaCl^, which contains a di-valent positive ion. The electro-positive colloid is most readily precipitated by K2SO4, which contains a di-valent negative ion. The tubes may now be warmed to 60° C, and the further effect noted. (d) By the addition of compounds with complex ions. It is found that colloids that carry a positive charge are often readily precipitated by compounds with a complex negative ion. Thus, proteins in acid solution generally carry a positive charge, and they are precipitated by phosphotungstic, phosphomolybdic, tannic, or ferrocyanic acids. Probably the complex ions are more readily adsorbed by the positively charged colloid than are the simple ions. The charge of the colloid thus being neu- tralised, precipitation of the complex takes place. If the colloid carries a negative charge it is often readily precipitated by compounds with a complex positive ion, such as the hydrochlorides of the alkaloids, aromatic bases, etc. G. The Concentration of Hydrogen ions. The only satisfactory method of expressing the "re- action" of a fluid is in terms of the concentration of hydro- gen ions per litre of the fluid. This concentration is so important as a factor in the physiological properties of fluids that the theory of the matter should be grasped by students at an early stage of their physiological studies. Pure distilled water is very slightly ionised into hydro- gen ions or hydrions and hydroxyl ions or hydroxidions. H2O ~ ^ H + OH. This dissociation proceeds to an equilibrium, in which, according to the laws of mass action, - — fn-pTT — ^ = a constant. (HjU) CH. I.J HYDROGEN ION CONCENTRATION 15 So (H) X (OH) = a constant x (Hfi). The brackets indicate the concentration per htre of gram-ions or gram-moles respectively. Since the mass of undissociated water is enormously .large compared to the mass of the free ions, it can be regarded as a constant, so (H) X (OH) = a constant. This constant varies considerably with the tempera- ture. At 21° C. it is IO-". Since the ions are equal in number, each has a concentration of io~' gram-ions per litre. It must be particularly noticed that the product and not the sum of the gram-ion concentrations is constant. If an acid be added to distilled water the acid is partially or completely dissociated into hydrogen ions, and the negative ions characteristic of the acid employed. In such a mixture the concentration of hydrogen ions per litre at 21° C. is greater than io~'' grams, and the solution is " acid." If (H) be increased to lO"* it follows that the concentration of hydroxyl ions per litre must be decreased to lo-^" gram-ions. For (H) x (OH) = iq-^*. If an alkaU be added to distilled water the base is dissociated into hydroxyl ions and certain positive ions. The concen- tration of hydrogen ions per litre at 21° C. is conse- quently less than 10-' grams, and the solution is " alkaline." A " neutral " solution is one in which (H) at 21" C. = 10-7. An " acid " solution is one in which (H) at 21° C. is greater than 10-''. An " alkaline " solution is one in which (H) at 21° C. is less than io~''. Acids differ markedly in the degree to which they are ionised in solution. " Strong " acids, like HCl or HNO,,, 16 THE PROPERTIES OF SOLUTIONS. [CH. I. are freely ionised ; whilst " weak " acids, like acetic acid, are only feebly ionised. By electrical measurements of the conductivity of the solution it has been shewn that o.i N.HCl is ionised to the extent of 84 per cent, at 18° C. If it were completely ionised there would be o-i gm. of hydrion per litre. As it is, only partially ionised, (H) is o-i X -^ = 0-086 = 8-4 X 10-2 at 18° C. 100 Similarly, o.iN. acetic acid is only dissociated to the extent of 1-36 per cent. So in this case (H) = o-i X — — = 0-00136 = 1-36 X iQ-^. 100 This method of expressing the hydrogen ion concentra- tion is not convenient. It is preferable to adopt the nota- tion of Sorensen, who introduced the symbol Ph to denote the " hydrogen-ion-exponent." Ph is the logarithm to the base 10 of (H), the negative sign being omitted. In other words Ph = -log,o (H). A few examples should make its meaning clear. o-iN.HCl has (H) = 8-4 x 10-2. Now log^o 8-4 = 0-92. So 8-4 X 10-2 = IO0-92-2 = lo-i-os. So Ph = i-o8. o-iN. acetic acid has (H) = 1-36 X io-« = lo^-^s-s = io-2-8«^ So Ph = 2-867. It will be observed that Ph decreases as the acidity in- creases. Also that if (H) is doubled, Ph is not halved, but only decreased by 0-301, since logio2 = 0-301 . It is important to note that the Ph of a solution cannot be determined by the ordinary method of titration. Let us consider the case of o-i N.HCl and o-i N. acetic acid. If these be titrated with o-i N.NaOH until they each give a pink with phenol phthalein, 10 cc. of each acid will require exactly 10 cc. of the alkali, and will therefore CH. I.] BUFFERS." 17 have apparently the same acidity. But actually the hydrochloric acid has an (H) over 60 times greater than the acetic acid. The reason for this is that the acetic acid is only very slightly ionised, the amount ionised being a certain proportion of the total acid present. As soon as the ionised part has been removed by the addition of a base, a further fraction of the previously un- dissociated acid is ionised. This process is repeated with further additions of alkali until the whole of the acid originally present has become dissociated, and its hydrogen ions have united with the hydroxyl ions of the base. The next trace of added alkali reacts with the indicator to give a pink colour. Titration therefore only gives us an index of the capacity of the solution to neutralise acids or alkalies ; it does not give us information concerning the potential of the hydrogen ions, i.e. the Ph. " Buffers." A single drop of 0-02 N.HCl added to a litre of pure water at 18° C. would cause a change in Ph from 7-07 to about 6. A trace of diffusible alkali from a glass bottle might change the Ph to 8, or even higher, whereas exposure to the COg of the air might cause a drop to about 6. Thus it is extremely difficult to maintain any constancy of Ph in such a solution. But with certain substances present the addition of a small amount of acid or alkali causes only a minimal change in Ph- Such substances are called " Buffers." Various solutions are used for this purpose, such as phosphates, citrates, borates, and acetates. Let us consider the case of a solution of sodium acetate, to which is added a small amount of hydrochloric acid. Both substances are freely dissociated so that the following ions are originally present, Na, CH3.COO, H, CI. Now acetic acid is a weak acid, which means that CH3.COO and H ions can exist together only in very low concentrations. We therefore get Na + CH3.COO + H + CI = CH3.COOH + Na + CI. Thus the H ions of the added hydrochloric acid nearly disappear, owing to the presence of the buffer sodium 18 THE PROPERTIES OF SOLUTIONS. [CH. I. acetate. It must be noted that they do not all disappear, for some of the acetic acid formed is dissociated into H and CH3.COO. In the animal body the proteins, sodium bicarbonate, and phosphates all function as buffers, and help to maintain a constancy in the hydrogen ion concentration of the tissue fluids. The effect of dilution on (H). With a weak acid of the type HA, the extent of dissociation is governed by the equation (H) X (A) , K (I) (HA) ^ ' (HA) is the concentration of the undissociated mole- cules per litre, and K is the. " Dissociation constant " of the acid. Since (H) = (A), we can write this (H)2 = K(HA) or (H) = ^K(HA). This indicates that if a solution of a weak acid be diluted four times (H) is halved ; if it be diluted 16 times it is reduced to one-fourth. In the presence of any considerable amount of the sodium salt, the effect of dilution is quite different. We can write equation (i) in the following form : (H) = aM (.) The sodium salts of weak acids are very freely dis- sociated, so that there is a relatively high concentration of A ions in the solution of the mixture. From equation (2) it will be seen that an increase of (A) must cause a decrease in (H). The dissociation of the weak acid being thus depressed it follows that practically all the acid is present in the undissociated form, so that we can assume that (HA) = (acid). Further, practically all the free ions arise from the dissociation of the sodium salt, so that (A) = (Sodium salt). CH. I.] HYDROGEN ION CONCENTRATION. 19 We can therefore write equation (2) as (H) = ^ (^"^) - - (3) ^ ' (Sodium salt) ^^' Since the sodium salt is not fully dissociated, except in high dilutions, it is more correct to write it l\{\ = K (acid) , . ^ ' a (Sodium salt) ^^^ where a is the degree of dissociation of the salt. It follows from this that the (H) of such a mixture is mainly conditioned by the relative concentrations -of the acid and of its salt, and is only very slightly affected by dilution, which does not alter the relative concentrations. This is of considerable importance, since a large number of physiological fluids can be regarded as mixtures of weak acids with their sodium or potassium salts, and so suffer little change in (H) on dilution. The determination of the hydrogen ion concentration. The most accurate method is an electrical one, involving expensive and intricate apparatus. It is too complicated to be described here. A valuable method that does not require elaborate apparatus is the " indicator," or " colori- metric " method. An indicator is a substance that varies in colour tone or in depth of colour with the Ph of the solution. Each indicator shows a colour change over a certain range of Ph. At some particular P^ the indicator may show an inter- mediate or faint tint. The solution is then said to be " neutral " to this indicator. It does not follow that the solution is neutral in the strict sense, i.e. (H) = (OH). The Ph of a solution " neutral to phenol phthalein " is about 9 ; that of a solution " neutral to methyl orange " is about 4, the (H) in the latter case being 100,000 times greater than in the former. The method adopted for the determination of Ph by indicators is to take standard solutions of certain substances 20 THE PROPERTIES OF SOLUTIONS. [CH. I. which can be mixed in various proportions to give a series of solutions of a known Ph, which have been accurately- determined by the electrical method. A given amount of a suitable indicator is added to a measured volume of the fluid, and also to equal volumes of the standard test solutions, contained in tubes or vessels as uniform as possible. The solutions that give exactly the same tints have the same hydrogen ion concentration, provided that this concentration is in the range of the indicator employed. The results are not as accurate as the electrical method, owing to the difficulty of exactly matching the tints and Source of Light. Coloured fluid. 4. Water, 6. Coloured fluid. A Coloured B Standard fluid 5. Standard + + + Indicator. Indicator. Indicator. X y z glass screen Eye. Fig. 3. Plan of arrangement of tubes in Cole and Onslow's Comparator. also of the effect of proteins, salts, and other substances on the colour developed. If the fluid be coloured it is obvious that this simple method can only give very approxi- mate results. Walpole overcame the difficulty by viewing the {standard solution + indicator) through a layer of the coloured fluid of the same depth as that of the {coloured fluid + indicator). A special instrument was devised for this purpose. Hurwitz, Meyer and Ostenberg used Walpole's principle, but employed test tubes held in a box CH. I. DETERMINATION OF P„ 21 or " comparator." Cole and Onslow somewhat improved this by using a comparator containing three pairs of tubes, the arrangement being diagramatically shown in fig. 3. The addition of a ground glass plate fixed to the comparator between the eye and the tubes has been found by the author very much to improve the apparatus, slight differences in colour being more readily detected. The standard solutions taken are such that the appear- ance seen through Y is either intermediate between that seen through X and Z, or identical with one of them. The colour changes, and the ranges of the most useful indicators are given below in Table I. Certain other interesting data are presented in chart form in Table II. Cole and Onslow's Comparator. The author can very strongly recommend the new sulphone-phthalein indicators introduced by Mansfield Clark, Lubs and Acree. For further information on the subject of the colorimetric method of determination of P^ the student is referred to an important series of papers by Clark and Lubs, " Journal of Bacteriology," Baltimore, Vol. II., pp. I, 109 and 191 (1917)- 22 THE PROPERTIES OF SOLUTIONS. [CH. I. TABLE I. Indicators. Those printed in heavy tsrpe are the most useful for ordinary work. TKADE NAME CHEMICAL COMPOSITION RANGE OFPh COLOUR CHANGE, Acid— alkaline. I. Methyl violet o-i to 3-2 Green-blue 2. Thymol blue Thymol - sulphone - phthalein 1-2 to 2-8 Red-yeUow 3. Toepfer's re- agent Di methyl amino - azo-benzene 2-9 to 4-2 Red-yellow 4. Brom-phenol- blue Tetra - brom - phenol - sulphone-phthalein 2-8 to 4-6 YeUow-blue 5. Methyl orange p - benzene - sulphonic - acid-azo-di-methyl- aniline 3-1 to 4-4 Red-yellow 6. Congo red 3-0 to 4-5 Blue-red 7. Methyl-red p dimethyl amino - azo benzene - carbonic acid 4-4 to 6-0 Red-yellow 8. Brom -cresol- purple Di - brom - - cresol - sulphone-phthalein 5-2 to 6-8 Yellow-purple 9. Litmus 5-4 to 7-8 Red-blue 10. Brom-thymol blue Di brom thymol - sulphone - phthalein 6-0 to 7-6 Yellow-blue II. Neutral red 6-8 to 8-0 Red-yellow 12. Phenol-red Phenol-sulphone- phthalein 6-8 to 8-4 Yellow-red 13. Cresol-red cresol sulphone - phthalein 7-2 to 8-8 Yellow-red 14. Thymol-blue Thymol - sulphone - phthalein 8-0 to 9'6 Yellow-blue 15. Phenol phthalein Phenol phthalein 8-3 to lo-o Colourless-red 16. Thymol- phthalein Thymol-phthalein 9-3 to IO-5 Colourless-blue CH., I.J INDICATORS. 23 Preparation of solutions. These are best prepared from standardised solutions of known strength.* The most convenient concentrations for ordinary work are 0-04 per cent, for (i), (2), (4), (8), (10), and (14). 0-02 per cent, for (6), {12), and (13). o-oi per cent, for (5). 0-02 per cent, in 60 per cent, alcohol for (3), (7) and (11). 0-04 per cent, in 60 per cent, alcohol for (15) and (16). Strong aqueous solution, dialysed against dis- tilled water for (9). Volume required. In most cases ten drops to 10 cc. of the solution are about right. But the amount varies with the range, colour of solution, etc. Thus 12 drops of no. (12) may be required at Ph = 6-9, and 'only 5 drops at Ph = 8-o. It is essential that exactly the same amount be added to the measured volume of the fluid and to the same measured volumes of the standard solu- tions. The most convenient and accurate method of adding the drops is to have the bottle of indicators fitted with rubber corks pierced with Dreyer's dropping pipettes (fig. 5). Colour filters for dichroic indicators. Brom- phenol blue and brom-cresol purple are dichroic. To get rehable results, especially with turbid fiuids, it is necessary, to compare the solutions by using a source of light from which the blue rays have been screened off. This can be done by painting a piece of trans- parent tracing paper with a strong acid solution of phenol red, prepared by mixing one part of the stock 0-6 per cent, solution with one part of the standard 0-2 M acid potassium phosphate. The paper, while stiU wet, is pinned across the front of a box containing one or two powerful carbon filament lamps. The ex- phenolredL— 002% Ph 6-aT0 8-4 YELLOW-RED^ Fig. 5. Bottle and Dreyer's Drop- ping Pipette for Indicator Solutions. * These can be obtained from Messrs Baird & Tatlock, 14, Cross Street, Hatton Gardens, E.G. 24 THE PROPERTIES OF SOLUTIONS. [CH. I. amination should be conducted in a dark room, or the external light should be cut off by using a dark cloth. Another method is to take an unexposed photographic plate, fix it in hypo in a dark room, wash for some hours in running water, stain by immersion in the dye, drying and mounting a piece on the comparator on the side towards the light, in place of the ground glass screen. Standard solutions of definite P^ The most convenient sets of solutions that have been worked out are those of Clark and Lubs. A constant volume (50 cc.) of a standard solution of acid potassium phthalate, acid potassium phosphate or of boric acid is measured into a 200 cc. measuring flask. A given amount (x cc.) of standard NaOH or HCl is then added, and the volume brought to the mark with distilled water. The Ph obtained with the different solutions are given in the tables below. If intermediate points are desired, they can be obtained from curves drawn from the points given. Preparation of solutions, 0-2 M acid potassium phthalate. Dissolve 40-828 gm. in dis- tilled water and make up to i htre. The salt should be recrystallised from distilled water and dried at 110° C. for some hours. 0-2 M acid potassium phosphate. Dissolve 27-231 gm. in dis- tilled water and make up to i htre. The salt should be recrystallised from distilled water and dried at 110° C. for some hours. 0-2 M Boric Acid in 0-2 KCl. Dissolve 12-4048 gm. of air dried boric acid and 14-912 gm. pure ignited KCl in distilled water and make up to i litre. Sodium Hydroxide. Dissolve 100 grams of the best NaOH in 100 cc. of distilled water in an Erlenmeyer flask of resistance glass. Cover the mouth of the flask with tin foil, and allow the solution to stand o\'er- night till the carbonate has mostly settled. Cut a hardened filter paper to fit a Buchner funnel. It must be noted that the paper shrinks a little in treatment, and must be cut rather large. Treat it with warm, strong [i;i] NaOH solution. Decant the soda and wash the paper first with absolute alcohol, then with dilute alcohol and finally with large quantities of distilled water. Place CH. I.] STANDARD SODA. 25 the paper on the Buchner funnel and apply gentle suction until the greater part of the water has evaporated. Now pour the con- centrated alkah upon the middle of the paper, spread it with a glass rod, and filter under suction. The clear solution is now diluted quickly with cold distilled water, that has been recently boiled to remove CO2, to make approximately N. NaOH (10 cc. per Utre makes about 0-2 N.). 10 cc. of this is withdrawn and roughly standard- ised against N. HCl. It is then diluted tiU it is approximately 0-2 N with C02-free water and the solution poured into a paraffined bottle, to which a burette and soda-lime guard tubes have been attached (see fig. 6). The solution is then accurately stan- dardised against weighed amounts of the pure acid potassium phthalate. To do this accurately weigh up about 1-5 gms. of the salt, dissolve in about 30 cc. of distilled water, add phenol phthalein and titrate with the alkali till a faint but dis- tinct and permanent pink is developed. A current of COj-free air should be blown Fig. 6. Paraffined bottle (A) for storing standard alkali. B and C are soda lime tubes. The burette is filled by sucking at C. through the solution during the titration. The apparatus shewn in fig. 35 is convenient for this purpose. If p be the exact weight of the phthalate taken, and s the volume of soda required, the normality of the soda is I -000 X p ~ = a. 204-14 X s Instead of using x cc. of 0-2 N, the amount of the standardised soda that must be employed is X X 0-2 cc. It is convenient to label the bottle with the factor 0-2 0-2 N Hydrochloric acid. This can be prepared from a freshly distilled 20 per cent, solution, and standardised against the standard soda, using methyl red as the indicator. 26 THE PROPERTIES OF SOLUTIONS. [CH. Series A. 50 cc. 0-2 M. acid potassium phthalate. X cc. 0-2 N. HCl. Diluted to 200 cc. [Thymol Mue. Indicators recommended (Brom-phenol blue. Ph a; Ph :*; Ph X 2-2 4670 2-9 22-8o 3-6 5-97 2-3 42-50 3-0 20-32 37 4-30 2-4 39-60 3-1 17-70 3-8 2-63 2-5 37-00 3-2 14-70 3-9 I -00 2-6 32-95 3-3 11-80 27 29-60 3-4 9-90 2-8 26-42 3-5 7-50 Series B. 50 cc. 0-2 M. acid potassium phthalate. X cc 0-2 N. NaOH. Diluted to 200 cc. Indicators reconunended Brom-phenol blue. Methyl red. Brom-cresol purple. Ph X Ph X Ph X 4-0 0-40 4-8 17-70 5-6 39-85 4-1 2-20 4-9 20-95 57 41-90 4-2 370 5-0 23-85 5-8 43-00 4-3 5-17 5-1 27-20 5-9 44-55 4.4 7-50 5-2 29-95 6-0 45-45 4-5 9-60 5-3 32-50 6-1 46-20 4-6 12-15 5-4 35-45 6-2 47-00 47 14-60 5-5 37-70 6-3 48-10 CH. I.] STANDARD SOLUTIONS. 27 Series C. 50 cc. 0-2 M. acid potassium phosphate. X cc. 0-2 N. sodium hydroxide. Diluted to 200 cc. fBrom-cresol puq)le. Indicators recommended JBrom-thymol blue. Phenol red. Ph X Ph a; Ph X 5-8 372 6-6 17-80 7-4 39-50 5-9 470 67 21-00 7-5 41-20 6-0 570 6-8 23-65 7-6 42-80 6-1 7-40 6-9 26-50 77 44-20 6-2 8 -60 7-0 29-63 7-8 45-20 6-3 10-19 7-1 32-50 7-9 46-00 6-4 I2-6o 7-2 35-00 8-0 46-80 6-5 i6-oo 7-3 37-40 Series D. 50 cc. 0-2 M. boric acid in 0-2 M. potassium chloride. ;*; cc. 0-2 N. sodium hydroxide. Diluted to 200 cc. fCresol red. Indicators recommended [Thymol blue. Ph X Ph X Ph X Ph X 7-8 2-61 8-4 8-50 9-0 21-30 9-6 36-85 7-9 3-30 8-5 10-40 9-1 24-30 97 39-00 8-0 3-97 8-6 12-00 9-2 26-70 9-8 40-80 8-1 4-80 87 14-30 9-3 29-95 9-9 42-50 8-2 5-90 8-8 16-30 9-4 32-00 lO-O 43-90 8-3 7-30 8-9 19-00 9-5 34-50 28 THE PROPERTIES OF SOLUTIONS. [CH. I. Series E. Sodium acetate and acetic acid. The following series is given as being convenient for certain experiments. It should be noted that the Pjj of the solutions is only very slightly changed by considerable dilution with water.* Preparation of Solutions. N. acetic acid is prepared by titration against N. soda. 0-2 N. acetic acid is prepared from this by diluting 200 cc. to 1000 cc. with distilled water. o-2 N. sodium acetate is prepared by mixing 200 cc. of the N. acetic acid with 200 cc. of the N. soda employed and diluting to 1000 cc. with distilled water. Take :*; cc. of the sodium acetate, and add (io-;t) cc. of the 0-2 N. acetic acid. Ph X (io-a;) Ph X [lo-x) 3-8 1-2 8-8 4-8 5-95 4-05 3-9 1-5 8-5 4-9 6-5 3-5 4-0 1-8 8-2 5-0 7-0 3-0 4-1 2-2 7-8 5-1 7-45 2-55 4-2 2-65 7-35 5-2 7-85 2-15 4-3 3-1 6-9 5-3 8-25 1-75 4-4 3-7 6-3 5-4 8-5 1-5 4-5 4-25 5-75 5-5 8-8 1-2 4-6 4-8 5-2 5-6 9-05 0-95 47 5-4 4-6 5-7 9-25 0-75 8. The detehnination of the Pq of urine. Apparatus and reagents required. (i) A number of clean, dry test-tubes of thin clear glass and of uniform bore. | inch is a suitable external diameter. (2) A comparator for holding the tubes. Tliis is shown in figure 4. * See page 18. CH. I.] DETERMINATION OF Ph. 29 (3) A series of buffer solutions of known Ph prepared according to directions given above. It is convenient to have a series of these prepared and contained in bottles fitted with a rubber stopper, through which passes the stem of a 5 cc. pipette. (4) Solutions of appropriate indicators (see page 23). Methyl red, brom-cresol-purple, brora-thymol blue cover the range of the majority of specimens of urine. (5) A screen to cut out blue rays when using brom-cresol-purple (see page 23). Method. To 5 cc. of the filtered urine add 5 drops of methyl red. If the mixture is red, the Pi^ is in the neighbourhood of 5. If it is yellow, the Pjj is nearer 6. In the latter case treat another 5 cc. with 5 drops of brom-cresol-purple. A deep purple tint suggests that the Ph is higher than 6, in which case it may be necessary to use brom-thymol-blue. Having roughly obtained the range and the necessary indicator, place about 5 cc. of the specimen into two of the special tubes and place them in the holes marked 2 and 6. Into a tube in 4 place some distilled water. Measureexactly 5 cc. of the urine into another tube, and to it add 5 drops of the indicator (measured with a Dreyer's dropping pipette, fig. 5). Mix by rotating the tube between the palms of the hands, and place the tube in the hole marked 3. Measure 5 cc. of one of the buffer solutions into a tube, add 5 drops of the indicator, mix, and place the tube in the hole marked i. Hold the comparator to the source of light with the ground glass screen towards the observer and note the appearance opposite the slots x and y. It will then be ascertained whether the buffer chosen is acid or alkaline to the urine. In either case another buffer solution must be taken, the indicator added, and the tube placed in the hole 5, and the tubes examined again. This procedure must be repeated until two solu- tions are found of such a Ph that the colour as seen through y is intermediate between those seen through x and z, or that through y is identical with one of them. The Ph of the solutions finally employed should not differ by more than o-i. Note. — In measuring the indicator solutions it is essential to hold the dropping pipette vertical, to ensure the delivery of equal drops. 30 THE PROPERTIES OF SOLUTIONS. [CH. I. H. Ampholytes or amphoteric electrolytes. These are substances which can function as acids by forming salts with bases, and also as bases by forming salts with acids. The amino acids, such as glycine, are examples. Glycine can form a sodium salt, CHg.NHg.COONa, and also a hydrochloride, HCI.H2N.CH2.COOH. In strong acids it behaves as a base ; in strong alkalies as an acid. In neutral solutions it is a feeble electrolyte, and is partially dissociated into H and a negative ion (anion). H2N.CH2.COOH ^ T H2N.CH2COO + H...(i) and partially into OH and a kation. HO.H3N.CH2.COOH fZZ^ HO + H3N.CH2.COOH...(2) If a strong acid, such as HCl, be added the dissociation (i) is decreased, ia the same way as the dissociation of all weak acids is decreased by an increase in the hydrogen-ion concentration. On the other hand the number of kations formed is increased, since such a salt as glycine hydrochloride is freely dissociated. If a strong base, like NaOH, be added the dissociation (2) is depressed, and there is an increase in the number of anions of the ampholyte, due to the free dissociation of the sodium salt that is formed. For every ampholyte there is some particular concen- tration of hydrogen ions at which the dissociation (i) is equal to the dissociation (2). This is known as the " iso- electric point." Since the proteins are ampholytes, the conditions of a substance at its iso-electric point are of some interest. They are : (i) The sum of the anions and kations is at a minimum. (2) The concentrations of the anions and kations are equal. (3) If an ampholyte be added to a solution, whose [H] is greater than its iso-electric point, it functions as a base, and therefore decreases the [H] of the solution. If it be CH. I.] AMPHOLYTES. 31 added to a solution, whose [H] is less than its iso-electric point, it functions as an acid. If the (H) of a solution is not altered by addition of an ampholyte, then the (H) of the solution must be equal to the iso-electric point of the ampholyte. (4) The solubihty of an ampholyte is at a minimum at its iso-electric point. If a colloid the ampholyte flocks most readily at this point. (See Ex. 6.) (5) At its iso-electric point, a colloid is electrically neutral. The significance of the iso-electric points of various enzymes is discussed on p. 184. The iso-electric points of certain substances of physiological interest are shewn in Table II., p. 32. 32 CHART OF INDICATORS AND REACTIONS. [CH. I. TABLE II. 10- RANCE OF PRINCIPAL INDICATORS REACTIONS OF FLUIDS OPTIMUM REACTIONS ISOELECTRIC POINTS Ph 10 8-- 5-- 4-- fPancreatic juice (8'3) ~|lnteptinat contents (83) •JBIood (7-4) -Human niijk (7.1) -Pure water — — - ■Saliva (6.9) .>Cow's milli (6'7) -Urine (60) ■Infants' gastric juice (5'0)- ■0-0001 N.HCI (4-01) - ■0001 N.acetic (3-87) -001 N.acetic (3-37) -0-dOl N . HCI (301) ■0-1 N.acctiaaciil(2-«7) ■N.acetic (2-37) •ODl N.HCI (2-02) II Adult gastric juice (0-9 tot -6) 0-1 N.HCUl-08) >Trypsin on fibrin (80) ► Erepsift (78) -Histidine (7-2) — —True neutrality— — (Maltase (6-7) ■JPtyatin (6-7) (Trypsin on casein (6-7) /; ■Protease of Taka- Jiastase (51) ->Invertasc (45) -Pepsin (1-4) 'Alanine (6-7) OxyhxmoRlobin (6-7) "Glycine (66) ^Tyrosine (5-41) ^ Scrum globulin (5-4) \|Serum albumin I (denaturised) (5-4) -Serum, albumin (4-7) rCascin (4-6) OGelatine (4*6) ^Phenyl-alanine (4-48) -Aspartic acid (2'9) CHAPTER II. THE PROTEINS. A. Definition. Proteins are nitrogenous compounds found in the fluids and tissues of afl living organisms. Chemically, they are composed of a number of amino acids (see p. 67), con- densed together in a characteristic way so that the whole molecule is generally neither very acid nor very basic. Their chemical properties are dependent on the presence of these amino acids. Their physical properties are mainly due to the fact that they form colloidal solutions (p. i). The percentage composition varies very considerably in different proteins. The following can be taken as a rough average : — c = S3 per cent = 23 yt yj N = 16 )) }> H = 7 }} yr S = I n M 100 B, . Classification. It is not possible at present to give a rational scheme, for we have not sufficient data of a chemical nature by means of which we can characterise the individual proteins. The classification adopted here is based on physical and chemical properties, and closely follows the official classi- fication of the American Physiological Society. Where the British Society uses a different name this is indicated by (B). 34 THE PROTEINS. [CH. II. 1. Protamines. Basic substances forming stable salts with mineral acids, and containing a high percentage of nitrogen. On hydrolysis they yield only a few of the amino acids, and these are mainly the bases. They occur in the heads of ripe spermatozoa and in ova. 2. Histones. Similar to the protamines, but less rich in nitrogen and the basic amino acids. They are, however, more basic than the majority of the proteins, and are precipitated by ammonia. They are found in unripe spermatozoa, the stroma of red blood corpuscles, and in Ijmiphoid tissue. 3. Albumins. Soluble in distilled water and coagulated by boiling. 4. Globulins. Insoluble in distilled water, soluble in dilute salt solutions. Coagulated by boiling. Groups 3 and 4 are sometimes known as " native " proteins. 5. Glutelins. Found in abundance in vegetables. Insoluble in neutral solvents, but soluble in acids and alkalies. 6. Prolamines [GHadins (B)]. Also found in vegetables, but distinguished from the glutehns by their solubility in 75 per cent, alcohol. 7. Albuminoids [Scleroproteins (B)]. Found in the skeletal and connective tissues of animals. They are characterised by their insolubiHty in most reagents. • Examples are keratin, elastin, and collagen (the anhydride of gelatine). 8. Phospho-proteins. Rich in phosphorus. They must be carefully distinguished from the nucleoproteins. Examples are the casein [caseinogen (B)] of milk and viteUin of egg yolk. 9. Conjugated Proteins. Proteins united to a non-protein group. (i.) Chromoproteins. Protein + pigment molecule, e.g. haemo- globin, (ii.) Glycoproteins [Glucoproteins (B)]. Protein -f- carbohy- drate group, e.g. mucin, (iii.) Nucleoproteins. Protein + nucleic acid. These are prob- ably indefinite salts of nucleic acid with proteins (see p. 60). CH. 11.] GENERAL REACTIONS. 35 10. Hydrolysed Proteins. Formed by the action of acids, alkalies and certain enzymes on the native proteins. (i.) Metaproteins. Soluble only in acids and alkalies, (ii.) Proteoses or albumoses. Soluble in water, not coagulated by heat, precipitated by ammonium sulphate. (iii.) Peptones. Like the albumoses, but not precipitated by ammonium sulphate, (iv.) Polypeptides. Simple peptones, formed of mon-amino acids only. C. General Reactions. For the following reactions use egg-white that has been well beaten with 6 times its volume of water or serupi that has been diluted ten times with water. (i.) The proteins give certain colour reactions (see pages 38 to 41). (2.) They are precipitated by the salts of the heavy metals. The salts that are most used are lead acetate, mercuric chloride and nitrate, ferric chloride, copper sulphate, and zinc sulphate. The mechanism of the precipitation is somewhat complex, and probably varies for different salts and different concentrations. In a good many cases it seems to be due to the adsorption of the metallic kation by the negatively charged colloidal protein. For this reason the precipitation is best obtained when the reaction of the medium is somewhat alkaline, the protein then being negatively charged (see p. ii). Also the precipitate is often soluble in acid. It is often soluble in an excess of the metalhc salt, probably because the charge on the protein becomes positive owing to the adsorption of the excess of positive ions. 9. Treat 3 cc. of the solution with a few drops of mercuric nitrate. A white precipitate is obtained. This will partially or completely dissolve in a saturated solution of sodium chloride, pro- vided that the solution does not contain free acid. The solubiUty in sodium chloride is due to the fact that mercuric chloride is formed. This salt differs from the nitrate in that it is only feebly dissociated. 36 THE PROTEINS. [CH. II. 10. Treat 3 cc. of the protein solution with ferric chloride, drop by drop. A precipitate is formed soluble in excess. 11. Treat 3 cc. of the protein solution with a solution of lead acetate or basic lead acetate. A white precipitate is formed. (3.) The proteins are precipitated by the so-called " alka- loidal reagents." These include phosphotungstic, phos- phomolybdic, ferrocyanic, tannic, picric, metaphosphoric, and sulphosalicylic acids, and Briicke's reagent (potassio- mercuric iodide). It is possible that the precipitation is due to the adsorp- tion of the complex negative ions by the positively charged colloidal protein. It is suggestive that the substances are only effective in acid solution, in which the proteins are positively charged. The precipitating action of these re- agents on the peptones varies somewhat. As a rule they are not so readily precipitated as the albumins and globu- lins. 12. Treat 3 cc. of the solution with two or three drops of strong acetic acid and two drops of potassium ferrocyanide. A white precipitate is formed. Boil. The precipitate does not dissolve. Notes. — i. Primary proteoses are also precipitated by ferrocyanic acid, but the precipitate produced dissolves on warming and reappears on cooling (Ex. 55). 2. The precipitate and fluid often become coloured blue-green on boiling. This is due to a decomposition of the hydroferrocyanic acid on boiling it witii certain organic substances, such as proteins. 13. Acidify some of the solution with hydrochloric acid and add a few drops of a freshly prepared solution of tannic acid, or of Alm^n's reagent. A white or brown precipitate is usually formed. Note. — ^Almfen's reagent consists of 4 gm. of tannic acid in 8 cc. of strong acetic acid and 190 cc. of 50 per cent, alcohol. 14. Treat 3 cc. with an equal volume of Esbach's solution. A yellowish precipitate is formed. Note. — Esbach's solution is prepared by dissolving 10 grms. of picric acid and 10 grms. of citric acid in water and making the volume up to a litre. It is extensively used for the determination of albumin in urine (Ex. 421). CH. II.] PROTEIN PRECIPITANTS. 37 15. Acidify the solution with dilute hydrochloric acid and add a few drops of potassio-mercuric iodide (Briicke's reagent). A white precipitate is formed. Note. — Briicke's reagent is prepared by dissolving 50 grms. of potassium iodide in 500 cc. of distilled water, saturating with mercuric iodide (120 grms.), and making up to i litre. 16. Acidify a few cc. of the solution with dilute hydrochloric acid and add a few cc. of a 2 per cent, solution of phosphotungstic acid. A white precipitate is produced. 17. To a few cc. of the solution add a drop or two of a freshly prepared 25 per cent, solution of metaphosphoric acid. A white precipitate is produced. Note. — Metaphosphoric acid (HPO3) is used by Folin for removing pro- teins from blood and urine in certain quantitative methods. The solution must be freshly prepared, as on standing it slowly passes over into ortho- phosphoric acid (H3PO4), which has no precipitating action on proteins. 18. To a few cc. of the solution add a small amount (a large " knife point ") of sulpho^alicylic acid, or a drop or two of a strong (20 per cent.) solution. A white precipitate is obtained. Note. — The reagent is of considerable value for Hie detection of albumin in urine. (See Ex. 371.) It can be prepared by dissolving 13 grm. salicylic acid in 20 grms. HjSO, by warming, and, after cooUng, adding 67 cc. of water. (4.) The proteins are precipitated by strong alcohol. The albumins and globulins are rapidly changed by alcohol at room temperature into modifications that are insoluble in water, salt solutions, dilute alkalies or acids. That is, they are coagulated. The proteoses and peptones, the phosphoproteins, and gelatin are precipitated by alcohol, but the precipitate redissolves in water or dilute alkalies. 19. Place about 4 cc. of serum in a test-tube and cool to o" C. by means of a freezing mixture. Fill the tube with strong alcohol that has previously been cooled to about 8° C, and mix. A white precipitate of the proteins is formed. Filter at once and treat the precipitate with water. It dissolves. 38 THE PROTEINS. [cH. II. 20. Allow a few drops of seram to fall into about lo cc. of strong alcohol at room temperature. A white precipitate is formed. Shake well and allow to stand for half an hour. Filter and treat the precipitate with water. It does not dissolve. D. Colour Reactions. 21. The Xanthoproteic reaction. To 3 cc. of the - protein solution in a test-tube add about one cc. of strong nitric acid. A white precipitate is formed (see Ex. 40). Boil for a minute. The precipitate turns yellow and partly dissolves to give a yellow solu- tion. Cool under the tap and add strong ammonia or soda till the reaction is alkaline. The yellow colour is turned to orange. Notes. — i. The essential features of the reaction are that a yellow colour is obtained when the solution is boiled with strong nitric acid, and that this yellow colour is intensified when the solution is made alkaline. 2. The precipitate is due to the formation of metaprotein by the action of nitric acid on albumins or globulins, this metaprotein being insoluble in strong mineral acids. It follows that proteoses,and peptones, etc., do not give the precipitate with nitric acid. 3. The yellow colour is due to the formation of a nitro-compound of some aromatic substance, i.e. u, substance containing the benzene ring. 4. The aromatic substances in the protein molecule that are responsible for the reaction are tyrosine, tryptophane and phenyl alanine. 5. Oleic acid, oUve oil and most vegetable oils give a well-marked xantho- proteic reaction. 6. To test for traces of proteins proceed as follows : Boil with nitric acid and divide into two portions. Cool one portion and make it alkahne with ammonia. Compare the colour of the two portions. The alkaline tube will shew a faint yellow colour when only the merest trace of protein is present. 22. Millon's reaction. Treat 5 cc. of the protein solution with half its volume of Millon's reagent. A white precipitate is formed. Cautiously heat the mixture. The precipitate turns brick-red in colour, or disappears and leaves a red solution. Notes. — i. The essential feature of the reaction is the red colour on heating. The white precipitate in the cold is due to the action of the mercuric nitrate on the proteins. (See Ex. 9.) 2. A white precipitate is also obtained witli solutions of urea. (See Exs. 341 and 342.) 3. Sulphates give a white precipitate of mercurous sulphate. CH. 11.] COLOUR TESTS. 39 4. The reagent is prepared by dissolving one part by weight of mercury in twice its weight of concentrated nitric acid (Sp. gr. i-42). The mixture is slightly warmed towards the end. It is then treated with twice its bulk of distilled water, allowed to settle over-night, and filtered. It contains mercurous and mercuric nitrates, excess of nitric acid, and a small amount of nitrous acid. 5. The reaction should never be attempted with a strongly alkaline fluid, since the alkaU will precipitate the yellow or black oxides of mercury. 6. If an excess of the reagent be employed the red colour is often dis- charged on boiling. 7. The reaction is given with all aromatic substances that contain a hydroxyl group attached to the benzene ring. Thus it is given by phenol, salicylic acid, and naphthol, but is not given by benzoic acid. 8. The aromatic substance derived from protein that is responsible for the reaction is tyrosine. CeH,<^°g^ j;-^;-^^^;^^^;^ |^j. {See Ex. 92.) 23. The glyoxylic reaction. (Hopkins and Cole.) Treat 2 or 3 cc. of the fluid with the same bulk of " reduced oxalic acid " ("glyoxylic reagent"). Mix and add an equal volume of con- centrated sulphuric acid, pouring it down the side of the tube. A purple ring forms at the junction of the fluids. ^ Mix the fluids by shaking the tubes gently from side to side. The purple colour spreads through the whole fluid. Notes. — i. The " glyoxylic reagent " is prepared by one of the follow- ing methods : — A. Treat half a Utre of saturated solution of oxaUc acid with 40 grammes of 2 per cent, sodium amalgam in a tall cylinder. When all the hydrogen has been evolved the solution is filtered and diluted with twice its volume of distilled water. The solution now contains oxalic acid, sodium binoxalate, and glyoxylic acid (COOH.CHO). It should be kept in a, closed bottle containing a little chloroform. B. In a flask place 10 grammes of powdered magnesium and just cover with distilled water. Slowly add 250 cc. of saturated oxahc acid, cooling under the tap at intervals. Filter off the insoluble magnesium oxalate, acidify with acetic acid, dilute to one litre with distilled water, and bottle as above. 2. The reaction does not succeed in the presence of nitrates, chlorates, nitrites, or excess of chlorides. 3. The colour is not well seen if the protein is mixed with certain carbo- hydrates {e.g. cane-sugar), owing to the char produced by the strong sulphuric acid. 4. It is important to use pure sulphuric acid for this test. It sometimes fails owing to the presence of impurities in the acid employed. At the same time it must be admitted that a very minute trace of ferric chloride does sometimes increase the intensity of the colour. 40 THE PROTEINS. [CH. II. 5. In performing the test on a solid substance, like fibrin, or keratin, a small amount of the material should be heated with a few cc. of the reduced oxalic acid and an equal volume of strong sulphuric acid. The mixture is shaken, and as the protein dissolves in the strong acid both the fluid and the solid particles assume a purple colour. 6. The substance in the protein molecule that is responsible for the reaction is tryptophane (indol-amino-propionic-acid) CjiHuNjOj. CH NH // \C HC HC C CHj.CH OOOH 'CH w CH NH 7. A similar reaction can be obtained by using a very dilute (1:250) solution of formaldehyde containing a trace of an oxidising reagent hke ferric chloride. The authors of the original reaction regarded this test (Rosenheim's) as being different from the glyoxyUc test, though the whole question is still confused. 8. The author has shown that many substances, especially aldehyde?, react with tryptophane to yield coloured products in the presence of strong HCl or H2SO1. Most of these reactions only succeed in the presence of an oxidising reagent, and are possibly due to a reaction with some oxidation pro- duct of tryptophane. 24. The biuret reaction (Piotrowski's reaction). Treat about 3 cc. of the solution with i cc. of 40 per cent, sodium hydroxide. Add a single drop of a i per cent, solution of copper sulphate. A violet or pink colour is produced. Notes. — i. The reaction is of especial importance in testing for proteoses and peptones, which give a rose colour. It is generally stated that other pro- teins give a violet colour, but usually egg-albumin gives a distinct rose tint. 2. It is important to avoid an excess of copper sulphate, the blue copper colour obscuring the violet or rose tint. 3. The test cannot be applied in the presence of a large amount of magnesium sulphate, owing to the precipitation of magnesium hydroxide by the alkah. 4. If the solution contains much ammonium sulphate it must be treated with a large excess of strong sodium hydroxide, as directed in Ex. 57. 5. The reaction is given by nearly all substances containing two O H II I — C— N— CH. 11.] COLOUR TESTS. 41 groups attached to one another, to the same nitrogen atom, or to the same carbon atom. Thus it is given by CONHj CONH. CONH. I I I CONH, NH CH, I I CONHj CONH2 Oxamide. Biuret (See Ex. 345). Malonamide. The cause of the reaction with proteins is the presence of one or more groupings of the last type, formed by the condensation of the carboxylic group of an amino-acid with the amino group of another amino-acid. The linkage thus formed is known as the " peptide " linkage. Thus it would be given by the polypeptide, glycyl-alanyl-tyrosine CeH^.OH NHa.CHj.CO. Glycyl- CH3 NH.CH.CO, -alanyl- CH^ NH.CH.COOH. -tyrosine. 25. The sulphur reaction. Boil a little undiluted egg-white or serum with some 40 per cent, sodium hydroxide for two minutes, and then add a drop or two of lead acetate. The solution turns deep black. Notes. — i . This reaction is due to the fact that the sulphur of the protein is hberated as sodium sulphide when boiled with the strong alkali. The sulphide gives a black colour or precipitate of lead sulphide when the solution is subsequently treated with lead acetate. 2. The reaction does not succeed with caseinogen, peptones, and certain other proteins. 3. The sulphur in the protein is mainly combined as CH,.SH CHj.S.S.CHj I I I CH.NH, or CH.NH. CH.NHj I I I COOH COOH COOH Cystein Cystine 26. Molisch's reaction. Treat 5 cc. of the diluted solution with three or four drops of a i per cent, solution of alpha-naphthol or of thymol in alcohol. Mix, and run about 5 cc. of concentrated sulphuric acid under the fluid. A violet ring is formed at the junction of the two Uquids. Notes. — i. The reaction is due to the presence of a carbohydrate group (glucosamine) in the protein. This is converted by the acid to furfurol, which condenses with the alpha-naphthol or the thymol to give the purple colour. (See notes to Exs. no and 114.) 2. A green ring is often seen in addition to the violet ring. This is due to the action of the sulphuric acid on the alpha-naphthol. 42 THE PROTEINS. [cH. II. E. The heat coagulation of albumins and globulins. These proteins are placed in a class by themselves, because they exhibit most characteristically the phenome- non of heat coagulation. A proper understanding of the conditions governing this phenomenon is so important that students are urged to study them attentively. The following matter should be re-read after the section on metaproteins has been studied. When a solution of albumin or globulin is heated under certain conditions the protein separates in a form which is insoluble in water, dilute salt solutions, acids and alkalies. This is the phenomenon known as " heat coagulation." The term " coagulation " is used as an indication of an irreversible change and to distinguish the condition of the protein from that of " precipitation," in which re-solution can be brought about by a change of reaction, salt content, etc. The two most important conditions affecting heat coagulation are reaction and salt content. It will be found later that albumins and globulins are readily converted into metaproteins by treatment with acids or alkalies, the con- version being much accelerated by a rise in temperature. The metaproteins are soluble in dilute acids or alkalies, but are insoluble in the neutral condition, i.e. in water or neutral salt solutions. An important fact about them is that if a precipitate of metaprotein is boiled it is " coagu- lated," that is, it will not redissolve in dilute acids or alkalies. The conversion of albumin or globulin into meta- protein is called " denaturation," and is a necessary ante- cedent of heat coagulation. This process is best regarded as an hydrolysis, which takes place at all reactions, but most rapidly in either acid or alkaline solutions. At boil- ing point the change is practically instantaneous, no matter what the reaction may be. Should the reaction be at the iso-electric point of the denaturised protein either the whole or CH. II.] HEAT COAGULATION. 43 a considerable part of this is aggregated into flocks (see p. II ). At high temperatures these flocks are coagulated, that is, they do not redissolve on altering the reaction of the fluid. The main effect of neutral salts is to aid the aggrega- tion of the denaturised protein. It often happens that more than one denaturised protein is formed by heating a solution containing albumins and globulins. The iso- electric points of these proteins may differ, so that a certain proportion of the protein will remain non-coagulated at any given reaction. This seems to be true, even in the case of the denaturised protein formed from a pure protein. The addition of a neutral salt will tend to cause flocking of this " soluble " portion of the denaturised protein, and these flocks will be coagulated should the temperature be high enough. The non-coagulated dispersed protein carries an electric charge, which varies with the reaction, being posi- tive in acid solutions and negative in alkaline solutions. We have seen that electro-positive colloids are flocked by negative ions, and electro-negative colloids by positive ions. Further, that this flocking power of the ions is much greater with di- and tri-valent ions than with mono-valent ions. It follows, therefore, that the ideal conditions for the maximal heat coagulation of an albumin or globulin are that the reaction should be at the iso-electric point of the denaturised protein formed, and that there should be present di- or tri-valent ions both positive and negative. These conditions are met by having the boiling solution very faintly acid to litmus and adding a trace of calcium chloride or magnesium sulphate. It is advisable to add the salt, to boil the solution, and then to change the reaction slowly by the addition of dilute sodium carbonate or i per cent, acetic acid, so that the final reaction is just acid to litmus. The exact point and procedure can only be determined by experience since it varies considerably with the concentration of the protein, etc. 44 THE PROTEINS. [CH. II. For the following five exercises use serum that has been diluted with lo volumes of distilled water. 27. Boil 5 cc. in a test-tube that has been previously rinsed with distilled water. The solution becomes opalescent, but usually no definite coagulum is formed. Cool the tube and add i per cent. acetic acid drop by drop. A precipitate of metaprotein is formed soluble in excess of acid. NoTB. — The reaction of the mixture after boiling is distinctly alkaline to the iso-electric point of the denaturised proteins formed. These are precipi- tated by bringing the reaction of the solution to the iso-electric point by the addition of acetic acid, but are redissolved by an excess. 28. To 5 cc. add two drops of i per cent, acetic acid and boil. A white flocculent coagulum is formed. Cool the tube and add two or three drops of strong nitric or acetic acid. The coagulum does not dissolve. Notes. — i. The amount of acid added is such that, after boiling, the reaction is near to the iso-electric point of the denaturised proteins. These are therefore precipitated and then coagulated. 2. The addition of the strong acid is to ensure that the precipitate that appears on boiUng does not consist of calcium or magnesium phosphate, which is soluble in dilute nitric acid. That such a phosphatic precipitate can be formed on boiling certain solutions is shown by the following experiment. Treat a solution of calcium chloride with sodium phosphate and then with excess of sodium carbonate. A precipitate of Ca3(POi)2 appears. Add acetic acid drop by drop till the precipitate j ust dissolves owing to the formation of the acid phosphate. Boil the solution for half a minute. A white precipitate appears. Add a drop or two of nitric acid. The precipitate dissolves. The appearance of the precipitate of Caj(P0i)3 on boiling is due to the alteration of reaction as the CO2 is evolved. 29. Treat 5 cc. of the solution with 0-4 per cent, hydrochloric acid, drop by drop, until the precipitate obtained by the first drop or two has redissolved (about five drops are usually necessary). Boil. The solution remains clear. Cool the tube and add 2 per cent. sodium carbonate, drop by drop. A precipitate of metaprotein is formed which redissolves in excess. Note, — The precipitate that first forms consists of globuUn (see Ex. 32). On adding enough HCl to redissolve this the reaction is such that it is acid to the iso-electric point of the denaturised proteins. These are precipitated by an alkali and redissolve in an excess. 30. To 5 cc. add two drops of 2 per cent, sodium carbonate and boil. The solution remains quite clear. Cool the tube and add I per cent, acetic acid, drop by drop. A precipitate of metaprotein is formed, soluble in excess. CH. II.J ALBUMINS AND GLOBULINS. 45 Note. — The results in this exercise are similar to those obtained in Ex. 27, except that the increased alkalinity prevents the formation of the opalescence obtained in the absence of added alkali. 31. To 5 cc. add a drop of 2 per cent, calcium chloride and boil. A considerable coagulum is obtained. Note. — Though the solution is alkaline to the iso-electric point, the di- valent positive calcium ion precipitates a certain proportion of the negative colloidal protein. F. The properties of albumins and globulins. Globulins are generally insoluble in distilled water, but soluble in dilute acids and alkalies, and in weak solutions of neutral salts. A neutral solution in a dilute salt is coagulated on boiling. A solution in dilute acid or alkali is converted into a solution of metaprotein on boiling. If the globulin be dissolved in a minimum amount of a neutral salt solution and the solution be diluted with several volumes of distilled water, the globulin is partially precipitated, for a certain concentration of salt is necessary to keep the globulin in solution. If the globulin be dis- solved in dilute acid or alkali, there is no precipitation on dilution. The globulins are completely precipitated by full saturation with magnesium sulphate or by half-satura- tion with ammonium sulphate, i.e. by treatment of the solution with an equal volume of a saturated solution of ammonium sulphate. Albumins are soluble in distilled water, dilute salt solutions, dilute acids and alkahes. A neutral solution in water or salt is coagulated on boil- ing. A solution in dilute acid or alkali is converted to a solution of metaprotein on boiling. Solutions of albumins are not precipitated by saturation with magnesium sulphate nor by half-saturation with 46 THE PROTEINS. [oH. II. ammonium sulphate if the reaction of the solution be neutral or alkaline. They are partially precipitated by solutions of these substances in the presence of acid. They are completely precipitated by full saturation with ammonium sulphate from a neutral, acid, or alkaline solution. The solubility in water of the globulins of blood serum is apparently raqdified by the presence of certain " lipines " (see p. 153). If the serum globulins be precipitated by half saturation with ammonium sulphate, the pre- cipitate dissolved in water, as described in Ex. 36, and the solution thoroughly dialysed, it will be found that only a portion of the protein is precipitated by the dialysis. The fraction that remains soluble in water has been called " pseudo-globulin " to distinguish it from the water-insoluble fraction or " eu-globuUn." It was formerly believed that " pseudo-globuUn " was an albumin and that it was impossible to separate the globulins from the albumins by half saturation with ammonium sulphate. Recent work by Hartley on the globulins of serum and by Eaistrick on those of milk have demonstrated, however, that the distribution of nitrogen as mon-amino acids and as bases is practically the same for the two globulin fractions, which difier appreciably from the albumin fraction in this respect. It is probable that the insolubility of the " eu-globulin " in water is due to its association -with lipoid. 32. Dilute 5 cc. of serum with 50 cc. of distilled water. A faint cloud of serum globulin is formed. Cautiously add 0-4 per cent, hydrochloric acid or i per cent, acetic acid until the cloud has reached its maximum density. Divide into two portions A and B. To A add a couple of drops of a saturated solution of ammonium sulphate. The solution becomes quite clear. To B add a couple of drops of strong acid. The cloud disappears. Note. — The globulin of the serum is held in solution both by salts and alkahes. Dilution alone produces a very small precipitate, but if the solution be now treated with j ust sufficient acid to neutralise the alkaU, a much larger fraction of the globuhn is thrown down. This globulin is soluble in dilute neutral salts, or in an excess of acid. 33. Prepare a suspension of globulin by the following method. To 15 cc. of serum in a beaker add 2 cc. (about 30 drops) of i per cent, acetic acid and 100 cc. distilled water. Stir and allow the mixture to stand for about 20 minutes. A precipitate of globulin settles down. Very carefully pour off the supernatant fluid and divide the suspended globulin into two equal portions in clean test- tubes. With these perform the two following exercises. CH. II.] GLOBULINS. 47 34. Add a 5 per cent, solution of sodium chloride, drop by drop, till the globuKn has just dissolved. It is not easy to get a crystal clear solution, probably owing to the presence of a trace of some other protein (nucleo-protein). Divide the solution into three por- tions, (a), {b) and (c). (a) Boil. The protein is coagulated. (6) Dilute with about five volumes of distilled water. The globulin is partially reprecipitated. (c) Treat with an equal volume of saturated ammonium sulphate solution. The globulin is reprecipitated. 35. Add 0-4 per cent. HCl, drop by drop, till the globulin has^MS^ dissolved. Divide the solution into three portions, (d), (e) and (/). (d) Add 2 per cent, sodium carbonate solution till the globulin is partially reprecipitated (one or two drops only are necessary). Now add a few drops of 5 per cent, sodium chloride. The precipitate of globulin redissolves. (e) Boil the solution. The protein is not coagulated. Cool under the tap and add enough 2 per cent, sodium carbon- ate to precipitate the metaprotein that has been formed by boiling. Now add a few drops of 5 per cent, sodium chloride. The precipitate of metaprotein does not dissolve. (/) Dilute with about five volumes of distilled water. The globulin is not thrown out of solution. 36. Mix about 10 cc. of undiluted serum with an exactly equal quantity of a saturated solution of ammonium sx^phate. A thick white precipitate is formed consisting of the whole of the globulin. Filter through a dry filter paper into a dry test-tube. Label the filtrate A. Scrape the precipitate off the paper and treat it with distilled water. The precipitate dissolves, the ammonium sulphate adhering to it forming a dilute salt solution which allows the globuhn to go into solution. Boil a portion of this solution. A heat- coagulum is formed. 37. Filtrate A contains serum-albumin in the presence of half-saturated ammonium sulphate. Apply the following tests : {a) Boil a portion. A heat-coagulum is fonried. 48 THE PROTEINS. [CH. II. (b) To another add one drop of strong acetic acid. A white precipitate of serum-albumin is formed. (c) Grind the remainder in a mortar with solid ammonium sulphate, till the fluid is saturated. A white precipitate of serum-albumin is formed. Filter oif the precipitate and test the filtrate for proteins either by boiling or by the glyoxyKc or xanthoproteic reactions. Proteins are absent, showing that all the proteins of serum are precipi- tated by complete saturation with ammonium sulphate. Note. — A certain test for albumin in a solution is to half-saturate it with ammonium sulphate, filter off any precipitate that may be present and boil the filtrate. A heat-coagulum indicates albumin. 38. Serum has been dialysed in collodion sacs (see p. 2) for 24 hours against distilled water in a tall cyhnder. Note the heavy precipitate of serum-globulin that has fallen to the bottom of the sac. Pipette off some of the clear fluid Eind add an equal volume of saturated ammonium sulphate. A precipitate of " pseudo-globuUn " (see note on p. 46) is obtained. Now pipette off some of the deposit, add about two volumes of distilled water, and divide into three portions, A, B, and C. To A add a couple of drops of saturated ammonium sulphate. To B add a drop of dilute soda. To C add a drop or two of dilute HCl. The globulin dissolves in each case. 39. Dilute 3 cc. of serum with about five times its volume of tap water, add a drop of 2 per cent, calcium chloride, and boil the mixture in a boiling tube. Add one drop of i per cent, acetic acid and boil again. Continue this procedure until a definite coagulum has formed, and the fluid between the flocks appears to be clear when examined in a thin layer. Filter. The filtrate should run through the paper rapidly and be crystal clear. If it filters slowly or comes through opalescent, repeat the experiment until the desired result is obtained. Test the filtrate for proteins by MiUon's and the xanthoproteic tests. Only insignificant traces should be found. Note. — This is the method usually adopted for removing albumins and globulins from solution, but it must be noted that it is almost impossible to remove the last traces by this procedure. If it is necessary to do so, colloidal iron (see Ex. 310), metaphosphoric acid (see Ex. 17), or some otlier reagent must be employed. The objection to the use of such reagents is that they are apt to precipitate the proteoses, peptones, etc. CH. II.J EGG-WHITE. 49 40. The action of mineral acids on albumias and globulins. (Heller's test.) Place a few cc. of strong nitric acid in a narrow test-tube. By means of a pipette add an equal volume of dilute ■ serum or egg-white, inclining the tube during the addition so that the protein solution is "layered" on the surface of the acid. A white ring appears at or immediately above the junction of the two fluids. Note. — This is one of the most important tests for albumins in urine. The reaction is also given by HCl and HjSOj, but not so readily as by HNO3. The primary proteoses also give a precipitate but this is soluble on warming. G. The chemistry of egg-white. 41. In egg-white which has been well beaten with a whisk (to break up the containing membranes), and diluted with four times its volume of distilled water, note a precipitate of ovo-mucin and globulin. Perform the following tests : («) Take the reaction to litmus.. It is alkaline. (b) Cautiously neutralise with dilute acetic acid. A sUght increase in the precipitate of ovo-mucin and globulin is noticed. Remove this by filtration if necessary, and with the filtrate perform the following reactions : (c) Boil a portion. A coagulum is formed, indicating the presence of either a globulin or an albumin. {d) Make another portion very faintly alkaline by the addition of a drop or two of 2 per cent. NajCOg. Now add an equal bulk of saturated (NH4)2S04. A shght precipitate of globuhn or albumin is formed. Filter this off, and boil a portion of the filtrate with a drop of i per cent, acetic acid. A coagulum of albumin is formed. Saturate the remainder of this filtrate with ammonium sulphate by grinding with the sohd in a mortar. A precipitate of albumin is formed. (e) Completely remove the globuhn and albumin by boiling. Filter and apply Millon's or the xanthoproteic protein test to the filtrate. Protein is found in small quantities. 50 THE PROTEINS. [CH. II. This protein is known as ovo-mucoid. It is not coagu- lated by boiling, nor precipitated by acetic acid. It is precipitated by saturation with ammonium sulphate, and also by strong alcohol. 42. The crystallisation of egg-albumin. (Hopkins' method.) Separate the white from a number of new-laid eggs, taking care not to allow any of the yolk to mix with the white. Measure the egg-white and churn it up with an exactly equal volume of a neutral fully- saturated solution of ammonium sulphate by means of a whisk, adding the sulphate in portions and mixing thoroughly after every addition. Notice the strong smell of ammonia that is evolved. Filter the mixture through a large pleated filter-paper. Measure the filtrate. Take 100 cc. of it and cautiously treat it with 10 per cent, acetic acid from a burette, noting the original level of the acid in the burette. Add the acid a drop or two at a time, shaking gently the whole time, imtil the precipitate produced at each addition no longer dissolves on shaking, and the whole mixture is rather opales- cent. This point is usually somewhat difiicult to determine, owing to the large number of air-bubbles that become suspended in the fluid and closely resemble a fine precipitate. When you are satisfied that a permanent precipitate has been produced, run in i cc. of the acid in addition to the amount already added. A heavy white precipi- tate is thus produced. Note the amount of acid that has been used for the portion of 100 cc, and treat the remainder of the filtrate with a corresponding amount of acid. Mix the two portions thoroughly and allow to stand overnight. Note that the precipitate has in- creased somewhat in amount. Mount a drop of the suspension on a slide, cover with a slip, but do not press. Examine under the high power of the microscope, and note the aggregation of very fine needles. Tl]|e albumin can be recrystalUsed by filtering, dissolving in as small an amount of water as possible, filtering again, and cautiously adding to the filtrate saturated ammonium sulphate till a faint permanent precipitate is produced. If the mixture be allowed to stand for some hours the albumin will separate out as fine needles. NoTES.^i. For the experiment to succeed it is absolutely essential that all the eggs employed be perfectly fresh. One rather stale egg may interfere with the crystallisation of a large number of fresh eggs CH. II.J METAPROTEINS. 51 2. It is important to add exactly the amount of acetic acid mentioned, that is, one per mille above the amount required to give a faint permanent precipitate. 3. The same method can be employed for the crystallisation of serum- albumin from the perfectly fresh serum of a horse, ass or mule. H. The Metaproteins. The metaprateins are derived from the albumins and globulins by hydrolysis. This can be effected rapidly by dilute acids and alkalies at temperatures over 60° C. (see Exs. 29 and 30) : more slowly at body temperature. They are formed immediately by the action of strong mineral acids at room temperature. They are insoluble in water, strong mineral acids, and all solutions of neutral salts, but are soluble in dilute acids or alkalies in the absence of any large amount of neutral salts. They are not thrown out of solution (in acid or alkali) by boiling. But if such a solution be neutralised or precipitated by the addition of an excess of a neutral salt, the suspended metaprotein is coagulated on boiling, so that it will no longer dissolve in acid or alkali. Piepaiation. Egg-white or serum is diluted with ten times its volume of either 0-4 per cent, hydrochloric acid or o-i per cent, sodium hydroxide and the mixture placed in a water bath or incubator at 40° C. for about twenty- four hours. The albumins and globulins are hydrolysed to metaprotein. 43. Neutralise about 25 cc. with 2 per cent, sodium carbonate, or 0'4 per cent. HCl, depending on the original reaction of the fluid. A bulky precipitate of metaprotein forms. The acid or alkali should be added until the maximum amount of precipitate is produced. The reaction then will probably be very sUghtly acid to litmus. Filter. The filtrate generally comes through very slowly. When as much as possible of the fluid has been removed in this way transfer the fluid on the paper to a small beaker, open the paper, and add the precipitate to the fluid that has been poured off, dilute with a httle water, and divide the suspension into six equal portions and with them perform the following six exercises. 44. Add some 0-4 per cent. HCl. The precipitate dissolves. Neutralise vyith sodium Cc^rbojic^te ; the precipitate reappears, 52 THE PROTEINS. [CH. II. 45. Add concentrated HCl drop by drop. The precipitate dissolves with the first drop, but generally reappears when an excess is added (see Ex. 21, note 2, and Ex. 40). 46. Dissolve in a little 0-4 per cent. HCl. Boil the solution : a coagulum is not formed. Cool under the tap and neutraUse with 0-2 per cent. Na^COs. A precipitate is formed which is soluble in an excess of the alkali. 47. Boil. Cool, and add some 0-4 per cent. HCl. The precipitate does not dissolve, i.e. metaprotein is coagulated when boiled in suspension. 48. Add a saturated solution of ammonium sulphate drop by drop. The precipitate does not dissolve in any dilution of the salt. The insolubihty in dilute solutions of neutral salts is an important distinction between metaproteins and globulins (see Ex. 32 and 34). 49. Dissolve in a httle 0-4 per cent. HCl. Treat the solution with an equal volume of saturated amrnonium sulphate solution. The protein is precipitated. I. The Albumoses or Proteoses and Peptones. These hydrolysed proteins are obtained by the further action of acids or alkalies on globulins, albumins and meta- proteins. They are best formed by the action of pepsin and hydrochloric acid on these proteins. Peptone is the end product of gastric digestion. They are prepared on a commercial scale and sold as — (i.) Witte's peptone, which is prepared from fibrin and consists of a mixture of albumoses and peptone. (ii.) Savory and Moore's peptone, which is prepared from meat, and only contains traces of albumoses. The following scheme indicates the successive steps CH. II.] PROTEOSES AND PEPTONES. 53 in the digestion of fibrin by pepsin and 0-2 per cent, hydrochloric acid : — Fibrin I Soluble Globulin I IVIetaprotein I Primary albumoses Secondaiy albumoses Proto-albumose : Hetero-albumose. Thio-albumose : Synalbumose, etc. Peptones. The following scheme shews the method adopted for the isolation of certain of the albumoses : — ■ Neutral Witte's peptone, treated with equal volume of saturated ammonium sulphate solution. Precipitate : dis- solved in water. Treated with 2 volumes of strong alcohol. Ppt. Hetero- albumose. Filtrate. Proto- albumose. Filtrate, treated with half its volume of saturated ammonium sulphate. Ppt. dissolved in water. Treated with 2 volumes of alcohol. - Ppt. Thio- albumose. Filtrate Saturated with ammo- nium sulphate. Ppt. dissolved in water. Treated with 2 volumes of strong alcohol. Ppt. Neglect. Filtrate. Treated with I vols, of alco- hol. Ppt. Synalbumose. - Filtrate. Peptones The primary allbumoses are soluble in water, dilute acids, alkalies and salt solutions. Their solutions are not coagulated on heating. They are precipitated by half- saturation with ammonium sulphate. They give a pre- 54 THE PROTEINS. [CH. II. cipitate, that disappears on warming and reappears on cooling, either with nitric acid or potassium ferrocyanide and acetic acid. They also give a precipitate in the cold with copper sulphate. They give all the ordinary protein colour reactions, with the exception of Molisch's. The secondaxy albumoses have somewhat similar pro- perties to those of the primary albumoses : but they are not precipitated by nitric acid, ferrocyanic acid, or copper sulphate. They require more than half-saturation with ammo- nium sulphate to precipitate them, but are completely precipitated by full saturation. Thio-albumose gives all the protein colour reactions and is particularly rich in sulphur (hence its name). Synalbumose gives the protein reactions, with the exception of the glyoxylic test. The peptones are very soluble proteins of rather a low molecular weight, so that they slowly diffuse through parchment membranes. They are the only proteins not precipitated by full saturation with ammonium sulphate. They fail to give precipitates with Esbach's and Briicke's reagents or ferrocyanic acid, but are precipitated by other protein precipitants, as tannic acid, phosphotungstic acid and lead acetate. For the following reactions make a 5 per cent, solution of " Witte's peptone " in hot water, just acidify with acetic acid and filter from a small amount of insoluble material (nuclein?). The solution contains all the albumoses and peptones. 50. Dilute a small amount with three or four times its bulk of water, and to portions of this apply the usual colour reactions for protein. They are all obtained. Note, in particular, that the biuret test gives a rose colour. 51. Boil the solution with a trace of acetic acid : it does not form a coagulum. CH. II.] PROTEOSES AND PEPTONES. 55 52. Add a little tannic acid : a white precipitate is formed. 53. Add a little Esbach's or Briicke's solution : a yellow or white precipitate is formed. 54. Add a little lead acetate solution : a white precipitate is formed. 55. To ID cc. of the 5 per cent, solution in a small beaker add 10 cc. of a saturated solution of ammonium sulphate. A white precipitate of the primary albumoses is formed. Stir the mixture vigorously for a short time with a glass rod that has one end covered with a small piece of rubber tubing : allow to stand for a few minutes. The precipitate will usually gather together and can be almost completely collected as a gummy mass on the end of the rod. Transfer it to about 5 cc. of hot water. The precipitate dissolves. Cool the solution and divide it into three portions. (a) Add a drop of strong acetic acid and two drops of potassium ferrocyanide. A white precipitate is formed, which dis- appears on heating and reappears on cooling. (b) To another portion add a few drops of strong nitric acid. A white precipitate is formed, which disappears on heating and reappears on cooling. (c) To the third portion add a drop of copper sulphate solution. A white precipitate is formed. 56. The fluid from which the main mass of primary albumoses has been removed is filtered and treated in a beaker with a single drop of sulphuric acid, and then with ammonium sulphate that has been finely powdered in a mortar. The mixture is stirred vigorously till the fluid is saturated with the salt. A flocculent precipitate of the secondary albumoses (deutero-albumoses) is formed. Collect this on the rod as before, dissolve in a Uttle water, divide the solution into three parts, and repeat the three tests already performed with the primary albumoses. A precipitate is not formed by any of the reagents. 57. The fluid from which the secondary albumoses have been removed contains peptone. Filter it, and treat a portion of the 56 THE PROTEINS. [cH. II. filtrate with twice its volume of 40 per cent, sodium hydroxide and a drop of I per cent, copper sulphate. A pink colour appears, due to the presence of peptone. Impoitant Note. — This large excess of strong NaOH most be added in order to decompose the (NHj);^©, with which the solution is saturated. The characteristic rose colour is only obtained when the alkaUnity is due to NaOH, ammonia being quite inefficient. 5 cc. of saturated (NH4)2S04 solution contains about 3-75 grms. of the salt. This requires 2-27 grms. of NaOH. 10 cc. of 40 per cent. NaOH, containing 4 grms. of NaOH, is thus sufificient. 58. Evaporate a small portion of the original fluid to complete dr57ness, fuiishing the process on a water bath in order to prevent charring. Rub up the residue with successive small quantities of strong alcohol (95 per cent.). Add the extracts together, filter and evaporate them to dryness on a water bath. Dissolve the residue from this evaporation in a httle water and test for proteins by the various colour tests. Only insignificant traces are present, showing that albumoses and peptones are insoluble in strong alcohol. Note. — It is frequently desirable to remove all proteins from a solution before testing for certain substances, e.g. sugars, bile-salts, urea, etc. In the case of albumoses and peptones this can only be effected by the method described above, advantage being taken of the solubiUty of sugars, etc., in alcohol, and the insolubility of all proteins in the same. The aqueous solution prepared in this way will be spoken of as " an alcoholic extract." Peptones. Use a 2 per cent, solution of Savory and Moore's peptone, which is usually free from albumoses. 59. Apply the usuelI colour reactions for proteins. They are all obtained. Note. — The glyoxyUc reaction may not be very intense, owing to the presence of chlorides in the preparation. Pure peptone, when freed from chloride by appropriate means, gives a very good glyoxyUc reaction. 60. Add a drop or two of strong acetic acid and a drop of potassium ferrocyanide. No precipitate is produced, showing that the primary albumoses are absent. 61. Add a little Esbach's or Briicke's solution. A very sUght or no precipitate is formed, if the solution be free from albumoses. 62. Saturate a portion with ammoniuutn sulphate. No precipi- tate, or only a slight one, is produced, showing that albumoses are absent. CH. II.] MUCIN. 57 63. Treat 5 cc. of the filtrate from Ex. 62 with two volumes of 40 per cent. NaOH and a drop of copper sulphate. A pink colour is formed. 64. Add a few drops of a solution of tannic acid. A white precipitate is formed. 65. Add a few drops of a solution of lead acetate. A white precipitate is formed. J. The Gluco-proteins. These bodies are conjugated proteins, the protein being united to a carbohydrate group. They consist of the mucins and mucinoids or mucoids. The mucins are found in connective tissue and are secreted by certain of the saHvary glands and various parts of the alimentary canal, notably the large intestine. Their solutions are viscous. They are soluble in dilute alkalies and are precipitated from solution by acetic acid, the precipitate being insoluble in excess of acetic acid. They are also soluble in o-i per cent, hydrochloric acid. On hydrolysis with acids the sugar group is split off and will reduce Fehling's solution. The mucoids are not so viscous and not so readily precipitated by acetic acid, the precipitate being soluble in excess. They are found in ovarian cysts and in white of egg [see Ex. 41 (e)]. Fieparation of Hucin. Mince the submaxillary gland of an ox, grind with sand and add o-i per cent. NaOH (i litre to 50 grams of the moist gland). Shake well in a large bottle from time to time and leave for about half an hour. Strain through muslin and filter through coarse filter-paper. (This crude solution should not be prepared too long before use, as mucin loses its characteristic properties if left standing with alkalies.) 66. Add acetic acid drop by drop. A stringy precipitate is formed, insoluble in excess of the acid. 67. Remove the precipitate on a glass rod, wash with water, and apply the usual colour reactions for proteins, e.g. xanthoproteic, glyoxyhc, and MUlon's. They are all given by mucin. 58 THE PROTEINS. [CH. 11. 68. Treat some of the precipitate with o-i per cent. HCl. The mucin dissolves. 69. Treat some of the precipitate with 2 per cent. NajCOj. The mucin dissolves. K. The reactions of certain Albuminoids. Gelatin is found in the body in the form of its anhy- dride, collagen. This occurs in v^^hite fibrous tissue and in the organic substance of bones, and can be converted into gelatin by boiling v^^ith a dilute acid. Dried gelatin swells in cold water, but is quite insoluble in it. On warming, a more or less viscid solution is obtained, which solidifies to a jelly on cooling provided the concentration be greater than I per cent. This process is reversible on warming and cool- ing. It is precipitated by half-saturation with ammonium sulphate, by tannic acid, phosphotungstic acid, Esbach's and Briicke's reagents, but not by normal lead acetate. On complete hydrolysis it yields a high percentage of its nitrogen in the form of glycine, but only traces in the form of the aromatic amino-acids, tyrosine, or trytophane, and none as the sulphur-containing compound, cystine. There- fore its solutions fail to give the glyoxylic, Millon's and sulphur colour tests for proteins, and only give a shght xanthoproteic test, which is due, either to an impurity or to a small amount of phenyl-alanine. 70. Break gelatin up into small pieces and add a small amount of cold water. The gelatin does not dissolve. Immerse the test- tube in a beaker of boiling water and leave it for a short time. The gelatin dissolves. Cool the tube under the tap : the gelatin sets to a jelly. Perform the following tests with an approximately i per cent, solution of gelatin : (a) Xanthoproteic reaction : slight. (b) Millon's reaction : very slight, showing absence of tyrosine from gelatin molecule. (See notes to Ex. 22.) (c) GlyoxyHc reaction : not obtained, showing absence of tryptophane. (Ex. 23.) CH. 11.] GELATIN AND KERATIN. 59 {d) Biuret reaction : violet colour. {e) Sulphur reaction : not obtained, showing absence of cystine. (Ex. 25.) (/) Add acetic acid : no precipitate. (§■) Add acetic acid and potassium ferrocyanide : very shght or no precipitate. [h) Add tannic acid : white precipitate. (i) Add lead acetate : very slight or no precipitate. (j) Half saturate with ammonium sulphate. The whole of the gelatin is precipitated, as shown by a negative biuret test in the filtrate (distinction from peptones). {k) Add Esbach's or Briicke's solution ; yellow or white precipi- tate (distinction from peptones). Keratin, An insoluble body found in the hair, skin, nails, and horns. Remarkable for the high percentage of cystine it yields on acid hydrolysis. 71. Perform the following tests by using horn shavings, or hair. Note insolubility in hot or cold water, dilute acids, and dilute alkahes. {a) Xanthoproteic reaction : well marked, (b) Millon's reaction : well marked. (c) Glyoxylic reaction : well marked. (d) Biuret reaction : not obtained, owing to insolubihty. (e) Sulphur reaction : well marked. CHAPTER III. THE NUCLEOPROTEINS, NUCLEINS AND NUCLEIC ACIDS. Nucleic acid is a complicated organic acid containing phosphorus, which is found widely distributed in animal and vegetable tissues. It is a special constituent of the nuclei and is therefore most abundant in cellular organs, such as the thymus, the pancreas, the testis, and the lymphatic glands. Nucleic acid forms salt-like combinations with proteins, the amount and nature of the protein combining with the nucleic acid varying considerably. Such combinations are known as nucleoproteins. They are soluble in water and dilute salt solutions. They show acidic properties, being soluble in alkalies and precipitated by dilute acids. They dissolve to form an opalescent solution in excess of strong acetic acid. (Distinction from mucin.) A rather special form of nucleoprotein is nucleohistone, in which the nucleic acid is combined with the basic protein, histone. It has similar physical properties to those of the other nucleoproteins, but is precipitated as a calcium com- pound by 0-2 per cent, calcium chloride solution. On digesting nucleoprotein with pepsin and hydro- chloric acid, the greater part of the protein is removed as peptone, but a certain amount is still left combined with the nucleic acid. This compound is known as niiclcui. It is insoluble in water and dilute salt solutions, but is soluble in dilute alkalies. By hydrolysis of nuclein by pancreatic juice or better by dilute alkahes, the remainder of the protein is removed, and there is obtained nucleic acid. Nucleic acid is not hydrolysed by trypsin, but it is CH. III.] NUCLEIC ACID. 61 broken down by a variety of ferments found in the tissues. The final products of hydrolysis of thymus nucleic acid are Phosphoric acid. Purine bases, adenine, and guanine. Pyrimidine bases, thymine, and cytosine. An unknown hexose sugar. Yeast nucleic acid differs only in yielding uracil instead of thymine and a pentose sugar (J-ribose) instead of the hexose. As to the composition of the nucleic acids, it has been estabhshed that they consist of certain groups called nucleotides, which can be liberated by the action of enzjones found in the tissues, and called nucleotidases. There are apparently four nucleotides to the molecule of nucleic acid. The nucleotides consist of phosphoric acid-sugar-base, the latter being either a purine base or a pyrimidine base. By the action of an enzyme, called phospho-nuclease, on the mononucleotides the phosphoric acid is split off, leaving the carbohydrate attached to the purine or pyrimidine base. These compounds are known as purine — or pyrimidine — nucleosides. The nucleotides can, however, be attacked by another enzyme, purine — or pyrimidine — nuclease, which splits off the base from the phosphoric acid-sugar complex. The following scheme, suggested by Levene and Jacobs, may represent the structure of thymus-nucleic-acid. Purine-nucleoside Phosphoric acid — hexose — guanine I Phosphoric acid — Hexose — thymine Phosphoric acid — Hexose — cytosine I Ph gsphoric acid - hexose - ad enine Mono-nucleotide. For further information on the subject the student is referred to the valuable monograph by W. Jones.* * Nucleic Acids, by Walter Jones. (Longmans, Green & Co.. London, 1914-) 62 NUCLEOPROTEINS, NUCLEINS, NUCLEIC ACIDS. [cH. III. The purine bases are of especial interest in connection with the origin of uric acid. (6) (i) N— CH Purine is C5H4N4 or (2) HC {s)C- (7) -NH (3) N- SCH(8) -N -C~ (4) (9) The figures in brackets are used for indicating the position of substitution groups. The chief purines of physiological interest have either the (2), (6), or (8) H atoms replaced, so we can write purine as H...(2) H...(6) or simply .H...(8) Adenine is 6-aiTiino-purine, P— — NH2 N=C.NH, H H H or HC C— NH II II N— C— N- CH By the action of a deaminising enzyme, adenase, it is con- verted to hypoxanthine , or 6-oxy-purine, P^^H ^H Guanine is 2-amino-6 oxy-purine, P ■NH, OH CH. III.] NUCLEOPROTEIN. 63 It is converted by guanase into di-oxy-purine, or xanthine, /OH P^OH Hypoxanthine, or xanthine, are oxidised by xanthin oxydase into 2, 6, 8 tri-oxy-purine, or uric acid P^OH (see page 292). The pyrimidine bases are less compHcated than the purine bases. The pyrimidine ring is (i) N=CH (6) I I (2) HC CH (s) II II (3) N— CH (4) Uracil is 2-6-di-oxy-pyrimidine. Thymine is 2-6-di-oxy- 5 -methyl pyrimidine, or 5 -methyl uracil. Cytosine is 6-amino-2-oxy-pyrimidine. Practically nothing is known as to their behaviour in the body. 72. Preparation of nucleoprotein. Lymphatic glands of the ox or sheep, or the thymus of a calf are freed from fat, finely minced, ground with sand and extracted for twelve hours with ten times their weight of distilled water in a large bottle, a small amount of toluol or chloroform being added to prevent decomposition. The bottle should be shaken vigorously at frequent intervals to break up the gelatinous masses that sometimes form. The fluid is strained and centrifugalised to remove all debris (filtration being very slow). This fluid contains both nucleoprotein and nucleo-histone. 73. To a portion add dilute acetic acid till no more precipitate is produced, and place on the water-bath at 37° C. for a few minutes. 64 NUCLEOPROTEINS, NUCLEINS, NUCLEIC ACIDS. [cH. III. A heavy precipitate of nucleoprotein and nucleohistone is formed. Allow this to settle in a cyhnder : pour or pipette off as much of the supernatant fluid as possible, and filter the remainder. Note that the precipitate is soluble in dilute alkalies and is reprecipitated by acidification ; that it dissolves to an opalescent solution in excess of acetic acid (difference from mucin) ; and that it gives all the usual colour reactions of proteins. 74. To another portion add one-tenth of its volume of 2 per cent, calcium chloride and warm to 37° C. A white precipitate of nucleohistone is formed. Pour off the supernatant fluid, and to this fluid add dilute acetic acid drop by drop ; a white precipitate of nucleoprotein is produced. 75. Precipitate the nucleoprotein and nucleohistone from the remainder of the fluid by means of acetic acid as in Ex. 73. Collect the precipitate on a filter paper, allow it to drain well, and then transfer it by means of a spatula to a small thimble-shaped porcelain capsule. Heat carefully, first to drive off the water, and then to carbonise the residue. Add one-third of a crucible full of fusion mixture (K2CO3 two parts, KNO3 one part), and heat as strongly as possible till the mass fuses. Allow the melt to cool, and extract it with nitric acid (diluted with an equal quantity of distilled water) till the mixture no longer effervesces. Filter : treat the filtrate with about one-tenth of its volume of strong nitric acid and one- third its volume of ammonium molybdate ; boil for two minutes. A yellow precipitate of ammonium phospho-molybdate separates out, often on the sides of the vessel. The phosphorus of the nucleic acid has been oxidised to phosphoric acid. 76. Preparation of thymus nucleic acid. (After W. Jones.) To a boiling mixture of 2 litres of water, 100 grms. sodium acetate and 23 grms. of caustic soda, add in small successive portions i kilo, of trimmed and finely ground calves thymus. Immerse the vessel for two hours in boihng water, stirring occasionally. Dilute with one- third volume of water and make faintly but distinctly acid to htmus with 50 per cent, acetic acid. The amount of acid required is usually about 100 cc, but the final additions must be made extremely cautiously until a point is reached which allows of good filtration. CH. III.J GUANINE AND ADENINE. 65 If a portion does not filterwell after being boiled and dried on a paper heated with boiling water, the point must be reached by the addition of more acetic acid or of caustic soda. Now boil the bulk and filter, using a hot water fimnel. Concentrate the filtrate on a water bath to about 750 cc, and pour the warm solution slowly into i litre of 95 per cent, alcohol in a large beaker. Allow the mixture to stand over- night. The precipitated sodium nucleate settles to a spongy white mass. Pour off the supernatant fluid and squeeze out the remainder as far as possible by means of a spatula. Wash by decantation first with 80 per cent., and then with 95 per cent, alcohol. Squeeze out the last wash fluid as much as possible and transfer to a flask with 300 cc. of hot water, and heat on the water bath for 30 minutes. Add 10 cc. of 20 per cent, caustic soda, and filter from insoluble phosphates, using a hot water funnel. Acidify with acetic acid and pour into 700 cc. of 95 per cent, alcohol. AUow to stand over-night, wash by decantation with alcohol of increasing strength, and grind in a mortar with absolute alcohol until it has crumbled into a fine white powder. Transfer to a filter with absolute alcohol and dry in a sulphuric acid desiccator. The product should weigh over 30 grms., and consists of the soluble sodium salt of thjnnus nucleic acid. A 4 to 5 per cent, solution in warm water becomes gelatinous at room temperature, the viscosity being decreased both by acetic acid and sodium hydroxide. 77. Preparation of Guanine and Adenine from Nucleic Acid. Heat on a boiling water bath 50 grams, of commercial yeast nucleic acid for two hours with 200 cc. of 10 per cent, sulphuric acid in a flask fitted with a reflux condenser. Treat the hot fluid with strong ammonia. Guanine is precipitated. Continue to add the ammonia till the neutral point is reached, and then add an excess of 2 per cent, of the reagent. Allow to cool and filter. Reserve the filtrate A. Wash the guanine with i per cent, ammonia, adding the washings to A. Suspend the guanine in boiUng water and dissolve in a minimal amount of 20 per cent, sulphuric acid. Add a small amount of good charcoal, boil, and filter. Add ammonia as before to precipi- tate the guanine. Filter, dry at 40° C, and dissolve in 20 to 25 times its weight of boiling 5 per cent, hydrochloric acid. Upon 66 NUCLEOPROTEINS, NUCLEINS, NUCLEIC ACIDS. [cH. III. cooling the solution deposits needle clusters of guanine chloride, C6H5N5O.HCI.2H2O. The filtrate A is filtered again if necessary, and made faintly acid with 20 per cent, sulphuric acid. Boil and add 10 per cent, copper sulphate. The adenine cuprous compound is precipitated. Filter and wash. Suspend in water and decompose by sulphuretted hydrogen. Filter from copper sulphide and evaporate to dryness on the water bath. Dissolve in the smallest possible amount of hot 5 per cent, sulphuric acid, and allow to cool. Adenine sulphate (C5H5N5)2H2S04.2H20 is obtained in crystalline form. CHAPTER IV. THE PREPARATION AND PROPERTIES OF CERTAIN AMINO-ACIDS. Amino-acids are compounds in which a H atom of an alkyl group of an organic acid has been replaced by an amino-group. CH3 CH2.NH2 I I COOH COOH Acetic acid. A-mino-acetic acid [Glycine). All the physiological amino-acids have the amino group attached to the same carbon atom as that to which the carboxylic group is attached, i.e. they are a-amino-acids. CH3 CH3 CH2.NH2 CH2 CH.NH2 1 CH2 1 COOH COOH Propionic acid, a-amino- propionic acid {alanine). COOH ^-amino-propionic acid. The following are the most important amino-acids that have been obtained from proteins by hydrolysis. NHj GROUP I. Neutral ampholytes R - CH.COOH. Glycine (amino-acetic acid) CH2(NHa).C00H. 68 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. Alanine (a-amino-propionic acid) CH3.CH(NH2).COOH. Valine (a-amino-iso-valeric acid) S&^CH.CH(NH2).C00H. Leucine (isobutyl-amino-acetic acid) rS' ^CH.CH2.CH(NH2)COOH. Phenyl-alanine QH5.CH2.CH(NH2)COOH. Tyrosine (^-oxy-phenyl-alanine) ^OH (I) ^«"*^CH2.CH(NH2)COOH (4) Tryptophane (/3-indol alanine) C3H6N.CH2.CH(NH2)COOH. Cystine (dicysteine or di-/3-thio-a-amino propionic acid) Cri2 — o — S — Lrig I i CH(NH2) CH(NH2) I I COOH COOH GROUP II. Acid ampholytes. R - COOH. I CH(NH2).C00H. Aspartic acid CH2.COOH. (amino-succinic acid) | CH(NH2).C00H. Glutaminic acid CHg.COOH. (amino-glutaric acid) CH2 CH(NH2).C00H. CH. IV..1 AMINO-ACIDS. 69 GROUP III. Basic ampholytes. Lysine (a, e, di-amino caproic acid) CH2(NH2).CH2.CH2.CH2.CH(NH2).COOH. Arginine (^-guanidine-a-amino valeric acid) NH ^^C-NH.CH2.CH2.CH2.CH(NH2).COOH. Histidine (/3-iminazole-alanine) CH / '^ NH N I I CH=C— CH2.CH(NH2).COOH. General reactions ol the amino-acids. 1 . They form two classes of salts : (a) With acids, owing to the presence of the — NH2 group (see Ex. 80). (b) With bases, owing to the presence of the — COOH group (see Ex. 79). 2. When dissolved in alcohol and saturated with dry hydrochloric acid they form esters, which are bases (see Ex. 78). R.CH.NH2 R.CH.NH2 I +QH5.0H= I +HA COOH COOQHj This reaction is of considerable importance, as Fischer's method of separation of the amino-acids is based on the fractional distillation of the esters. 3. They combine with aldehydes to form methylene compounds, which are acids (see Ex. 260). 70 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. R.CH.NHj R.CH.N = CH^ I + H.CHO= I + H2O. COOH COOH. This reaction is the basis of Sorensen's method of estimating the production of amino-acids during tryptic digestion. 4. They form insoluble crystalline compounds with /3-naphthalene-sulphochloride. SOaCl^HjjN.CH.R ^ SO^.NH.CH.R ^ j^^j 5. They form moderately soluble copper salts. With the exception of tryptophane (see p. 9S) these can be readily crystallised from boiling water. The tryptophane copper salt is characteristically insoluble, even in boiling water. In the presence of even traces of other amino-acids it be- comes soluble in the solution of their copper salts. 6. They are acted upon by nitrous acid, yielding nitrogen gas (see Ex. 81). R.CH.NH2 HNO2 = R.CH.OH I -f I -H N^-FHaO. COOH COOH This important reaction is the basis of Van Slyke's gaso- metric method for the estimation of amino-acids in blood and tissues. 7. They are optically active with the exception of glycine, which does not contain an asjanmetric carbon atom (see p. 147]. Methods of separation. The proteins can be hydro- lysed by boiling acids, boiling alkalies, or by the action of certain enzymes, e.g. trypsin. The products are then separated by : — I. Fractional crystallisation of the amino-acids, or of their hydrochlorides. In this way tyrosine, leucine, cystine, and glutaminic acid hydrochlor- ide are obtained. CH. IVJ AkiNO-ACIDS. 71 II. Fractional precipitation, i.e. by adding a reagent to the mixture which forms an insoluble com- pound with only one or a few of the substances present. In this way, by the use of mercuric sulphate, tryptophane was first isolated, and cystine and tyrosine can also be obtained ; by the use of mercuric chloride histidine is separ- ated. III. Fractional £^js^i7/a^ioM of the esters. This method, introduced by Emil Fischer, led to the discovery of several of the amino-acids, and serves for the 4 quantitative estimation of some of them. Percentage amounts of some amino-acids in certain proteins : S? iz; §1 o 1-1 S- < M O M O d M O Sa Glycine 3-5 0-4 16-5 25-8 47 — Alanine 2-7 2-2 0-9 2-3 0-8 6-6 1-5 4-2 Leucine 20-0 18-7 10-5 6-0 2-1 21-4 8-0 290 Tyrosine 2-1 2-5 4-5 1-8 3-9 3-2 1-3 Tryptophane . . + + 1-5 I-O + + Cystine 0-3 0-7 0-0 — — 110 0-3 " Aspartic acid . . 1-5 2-5 1-8 0-9 0-6 + 0-3 4-4 Glutaminic acid 8-0 8-5 21-8 34-5 0-9 0-3 37 17 Lysine — — 5-8 3-9 — — 4-3 Arginine — — 4-8 3-0 8-5 c-3 — 5-4 Histidine — 2-6 1-2 0-4 ~ 110 The figures in heavy type draw attention to the reason why certain proteins are used for the preparation of particular amino-acids. 72 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. 78. Glycine. A The preparation of glycine ester hydrochloride from gelatin. (i.) To 300 cc. of pure concentrated hydrochloric acid in a round-bottomed flask add 100 grains, of ordinary glue. (ii.) Heat on a boiling water bath, with occasional shaking, until the glue has dissolved. Fig. 7. Heating on a sand bath under a reflux condenser. (iii.) Boil the mixture on a sand bath imder a reflux condenser for four hours (see fig. 7). (iv.) Transfer the dark product to a Utre distilling flask, and distil off the acid as completely as possible in vacuo, using the apparatus shewn in fig. 8. The fluid is placed in flask A, which is immersed in a water bath maintained CH. IV.] GLYCINE. 73 at 45° to 50° C. Join this flask to another distilHng flask B, by a tight-fitting rubber stopper. Connect the side neck, D, of this to an exhaust pump, and put the pimip in action. The screw clamp, C, is on a piece of Fig. 8. Distillation in vacuo.* pressure tubing attached to a glass tube drawn to a fine capillary that passes to the bottom of the flask. The screw must be tightened until the bubbles of air pass through the fluid at such a rate that they cannot quite be counted. The flask B is cooled by means of a stream of cold water, which passes on to it by means of a double T-piece. In the absence of this it is advisable to place a piece of filter paper on the flask to keep the stream of water well spread. * For alternative method see p. 99. 1A PROPERTIES OF CERl'AiN AMINO-ACIDS. t' CH. IV. (v.) When as much acid as possible has been removed, slowly open the screw C to abolish the vacuum. Cautiously dis- connect the rubber tubing at D before turning off the pump. With the pump connexions shewn in fig. 9, the abolition of the partial vacuum is readily accomphshed without danger. Fig. 9. Connexions of vacuum pump in author's laboratory. With the tap C in the position shewn a small amount of air is admitted through the narrow tube A. When C is turned through a right angle, full suction through E is obtained. (vi.) Disconnect the apparatus cind stopper the side neck of A with a cork. (vii.) To the thick viscous mass in the flask add 500 cc. of absolute alcohol and heat on a water bath under a reflux condenser until it has dissolved. (viii.) Allow the solution to cool somewhat, add 3 to 5 grms. of a good quality animal charcoal, boil on the water bath for 10 minutes, and filter hot into a flat-bottomed litre flask. CH. IV.] GLYCINE. 75 (ix.) Cool the filtrate, first under the tap, and then with ice, and pass in a stream of dry hydrochloric acid gas (see fig. lo) until the fluid is saturated. Fig. 10. Apparatus for saturating a fluid with dry hydrochloric acid gas. B is concentrated hydrochloric acid. A is concentrated sulphuric acid, which is allowed to drop slowly into the flask. C contains concentrated sulphuric acid to dry the gas. The dry gas can be introduced into the fluid by means of a Folin absorption tube, D, though this is not usually necessary. (x.) Boil on a water bath under a reflux condenser for 30 minutes , cool thoroughly, and allow it to stand over-night in a refrigerator. The glycine ester hydrochloride generally separates as a mass of colourless needles. Should this not occur, it is advisable to " sow " the fluid with a small quantity of the crystals obtained from another preparation, or rub the sides of the vessel with a glass rod. [A second crop of crystals can often be ob- tained by concentrating in vacuo and repeating processes (ix.) and (x.)]. 76 PROPERTIES OF CERTAIN AMINO-ACIDS. [cH. IV. (xi.) Filter on a small Buchner funnel (fig. ii), wash with a little ice cold absolute alcohol, and dry in a desiccator. C2H5.OH+HOOC.CH2.NH2.HCI = C2H5OOC.CH2.NH2.HCI.+H2O Glycine hydrochloride. Glycine ester hydrochloride Yield : 10 to 15 grams. B. The conversion of glycine ester hydrochloride into glycine. (i.) Weigh the glycine ester hydrochloride, place it in a 250 cc. round-bottomed flask and add 10 cc. of water. (ii.) Add 100 cc. of ether and cool in a freezing mixture. (iii.) Gradually add 33 per cent, caustic soda, shaking weU during the addi- tion, until the aqueous layer is neutral to litmus. About o-8 cc. are required for every gram of the hydrochloride. (iv.) Add powdered potassium carbonate, shaking vigorously between whiles, until the watery layer is a paste. By treatment with alkali the glycine ester hydrochloride is converted into glycine ester, which is soluble in ether. (v.) Pour off the ether, filter it, and place it in a stoppered flask. (vi.) Add another 50 cc. of ether to the residue, shake well, remove and filter the ether, adding it to that in the stoppered flask. Repeat this once more. (vii.) Dry the ether by shaking for 5 to 10 minutes with about 20 grams of anhydrous potassium carbonate. Decant the ethereal solution and remove the last traces of water by means of anhydrous sodium sulphate. This is prepared by strongly heating 25 grams, of sodium sulphate in a porcelain dish and cooling the warm melt in a desiccator. Allow the cool, finely powdered, sul- phate to stand with the ether for at least six hours. Fig. II. Buchner funnel and filter- ing flask. CH. IV.] GLYCINE. 77 (viii.) Filter off the ether and transfer it to a distilling flask. Connect this to another distilling flask as shewn in fig. 8. (ix.) Distil off the ether under reduced pressure, the receiving flask being thoroughly cooled (best done by packing with ice). (x.) When all the ether has been removed, change the receiving flask and distil over the glycine ester, at a temperature of 44° C. and a pressure of ii mm. mercury. The receiving flask must be well cooled. (xi.) Transfer the distillate to a round-bottomed flask, add lo times its volume of water, and boil on a sand bath under a reflex condenser until the alkaline reaction has dis- appeared. (xii.) Concentrate the solution in an evaporating basin on the water bath. Crystals of glycine are obtained. Properties of Glycine. It crystallises from vi^ater in hard, flattened, colourless prisms. On heating, it becomes brown at 228°, and melts at 232°-236°. The crystals have a sweet taste, from which fact the original name of glyocoll was derived (yXvKv^, sweet : KoXXa, glue). It is readily soluble in water (i part of glycine in 4-3 parts of cold water). It is insoluble in absolute alcohol, and in ether. When boiled with concentrated alkali ammonia is evolved. On treating the residue with hydrochloric acid, hydrocyanic acid is evolved, and oxalic acid is found to be present. Aqueous solutions give a red colour with ferric chloride, similar to that given by acetic acid. On shaking an aqueous solution with benzoyl chloride and sodium carbonate, hippuric acid is formed. QHg.COCI + HaN.CH2.COOH = CeHB.CO.NH.CHg.COOH + HCl. Benzoyl chloride. Glycine. Hippuric acid. 79. Preparation of the copper salt of glycine. To a solution of about 0-5 gram, of glycine in about 40 cc. of distilled water, add an excess of freshly precipitated, well washed cupric hydroxide. 78 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. Boil for 5 minutes and filter. Concentrate the filtrate in an evaporat- ing basin on a boiling water bath, and set the dish aside. Fine blue needles of the copper salt are formed, having the composition CH2.CH.NH2.COO\^„ „„ CH,.CH.NH,.COO ^^ ^"•^a'-'- Note, — The copper hydroxide is prepared by adding ro cc. of 20 per cent, copper sulphate to about 100 cc. of distilled water, and stirring in 16 cc. of N. sodium hydroxide, previously diluted with about 300 cc. of water. The precipitate is filtered and very thoroughly washed with cold distilled water until neutral to litmus. 80. Glutaminic Acid. A. Preparation of Glutaminic acid hydrochloride. (i.) To 100 grams, of gluten flour* in a 500 cc. round bottomed flask add 300 cc. of pure concentrated hydrochloric acid, and heat on the water bath until the gluten has dissolved. (ii.) Add 20 grams, of good decolourising charcoal, to remove the dark " humin substance " that is formed during the subsequent hydrolysis. (iii.) Boil on a sand bath imder a reflux condenser for 6 hours. (iv.) Dilute with an equal volume of water and filter. (v.) Evaporate the filtrate in vacuo to about 150 cc. (see pages 73 and 94). (vi.) Transfer the residue to a 300 cc. Erlenmeyer flask, cool thoroughly, and saturate with dry hydrochloric acid gas (see fig. 10). (vii.) Allow the flask to stand in the ice chest. After 24 to 48 hours a mass of crystals of glutaminic acid hydrochloride separates. Add an equal volume of ice cold alcohol. (viii.) Filter on a Buchner funnel through a piece of well washed linen (handkerchief) cut to fit the funnel. Drain the mother Uquor away as completely as possible. (ix.) Wash the crystals with small amounts of ice cold concen- trated hydrochloric acid. Yield: about 40 grams. * This can be obtained from Messrs, Bishop and Brooke, 21, Cock Lane. Snow Hill, London, E.C. CH. IV.J GLUTAMINIC ACID. 79 B. RecrystalUsation of the hydrochloride. (i.) Dissolve the crystals in about loo cc. of water, boil with a sufficiency of decolourising charcoal, and filter. (ii.) Saturate the cooled filtrate with dry hydrochloric acid gas, and allow to stand in the ice chest over-night. (iii.) Add an equal volume of ice cold absolute alcohol and filter through linen on a Buchner funnel. (iv.) Dry in a vacuum desiccator over potash and sulphuric acid. C. Preparation of glutaminic acid from the hydrochloride. (i.) Weigh the pure, dry, hydrochloride and dissolve it in a minimal amount of water. Add 5-44 cc. of N.NaOH for every gram, this being the amount required to remove the HCl according to the following equation. HCl I CH.(NH2).C00H CH(NH2).C00H I +NaOH = I +NaCl+H20. CH2.CH2.COOH CH2.CH2COOH (ii.) Evaporate the solution in vacuo at 40° to 50° to reduce the volume to 60 to 100 cc. (iii.) Transfer the warm solution to a beaker, and allow it to stand over-night in the ice chest. (iv.) Filter off the crystals on a Buchner, wash with a little cold water, and dry. Yield : 18 to 20 grams. Properties of Glutaminic Acid. It crystallises from water in rhombic tetrahedra, which on rapid heating melt at 2 1 3°. It dissolves in about 100 parts of cold water, but is much less soluble in alcohol. Since it contains two carboxyl and only one amino-group, its aqueous solutions are markedly acid to litmus. The hydrochloride forms 80 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. triclinic tables, which melt at about 193°. This salt is readily soluble in water, but only very slightly soluble in concentrated hydrochloric acid. The calcium salt is quantitatively precipitated by strong alcohol, provided that the solution be sufficiently concentrated (25 to 30 per cent.). On boiling an aqueous solution of the hydrochloride, it is largely converted into the internal anhydride, pyrollidone carboxylic acid. COOH COOH CH.NH, CH, CH2 - COOH CH.— NH CH, CH„ - CO + H,0 This change does not take place in the presence of strong hydrochloric acid, nor during concentration in vacuo at 40° to 45°. Glutaminic acid is especially abundant in vegetable proteins. It is for this reason that gluten flour, consisting of ghadins and glutelins, is used for its prepara- tion. Glutaminic acid is dextrorotatory, [oJd in water = + 12°. In 10 per cent, hydrochloric acid [oJd = + 3i"- 81. Action of nitrous acid. To a solution of glutaminic acid in water add a solution of nitrous acid, obtained by acidifying a strong solution of potassium nitrite with acetic acid. An evolution of nitrogen gas occurs. COOH COOH CH.NHa 1 CHa + HNO2 CH,.COOH CH.OH CHa + Ng + H^O CHij.COOH Oxy-glutaric Acid. [CH. IV. ASPARTIC ACID. 81 Aspartic Acid. 82. Preparation from Asparagine. Asparagine is the amide of aspartic acid, and it is converted to the acid by hydrolysis with acids. HCl I CH(NH2).COOH.H20 CH(NH2).C00H 1 + 2HCI = I + NH4CI. CH2.CONH2 CHj.COOH Crystalline asparagine Aspartic acid hydrochloride Mol.Wt. = 150. The hydrochloride is converted to aspartic acid by the addition of the calculated amount of sodium hydroxide. (i.) Into a 500 cc. round-bottomed flask introduce 15 grams, of crystalline asparagine (i/io gm. mol.) and 200 cc. of N.HCl (2/10 gm. mol.). (ii.) Boil gently on a sand bath under an efficient refiux con- denser for 6 hours. (iii.) Cool and add 100 cc. of N.NaOH (i/io gm. mol.), shaking during the addition. Set aside to crystallise in a cool place. (This soda is to convert aspartic acid hydro- chloride into free aspartic acid.) (iv.) Stir well and filter on a Buchner, and wash with small amounts of cold water. Reserve the filtrate and wash- ings for obtaining copper aspartate, or a further crop of crystals by evaporation. (v.) RecrystaUise by dissolving in the smallest possible amount of 50 per cent alcohol, filtering through a hot water funnel, and allowing to stand till quite cold. (vi.) Filter on a Buchner and dry in the air. Add the filtrate to that obtained in (iv.). Yield: about 12 grams, of the recrystallised product. 82 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. Properties of Aspartic Acid. It crystallises in small rectangular plates. It dissolves in about 360 parts of cold water and ig parts of boiling water. Like glutami- nic acid, its aqueous solutions are markedly acid to litmus. Solutions in alkalies are laevorotatory, those in hydro- chloric acid are dextrorotatory. The copper salt is very characteristic. It can be obtained by the method given in Ex. 79, or more readily by boiling a solution with some solid cupric acetate, and filter- ing the hot liquid. On standing, beautiful blue needles separate. This copper salt, which contains 4I molecules of water of crystallisation, is so sparingly soluble that it serves for the estimation of aspartic acid. Cystine. 83. Preparation from hair (or wool) by Folin's method. (i.) Heat 500 cc. of pure concentrated hydrochloric acid in a litre roimd-bottomed flask on a water bath. (ii.) Add 250 grms. of human hair (the sweepings from a hair- dresser's shop), or of washed wool (a piece of a pure woollen blanket). If hair is used it must be added in portions of about 50 grams, at a time, the hot mixture being well agitated after each addition. (iii.) Boil under a reflux condenser on a sand bath for 5 to 6 hours, or until the mixture no longer 5delds the biuret reaction. (iv.) To the hot mixture add solid sodium acetate to remove the free hydrochloric acid. The point is reached when a drop of the mixture added to about 2 cc. of water gives a reddish violet or brown, and not a deep blue with a few drops of a dilute (0-2 per cent.) solution of Congo red. Usually 500 to 600 grams, of the solid are required. Allow the mixture to stand over-night. (v.) Filter on a Buchner. The precipitate consists of cystine, together with a considerable amount of dirt and in- soluble debris. CH. IV.] CYSTINE. 83 (vi.) Transfer the precipitate to a porcelain beaker or dish, and boil with 150 cc. of 20 per cent, hydrochloric acid (by voliime). Filter. (vii.) Boil the residue with another 100 cc. of the hydrochloric acid, filter, and mix the two filtrates. (viii.) Boil these with 5 grams, of good decolourising charcoal (see p. 390), and filter. If the filtrate is not practically colourless, the solution must be boiled with more char- coal until a colourless or light yellow fluid is obtained. (ix.) Add a hot, concentrated, filtered solution of sodium acetate until the free hydrochloric acid is removed. (x.) Allow to stand till quite cold. (xi.) Filter off the cystine, wash with cold water, then with alcohol, and dry in the air. Yield : 8 to 10 grams. Properties of Cystine. It crystallises in characteristic hexagonal plates, which are only very slightly soluble in cold water (i part in 8840 parts of water) and in alcohol. It dissolves readily in mineral acids, but is insoluble in acetic acid. In normal acid urine it is soluble to the extent of I part in 2000. It is readily soluble in dilute alkalies and in ammonia. It is precipitated from its solution in sulphuric acid by mercuric sulphate (see p. 90). On reduction it yields cysteine CHg.SH. I CH.NH2 COOH. This is readily oxidised to cystine by atmospheric oxygen in ammoniacal solution. 84 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. 84. Dissolve a small amount of cystine in one or two cc. of 5 per cent, sodium hydroxide. Add a drop or two of lead acetate and boil for a minute. The solution is darkened owing to the formation of lead sulphide (see Ex. 25), Histidine. HC N CH NH, C.CHj CH COOK. NH 85. The preparation of histidine mono-hydrochloride. (i.) Place I litre of defibrinated ox or sheep blood (or preferably I Utre of the centrifuged corpuscular mass from blood) into a 2-litre, round-bottomed flask. (ii.) Add 500 cc. of pure concentrated hydrochloric acid, shaking well during the addition. (iii.) Heat on a boihng water bath, shaking at intervals, for 2 to 3 hours, imtil the precipitated blood proteins have re- dissolved. (iv.) Boil on a sand bath under a reflux condenser for 10 hours. (v.) Transfer to an evaporating basin and remove the greater part of the hydrochloric acid by evaporating on a boiling water bath in a flue chamber. (vi.) Add 40 per cent, caustic soda until the mixture is only shghtly acid to litmus paper. (vii.) Allow to stand over-night, and filter on the pump. Wash the precipitate with hot water, adding the washings to the main filtrate. CH. IV.] HISTIDINE. 85 (viii.) Place the solution in an evaporating basin, make it distinctly alkaline by the addition of caustic soda, and boil for 30 to 60 minutes to remove ammonia. This is tested by holding a moist litmus paper in the vapour. The removal of ammonia is hastened by adding a small volume of alcohol. (ix.) Pour the solution into about 5 litres of water contained in a large vessel. (x.) Add a hot saturated solution of mercuric chloride in water rmtil no further precipitate is obtained, keeping the solution sufficiently alkaline to ensure complete precipi- tation. Usually about 100 gms. of mercuric chloride are required. (xi.) Allow the mercury compound of histidine to settle over- night. (xii.) Syphon off the supernatant fluid. (xiii.) Filter off the precipitate on a large Buchner funnel and wash it with cold water. (xiv.) Transfer the precipitate to a porcelain dish and add hot hydrochloric acid (25 per cent, by volume) as long as any of the precipitate goes into solution, avoiding any large excess of acid. (xv.) Filter from the insoluble residue of calomel and wash this with cold water, adding the washings to the bulk of the fluid. (xvi.) Dilute the fluid to about 5 litres with distilled water. (xvii.) Dissolve 20 grams, of mercuric chloride in water and add this to the fluid. (xviii.) Make the fluid markedly alkaline by the addition of caustic soda. (xix.) Allow the voluminous precipitate to settle over-night. (xx.) Filter on the pump, drain, and wash thoroughly by grind- ing with water in a mortar and filter again. 86 PROPERTIES OF CERTAIN AMINO-ACIDS. [cH. IV. (xxi.) Grind the precipitate with a litre of water, transfer to a flask and decompose by means of sulphuretted hydrogen gas. As the precipitate is somewhat difficult to decom- pose it is necessary to pass the gas for 8 to lo hours. It must not be assumed the decomposition is complete as soon as the precipitate has blackened. On no account must the solution be heated. (xxii.) Filter off the mercuric sulphide, wash it with small quantities of hot water, adding the washings to the bulk of the fluid. (xxiii.) Concentrate the solution to a syrup in an evaporating basin on a boiling water bath. (xxiv.) Whilst still hot, add boiling 97 per cent, alcohol, with continuous stirring, until there is a faint permanent turbidity. AUow to stand over-night. (xxv.) Filter off the crystals of histidine hydrochloride, CeHaN302,HCl,H,0. (xxvi.) Repeat (xxiii.) to (xxv.) to obtain a second crop. Recrystallisation. (i.) Dissolve the crystals in twelve times their weight of 65 per cent, alcohol, by heating on a boiling water bath imder a reflux condenser. (ii.) Add a little charcoal and boil again. (iii.) Filter through a pleated paper on a hot water funnel, and allow the filtrate to cool. The histidine hydrochloride separates in the form of beautiful white glistening plates. Yield: 6 to 8 grams. Properties of histidine. Histidine is readily soluble in water, but very slightly soluble in alcohol. The aqueous solution has an alkaline reaction. It crystallises from alcohol in platelets, which melt with decomposition at about 253° C. The most convenient salt for the isolation of histidine is the monohydrochloride. It is readily soluble in water. CH. IV.] TRYPTOPHANE. 87 It crystallises from aqueous alcohol in platelets, which melt at 251°— 252° C. Even in very dilute solution histidine gives a volu- minous white precipitate with phosphotungstic acid. It is also precipitated by mercuric chloride in alkaline solution and by ammoniacal silver nitrate solution. 86. Totani's reaction for histidine. To a small knife point of the hydrochloride in a small beaker, add 2 cc. of 10 per cent. sodium carbonate. Dissolve a rather larger quantity of diazo- benzene-sulphonic acid in 4 cc. of 10 per cent, sodium carbonate in a test-tube, and add this to the solution in the beaker. A dark red colour is produced (Primary colouration). Make the solution distinctly acid with strong hydrochloric acid. The solution becomes orange coloured. Add zinc dust and aUow the reduction to proceed for about 15 minutes. Transfer a few cc. of the clear, colourless, supernatant solution to a test-tube, and render the solution alkahne by the addition of 25 per cent, ammonia. A characteristic golden yellow colouration is produced, which is permanent for a considerable time (Secondary colouration). Note. — ^Tyrosine gives a primary colouration that is identical with that described above. The secondary colouration is, however, a bright rose red, which gradually changes to a reddish brown. The reaction should be tried with the two substances simultaneously. Tryptophane. 87. Preparation. A. Digestion of the Casein. (i.) Weigh out 200 grams, of commercial casein.* (ii.) Gradually stir this into i litre of cold distiUed water in a large beaker, or enameUed vessel, avoiding the forma- tion of lumps as far as possible, (iii.) Transfer the viscous mass to a Winchester quart bottle by means of a large funnel with a short wide neck, (iv.) Wash out the mixing vessel with a jet of hot water and transfer this to the bottle, washhig the fimnel down with some more hot water. * " Insoluble Casein," i.e. casein without any added sodium carbonate or bicarbonate, is the best to use. It can be obtained from Messrs. Baird and Tatlock. 88 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. (v.) Add more hot water to make the volume up to about 2 litres and shake vigorously. (vi.) Adjust the reaction to Ph = about 8-i. lo cc. of the mixture is taken, treated with about lo drops of cresol red and titrated with o-2 N.NaOH from a microburette (or a I cc. pipette graduated in i/iooth cc.) until a reddish purple colour is obtained. The bulk is then treated with the corresponding amount of N. soda {i.e. 100 times the amoimt of o-2 N. used). Should the volume required exceed loo cc, 40 per cent, soda can be used, this being 10 N. The mixture is well shaken at frequent intervals after the addition of the soda. The reaction should now be alkaline to cresol red and acid to phenol phthalein. (vii.) Add 15 cc. of toluol (to prevent putrefaction) and 2 grams, sodium fluoride dissolved in about 10 cc. of hot water (to decrease the action of oxidases). Shake well. (viii.) Add 70 cc. of the pancreatic extract described on p. 212, or a corresponding amount of a commercial preparation of trypsin (" liquor pancreaticus ") and mix well. (ix.) Clean the inside of the neck of the bottle with a cloth, insert a cork, shake again, and stand the bottle in a water bath or air thermostat at 37° to 40° C. for 4 days, shaking the bottle every day without removing the cork. (x.) After 4 days add another 50 cc. of the pancreatic extract, and allow the digestion to proceed for another 4 days, making 8 days in aU. (xi.) Remove the bottle from the incubator, and allow it to stand at room temperature for 24 hours or longer. B. Filtration and acidification. (i.) Filter off the precipitate, which consists of t3n:osine, mi- digested casein, etc., reserving it for the separation of tyrosine described in Ex. 8g. CH. IV.J TRYPTOPHANE. 89 (ii.) Measure the filtrate and to every 86 cc. add 14 cc. of a 50 per cent, solution (by volume) of pure sulphuric acid. (This is prepared by gradually pouring 500 cc. of pure sulphuric acid into 500 cc. of distilled water, cooling thoroughly under the tap until the whole of the acid has been added. When quite cold the volume is made up to 1000 cc. with distilled water.) The mixture now contains 7 per cent, by volume of sulphuric acid. C. Separation of the mercuric sulphate precipitate. (i.) Add 250 cc. of a 10 per cent, solution of mercuric sulphate in 7 per cent, sulphuric acid (by volume). Mix well, and allow to stand over-night. (The mercuric reagent is prepared by grinding 100 grams, of mercuric sulphate with 500 cc. of distilled water, to which 70 cc. of pure concentrated sulphuric acid has been added, adding distilled water to make a volume of 1000 cc, and filtering if necessary.) (ii.) Filter off the bulky yellow precipitate of the mercuric sulphate compound of tryptophane on a Buchner funnel, reserving the filtrate for Ex. 90. (iii.) Wash the precipitate on the Buchner with cold 5 per cent, sulphuric acid (by volume) to remove the tyrosine. It is not necessary to remove the tyrosine completely at this stage. (iv.) Wash the precipitate with distilled water to remove the greater part of the sulphuric acid. D. Decomposition of the mercuric sulphate precipitate. (i.) Transfer the precipitate and paper to a wide-necked 500 cc. flask, washing the remainder of the precipitate in the Buchner into the flask by means of about 200 cc. of distilled water. Agitate thoroughly to get a good sus- pension of the precipitate. (ii.) Add 100 cc. of boiling water, to which 3 grams, of crystalline barium hydroxide has been added. Shake well. 90 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV (iii.) Test the reaction of the solution by means of litmus paper. If it is still acid add a hot solution of baryta until alkahne. (iv.) Pass in a stream of sulphuretted hydrogen gas, shaking at intervals, until the mixture is fully saturated. (v.) Heat on the water bath to about 50° C, shake well, and pass in more of the SHj if the odour of the gas is not perceptible, (vi.) Filter from the mixture of mercuric sulphide and barium sulphate. (vii.) Wash the precipitate with hot water, and squeeze the paper in a piece of mushn. Filter these washings, etc., through another small paper, and add them to the bulk of the fluid obtained in (vi.). /viii.) Add a few drops of 5 per cent, sul- phuric acid. If a white precipitate of barium sulphate is obtained, it indicates that an excess of baryta had been added, and that bcirium sulphide is present. Continue to add the dilute sulphuric acid until no further precipitate is obtained. Filter off the barium sulphate. If the addi- tion of the dilute sulphuric acid does not cause a precipitate, proceed directly to : — (ix.) Remove the sulphuretted hydrogen by a strong current of air, using the apparatus shown in fig. 12. (It is preferable to remove the SH^ by dis- tillation in vacuo at 45° C.) Fig- 12. Apparatus for removal of SHj by means of an air current. E. Removal of cystine and reprecipitation of the tryptophane. (i.) Measure the fluid and add 14 cc. of 50 per cent, sulphuric acid for every 86 cc. CH. IV.J TRYPTOPHANE. 91 (ii.) Cautiously add the mercuric sulphate reagent prepared as described in C (iii.) above. Add this luitil a slight definite precipitate is obtained. Usually about 15 cc. are required. Allow the mixture to stand for 10 minutes. Filter. (iii.) To the filtrate add 80 to 100 cc. of the mercuric reagent^ and allow the mixture to stand over-night. (iv.) Filter on a Buchner funnel, wash with cold 5 per cent, sulphuric acid, and then thoroughly with water. (v.) Suspend the precipitate and paper in 50 cc. of water. (vi.) Add 2 grms. of barium hydroxide dissolved in 70 cc. of boiUng water. (vii.) Decompose by SHj and proceed as in D (iv.) to D (ix.), taking care to have only a very slight amount of free sulphuric acid present. F. Removal of sulphuric acid (i.) Heat the fluid on a boihng water bath, and add a hot solu- tion of baryta to a point when no further precipitation can be seen. (ii.) Filter a portion until a clear filtrate is obtained, and test with a drop or two of the baryta solution. If no pre- cipitate is obtained, test another portion of the hot filtrate with a drop of dilute sulphuric acid. Should this fail to give a precipitate also, the correct point is reached, being that at which neither sulphuric acid nor baryta gives a precipitate. Baryta or sulphuric acid must be added imtil this condition is attained, the samples tested being added to the bulk. The process is much facilitated if the relative concentrations of the alkali and acid are roughly determined against one another. Filter to obtain a perfectly clear filtrate. (iii.) Add a single drop of 10 per cent, ammonia. 92 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. G. Crystallisation. (i.) Evaporate in vacuo (fig. 8) at a temperature of about 45° C. until the volume is reduced to rather less than 10 cc, best ascertained by measuring 10 cc. into the flask before the evaporation is commenced. (See page 99.)] (ii.) Disconnect the apparatus with the usual precautions. (See page 74.) (iii.) Heat for a short time on a boiling water bath, shaking the fluid round the flask to get as much as possible into solution. (iv.) Transfer to a small crystalHsing dish or beaker and add an equal volume of strong alcohol. (v.) AUow the dish to stand in a cool place over-night. (vi.) Filter off the precipitate, using a suction pump. (vii.) Wash out the flask and dish with small amounts of alcohol of increasing strengths, 65, 75, 85, and 95 per cent., using these for washing the crystals on the filter. (viii.) Dry the crystals in the air. H. Concentration of the mother liquors. (i.) Evaporate the mixed mother liquors and alcoholic washings in a boiling water bath, adding strong alcohol from time to time, until the crystalline precipitate that forms at the edge does not redissolve in the body of the fluid when stirred. (ii.) Set the dish aside for at least an hour. (iii.) Filter off the crystals and wash with small amounts of alcohol of gradually increasing strength. (iv.) Dry in the air. Yield: 0-6 to 2 grams. CH. IV.] TRYPTOPHANE. 93 I. Recrystallisation. (i.) Transfer the crystals to a small flask, fitteid with a reflux condenser. (ii.) Add a small amount of 70 per cent, alcohol, and heat in a boiling water bath. Very gradually add 70 per cent, alcohol (down the stem of the condenser) until the crystals have just dissolved. Add a large "knife-point " of decolourising charcoal and heat for five minutes. (iii.) Disconnect and rapidly filter through a small hot funnel into a small beaker. Allow to stand for at least an hour. (iv.) Filter on the pump, wash with 75, 85, and 95 per cent, alcohol. Dry in a vacuum desiccator, or in a warm oven at 80° to 90° C. There is a considerable loss on recry- stallisation. Properties of Tryptophane. It crystallises from aqueous alcohol in white glistening, six-sided plates. It is moderately soluble in cold water, but freely soluble in hot water. It is only sparingly soluble in absolute alcohol. On heating it changes colour at 220°, browns at 240°, and melts at 252° C. On heating still further, there is first an evolution of carbon dioxide, and then the formation of indol and skatol. Tryptophane is optically active, being laevorotatory in aqueous, but dextrorotatory in acid or alkaline solution. It gives colour reactions with a great variety of sub- stances. The most important of these is the reaction with glyoxylic and sulphuric acids (see Ex. 23). The investiga- tion of the cause of the similar colour reaction given by proteins led to the isolation of the amino-acid. It also gives colour reactions with most aldehydes in the presence of strong hydrochloric or sulphuric acids containing a trace of an oxidising substance, like ferric chloride. All these reactions are given by tryptophane when it is combined in a protein molecule. Free tryptophane gives a red-rose colour when treated with bromine water, the colouring matter being soluble in 94 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. amyl or butyl alcohol. This reaction is not given by combined tryptophane. Tryptophane is somewhat unstable. It is rapidly destroyed by boiling acids, especially in the presence of carbohydrates, yielding a dark, so-called " humin substance," or " melanin." It is oxidised by certain metallic salts, like copper sulphate, silver nitrate, gold chloride and ferric chloride, yielding indol aldehyde HC HC HC /^r nC.CHO CH NH CH and other substances. On heating on an open vi^ater bath in aqueous solution it suffers decomposition. For this reason it is necessary to concentrate in vacuo, or to add considerable quantities of alcohol during the evaporation. It is much more stable in alkaline solutions and, in the presence of other amino-acids, can be boiled v^^ith strong baryta for days without appreciable loss. It must be noted, however, that the substance obtained after baryta hydrolysis is racemic (see p. 152). In dilute sulphuric acid tryptophane gives a lemon- yellow precipitate with mercuric sulphate, the precipitate being a compound of tryptophane sulphate with mercuric sulphate. The precipitating effect of phosphotungstic acid on tryptophane seems to vary with the quality of the acid ; some samples only give a precipitate in concentrated solutions, others none at all, whilst some preparations pre- cipitate it completely even from relatively dilute solutions, especially in the presence of other amino-acids. It is not precipitated by lead acetate nor by mercuric chloride in neutral solution. 88. A. To a small knife point of tryptophane dissolved in about 3 CO. of water, cautiously add bromine water. A rose-red or CH. IV.] TYROSINE. 95 reddish- violet colour is produced, which turns yellow on adding an excess of bromine. If the addition of the bromine water be stopped when the red colour has reached its maximum intensity, it will be found that the coloured product can be shaken out with a few cc. of amyl or butyl alcohol. B. To a smaU knife point of tr3^tophane add a few cc. of " reduced oxahc acid " (see Ex. 23). Add an equal volume of pure sulphuric acid and mix. A beautiful purple colour is produced. C. To a small knife point of tryptophane add about i cc. of water, 2 drops of a 5 per cent, solution of cane sugar, and 5 cc. of pure hydrochloric acid. Boil for about a minute. A deep purple solution is obtained. D. Dissolve about 0-05 gram, of tryptophane in 5 to 10 cc. of water by boiling in a test tube. Add as much freshly prepared, washed copper hydroxide (see note to Ex. 79) as will go on a large spatula and boil for i minute. Filter hot. The filtrate is not blue and gives no reactions for tryptophane. The precipitate contains the copper salt of tryptophane, which is characteristically insoluble except in the presence of even traces of other amino-acids. It is then soluble in their copper salts. Tyrosine. Tyrosine is the least soluble of the amino-acids, and for that reason is easily obtained from proteins. When casein is digested by trypsin, under the conditions described in Ex. 87, it generally happens that a considerable amount separates as a chalky vi^hite precipitate in 6 to 10 days. The amount separating varies with the activity of the ferment preparation, and on the particular sample of casein employed. If a good yield of tyrosine is especially desired it is advisable to concentrate the filtrate obtained in Ex. 87, B (i.) to about one-fourth, allow to cool over-night, filter ofif the precipitate on the pump, and purify by the method described below. The concentrated filtrate can be diluted, and used for the isolation of , tryptophane, but owing to its unstability, the yield of this latter amino- acid is very apt_to_be_poor. 96 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. 89. Preparation of Tyrosine from Casein. (i.) Use the mixed mass of calcium phosphate, undigested casein, tyrosine, etc., obtained in Ex. 87, B (i.). (ii.) Boil the precipitate with about 250 cc. of water to which has been added 5 cc. of pure sulphuric acid. The tyro- sine dissolves in the acid, whilst a considerable amount of the protein residue remains insoluble. (iii.) Filter through a pleated paper, passing the filtrate back through the paper until it is clear. Filtration is apt to be rather slow. (iv.) Heat the filtrate on a boiling water bath, and add 10 cc. of strong ammonia. The reaction should now be acid to litmus paper. Cautiously neutralise by the addition of ammonia and allow to cool. Tyrosine crystalUses out, generally contaminated with calcium phosphate, etc. (v.) Filter off the tyrosine on the pump. Suspend it in about 300 cc. of water in a flask, boil, and add 5 cc. of strong ammonia and boil for 15 minutes. (vi.) Filter from the calcium phosphate. (vii.) Neutralise the fluid with 5 per cent, sulphuric acid and allow to stand, (viii.) Filter off the tyrosine on the pump, and wash well with cold water. Wash with a little alcohol, and dry in the steam oven or in a warm incubator. go. The separation of tyrosine by fractional precipitation. (i.) Treat the filtrate obtained in Ex. 87, C (ii.) with 5 volumes •of tap water in a large vessel, mix well, and allow to stand over-night. Owing to the reduction in the concentration of sulphuric acid the tyrosine is precipitated as a com- pound with mercuric sulphate. (ii.) Syphon off the supernatant fluid and filter the precipitate on a Buchner. Wash well with water. (iii.) Suspend the precipitate in about 200 cc. hot water and decompose by a stream of sulphuretted hydrogen gas. (iv.) Boil and filter. Tyrosine is left in solution in dilute sulphuric acid. CH. IV.] " LEUCINE. 97 (v.) Neutralise to litmus paper with ammonia, and allow to stand. Filter off the crystals of tyrosine, wash with cold water, and dry in a warm oven. Properties of tyrosine. It crystallises from water in white needles which are characteristically arranged in sheaves. It is only very slightly soluble in cold water (i part in 2490 parts of water at 17° C), but more soluble in hot water (i part in 150 parts). It is i^nsoluble in ether, acetone and absolute alcohol. It is readily soluble in dilute alkalies and dilute mineral acids, but it is only very slightly soluble in dilute acetic acid, and practically insoluble in glacial acetic acid. Its melting point is rather indefinite, but on rapid heating is 3 14° to 31 8°. It is laevorotatory in aqueous acid and alkaline solutions. On being heated in a tube it loses CO2, and is convertec? to /)-oxyphenylethylamine. It is not precipitated by phosphotungstic acid. It is precipitated by mercuric sulphate, the precipitate being soluble in dilute sulphuric acid. 91. Momer's reaction. To a few cc of Momer's reagent add a trace of tyrosine and boil. A green colouration is produced. Note. — The reagent is prepared by mixing i cc. oi formalin with 45 cc of water and cautiously adding 55 cc. of strong sulphuric acid. 92. Millon's reaction. To a trace of tyrosine add a few cc. of water and a drop of dilute sulphuric acid and boil. Cool the solution and add a few drops of Millon's reagent. A precipitate is not obtained. Heat. A red colouration is obtained. Note. — For the preparation of Millon's reagent, see Ex. 22. In the presence of 5 per cent, sulphuric acid the red colour is produced in the cold. NH, Leucine. ^JJ^ ^ CH . CH, . CH . COOH a-amino-caproic acid (a-amino-iso-butyl-acetic acid). Leucine can be obtained by fractional crystallisation from a protein digest, being separated by concentration of the mother liquors left after the isolation of tyrosine. The 98 PROPERTIES OF CERTAIN AMINO-ACIDS. [CH. IV. following exercise is suggested owing to the great prepon- derance of leucine over tyrosine in the proteins employed. 93. Preparation of leucine from blood. (i.) To I litre of defibrinated blood in a 2 litre flask, gradually add 150 cc. of pure sulphuric acid, shaking well during the addition. A semi-solid mass of coagulated protein is obtained. (ii.) Heat on a boiling water bath for 12 to 16 hours, shaking well at intervals. (iii.) Add some pieces of a broken porous pot, and heat to boihng on a large sand bath. It is necessary to start with the fluid hot from a water bath and to repeatedly shake the mixture imtil it boils. Otherwise there is a risk of the flask breaking. The mixture must be boded for 10 to 14 hours. (iv.) To the hot fluid add a hot solution of baryta until the mixture is alkahne to htmus. About 500 grams, of baryta in about i J htres of boihng water are usually necessary, (v.) Filter on a Buchner funnel. (vi.) Make the filtrate acid to htmus with dilute sulphuric acid. Concentrate in a porcelain basin over a free flame to about 500 cc. and filter, (vii.) Render the filtrate faintly alkaline to htmus by the addition of ammonia and concentrate on a boihng water bath until a crystalline crust has formed. AUow to cool over-night, (viii.) Filter on a small Buchner funnel, pressing the mass of crystals firmly with a pestle to remove as much of the mother Uquors as possible. The filtrate may be further concentrated and a second crop of crystals obtained. (ix.) RecrystaUise from 70 per cent, alcohol, as described in Ex. 87, I. The product is apt to be contaminated with isoleucine NH2 NH2 (cH >CH.CH.C00h) andvahne f ^^^ ^ CH.CH.COOH ) CH. IV.] LEUCINE. 99 Properties of leucine. Pure leucine is only very slightly soluble in cold water, but when it is contaminated with other amino-acids it is easily soluble. It is insoluble in absolute alcohol, but is soluble in hot dilute alcohol, and can be freed from tyrosine by crystallising from hot alcohol. When pure it crystallises in pearly plates. When impure it is apt to crystallise in soft spherical masses, which have a slightly radiate structure. It melts at 297" with decomposition. When heated gently in an open tube it sublimes at a temperature below its melting point and emits a characteristic smell of amyl- amine. It is laevorotatory in aqueous solution, but dextrorotatory in solution in hydrochloric acid. To pump Fig. 13. Distillation in vacuo by use of a Claisen flask (A). Alcohol or more of the fluid can be added through E as the distillation proceeds. D is a tube drawn out to a fine capillary through which a small amount of air enters the boiling fluid to prevent excessive bumping. CHAPTER V. THE CARBOHYDRATES. There are several groups of these compounds, only a few of which, however, are of any physiological importance. A. The Monosaccharides, or Simple Sugars. B. The Compound Sugars (Di- and Tri-saccharides). C. The Polysaccharides. The first two groups are colourless, crystalline substances, readily soluble in water, and usually of a sweet taste. The polysaccharides are mostly amorphous, insoluble in water, and without a sweet taste. A. The Monosaccharides. A simple sugar is an aldehyde, or ketone, linked directly to at least one alcoholic group. Sugars containing the aldehyde group are known as aldoses, which therefore contain I H— C— O— H I H— C = as a characteristic group. Sugars containing the ketone group are known as ketoses, of which the group H— C— O— H C = I is characteristic. CH. v.] MONOSACCHARIDES. 101 The simple sugars contain from two to nine carbon atoms, and are called biases, trioses, tetroses, pentoses, hexoses, etc., depending on the number of carbon atoms they contain.* The pentoses, CjHjoOg, are widely distributed in nature. In plants they are found both in the free state and in the form of condensation products, known ,as pentosans (C5H8O4),,. The most important of the pentoses are the aldoses, arabinose and xylose, best obtained from the corresponding pentosans in gum arable and beech sawdust respectively. Ribose is a constituent part of the molecule of yeast nucleic acid. Pentoses are occasionally found in human urine (see p. 312). The hexoses, CgHiaOg, are of great physiological import- ance. Of the many that have been synthesised in the laboratory only the following are found in nature, and are of physiological interest : — ■ CHO CHO CHO CH2OH I . I I I H.C.OH HO.C.H H.C.OH CO 1 III HO.C.H HO.C.H HO.C.H HO.C.H H.C.OH H.C.OH HO.C.H H.C.OH H.C.OH H.C.OH H.C.OH H.C.OH I III CH2OH CH2OH CH2OH CH2OH d-glucose : (i-mannose d-galactose (i-fructose (dextrose, grape- (laevulose, fruit sugar) sugar) * This is not strictly true, for there exist substituted sugars in which one or more H atom is replaced by a methyl group. A methyl pentose thus con- tains six carbon atoms. 102 THE CARBOHYDRATES, [CH. V. It will be noticed that the first three are aldoses, whilst fructose is a ketose. The first three are stereo-isomers, differing only in the arrangement of the H and OH groups in space round the four central carbon atoms, all of which are asymmetric. (See p. I Si.) It therefore follows that these compounds are optically active, that is, their solutions can rotate the plane of polarised light.* In the above formulae they are represented as being aldehydes, but certain facts seem to indicate that they can exist in another form. Thus, if glucose be dissolved in water it is found that the solution at first has a much higher rotatory power than when it has been kept for some hours or has been boiled with a trace of alkali. This phenomenon is known as mutarotation. Also it is very much less active chemically than the above formulae warrants. These properties are explained by assuming that when first dissolved in water, glucose exists as a y-lactone, having the formulae H*C.OH • Ordinary glucose is dextro-rotatory. Fischer synthesised its laevo- rotatory optical ahtipode which he called /-glucose. All those sugars which are synthetically related to d-glucose or d-galactose he grouped into the d- class. Thus ordinary fructose he named d-fructose, though actually it is strongly laevorotatory. CH. v.] GLUCOSE. 103 In this state the *C atom is asymmetric, so that two forms of glucose are possible, called a- and /3-glucose. HO.C.H* *H.C.OH H.C.OH H.C.OH CH2OH a-glucose. CHgOH /8-glucose. Under certain conditions two forms of glucose can be isolated, one with a rotatory power [oJd = + 110°, the other with a rotatory power of [ajo = + i9°- When kept in solution there results an equilibrated mixture of Wd = + S2-S". It is possible that there are three com- pounds now in solution, the aldehyde and the two y-lactones. If the *H atom be replaced by some other group (generally aromatic), the compound formed is called an a- or /3-glucoside, which can be converted into glucose and another compound by hydrolysis with acids or certain ferments. The natural glucosides (phloridzin, salicin, etc.) are /3-glucosides. These glucosides are hydrolysed by the enzyme emulsin, which hydrolyses all 8-glucosides. Mal- tose (p. 113) is glucose-a-glucoside. It is not hydrolysed by emulsin, but is by maltase, which hydrolyses all the a-glucosides. Physical and chemical properties of the monosaccharides. They are white crystalhne solids, very soluble in water and alcohol. Insoluble in ether, acetone, and most of the organic solvents. Being adehydes or ketones, they are 104 THE CARBOHYDRATES. [CH. V. susceptible of being oxidised to various acids, thus reducing certain oxidising reagents. This reaction only takes place in hot alkaline solutions, and is of great value as a test for these sugars, and especially as a basis of various methods of estimation. They react with phenyl hydrazine in excess to give insoluble crystalline bodies called osazones. These are of the greatest value in determining the presence of and in characterising the monosaccharides, though not in dis- tinguishing them from one another. When heated with an alkah the monosaccharides become yellow and then brown, and finally decompose into a mixture of acids and resinous substances. They are reduced by sodium amalgam to hexahydric alcohols. Sorbite is formed from glucose, mannite from mannose and dulcite from galactose. Fructose yields a mixture of sorbite and mannite. These alcohols are of considerable interest, as they are used by bacteriologists for the differential diagnosis of certain pathogenic organ- isms. On oxidation glucose gives rise to three acids^ COOH CHO COOH I I I (CHOH)^ (CH0H)4 (CHOH)^ I 1 I CH2OH COOH COOH Gluconic acid. Glycuronic acid. Saccharic acid. Glycuronic acid is interesting physiologically, as it is frequently found in the urine in combination with various drugs, such as chloral, camphor, phenol, etc. These com- pounds protect the organism from the injurious effects of the drugs. On oxidation galactose gives inactive mucic acid, which is isomeric with saccharic acid. Being only slightly soluble its production is used as a test for the presence of lactose in urine, since lactose is hydrolysed by acids into galactose and glucose. CH. V.J GLUCOSE. 105 Glucose (dextrose or grape sugar) QHigOg. 94. Preparation of glucose from starch. To 700 cc. of distilled water add 40 cc. of pure HCl and boil. Mix 100 grams, of potato starch with 200 cc. of cold water, and slowly stir this into the boiling mixture. Wash in the remainder of the starch with another 100 cc. of water. Boil under a reflux condenser for 3 hours. To the hot solution add solid lead carbonate, a little at a time, till effervescence ceases (about 100 grams, are usually required). Cool and filter. Evaporate the filtrate to a thin syrup. Add an equal volume of hot 95 per cent, alcohol. Filter. Evaporate the filtrate to a thick syrup. Treat this with twice its volume of 93 per cent, alcohol and allow the solution to cool slowly. If a syrup falls out of solution on cooling, the alcohol is too strong, and a few drops of water should be added and the solution again heated to redissolve it. When the cooled solution no longer deposits any syrup add a crystal of glucose and set aside to crystal- lise. After crystalhsation is complete, which may take six or seven days, drain the crystals and dry by spreading them on a porous earthenware plate. To recrystallise dissolve the dried crystals in half their weight of water and add to the resulting syrup twice its volume of boiling 93 per cent, alcohol. Set the alcoholic solution aside to crystalhse, and dry the resulting crystals as before. Unless directions to the contrary are given use a 0-2 per cent, solution of glucose for the following exercises. 95. Boil 3 CC. with I cc. of 5 per cent, sodium hydroxide. The solution turns yellow. Note. — The yellow colour is due to the formation of caramel (a condensa- tion product) by the hot alkaU. 96. Treat two or three cc. of 5 per cent, caustic soda with four or five drops of a i per cent, solution of copper sulphate. A blue precipitate of cupric hydroxide, Cu(0H)2 is formed. Add to the mixture an equal bulk of the sugar solution. The precipitate dissolves. Boil the solution for a short time. The blue colour dis- appears, and is replaced by a yellow or red precipitate of cuprous oxide, CujO (Trommer's test). 106 THE CARBOHYDRATES. [CH. V. Notes. — i. The amount of copper necessary depends on the percentage of sugar present. If only a small amount of sugar be present a mere disappear- ance of the blue colour is all that may happen, or possibly the fluid may assume a faint yellowish-red tint. If excess of copper be added, the reduction is obscured by the blue cupric hydrate in solution, or the black precipitate of cupric oxide that is formed on heating this in the alkaline solution. It is always best to add the copper sulphate a few drops at a time, boiUng between each addition. 2. The reaction is a type of several that have been introduced for the detection of glucose, all of which depend on the fact that in alkaUue solution it has reducing properties when boiled. For this reason, glucose, and all sugars that have this property are sometimes spoken of as " reducing sugars." 3. The property that glucose and other sugars have of dissolving cupric hydrate is common to a large number of organic compounds, such as glycerol, Rochelle salt and sodium citrate. 97. Boil about 3 cc. of Fehling's solution (see Note i) in a test-tube. No change occurs. Add about 3 cc. of the glucose solution and boil again. A red precipitate of cuprous oxide is formed. (Fehling's test.) Notes. — i. Fehling's fluid is prepared as follows : (a) Dissolve 103-92 grams, of pure copper sulphate in warm distilled water and dilute to one litre. (b) Dissolve 320 grams, of potassium sodium tartrate (Rochelle salt) in warm water, add a little carboUc acid to prevent the growth of fungi, dilute to exactly a litre and filter. (c) Dissolve 150 grams, of sodium hydroxide in distilled water and dilute to I litre. For use take exactly equal quantities of a, b, and c, and mix. Though the individual constituents keep indefinitely, the fluid when prepared suffers decomposition, so that a reduction occurs on boiUng. For this reason the fluid should be prepared just before use, and must always be tested by boiUng before being used. The fluid is of such a strength that the copper sulphate in 10 cc. is just reduced by 0-05 grams, of dextrose. 2. The addition of the Rochelle salt is for the purpose of dissolving the cupric hydroxide that would otherwise be precipitated by mixing {a) and (c). 3. The test is much more dehcate and certain than Trommer's test, and should always be used in preference to it. 4. If the fluid that is being tested is acid, it should be neutralised. 5. Ammonium salts considerably interfere with Fehling's test owing to the ammonia liberated dissolving the cuprous oxide to a, colourless com- pound. If they are present a Httle extra alkaU should be added, and the mixture boiled for two or three minutes to allow of the evolution of the ammonia. 6. In testing for small amounts of glucose it is advisable to avoid an excess of FehUng's solution, owing to the excess of alkali tending to destroy the glucose before the latter can exert its reducing reaction on the copper. The neutral solution should be made faintly blue with Fehling's solution, and then boiled. CH. v.] GLUCOSE. 107 98. To 2 cc. of a I per cent, solution add 2 cc. of 40 per cent, sodium hydroxide. Heat to boiling and keep boiling for one and a half minutes. To the hot solution add half its volume of Fehling's solution. No reduction, or only a very slight one, is obtained. Note. — Glucose is completely destroyed by boiling with sodium hydroxide. 99. To 3 CC. of a I per cent, solution add a large " knife point " of anhydrous sodium carbonate Boil for i minute, cool under the tap. Add half its volume of Fehling's solution, and allow to stand without boiling. The FehUng's solution is reduced without boiling. Note. — The experiment indicates that by the action of alkalies glucose is converted to a material that will reduce Fehling's solution in the cold. Ex. 98 indicates that this material is destroyed by caustic alkalies. It will be seen later (Ex. 118) that the disaccharides, lactose and maltose, dififer from glucosein that they reduce Fehling's in the cold after being boiled with either sodium hydroxide or sodium carbonate. 100. To 5 cc. of Benedict's solution in a test-tube, add about eight drops of the sugar solution. Boil vigorously for one or two minutes and allow the tube to cool spontaneously. The entire body of solution will be filled with a precipitate, red, yellow, or green in colour depending on the concentration of the sugar. (Benedict's test.) Notes. — i. Preparation'ofJBenedict's solution for qualitative test. Dissolve 173 grams, of sodium citrate and 90 grams, of anhydrous sodium carbonate in about 600 cc. of distilled water by the aid of heat. Pour through a folded filter and make up to 850 cc. Dissolve 17-3 grams, of crystallised copper sulpTiate in 100 cc. of water and make up to 150 cc. Pour the carbonate citrate solution into a large beaker and add the copper solution slowly, with ronstant stirring. The mixed solution is ready for use and does not deteriorate on long standing. 2. Benedict's solution has certain advantages over Fehling's. For example, it is not so readily reduced by uric acid or urates, nor by creatinine. It is not reduced by chloroform, which is sometimes added to urine as a preservative. It does not destroy a small amount of sugar, as Fehling's does (see note 6 to Ex. 97). Also it can be used for testing urines for sugar in artificial light, since it is the bulk and not the colour of the precipitate that is of importance. 3. Though Benedict's test is much better than Fehling's for the detection of small amounts of glucose in urine, it is not quite so useful for other work. The author claims that his test (Ex. 104) is the most sensitive for general use. loi. To 5 cc. of the modified Barfoed's reagent in a test-tube add I cc. of the 0-2 per cent, solution of glucose and stand the tube in a beaker of boiUng water. After three and a half minutes remove the tube and examine it against a black background. A definite reduction is obtained. Repeat the experiment with i cc. of the 108 THE CARBOHYDRATES. [CH. V. solution diluted i in 5. A reduction may or may not be obtained, depending on the sensitiveness of the reagent (Barfoed's test, Hinkel and Sherman's modification). Notes. — i. The reagent is prepared by dissolving 45 grams, of neutral crystallised cupric acetate in 900 cc. of distilled water and filtering if necessary. To the filtrate add i -2 cc. of 50 per cent, acetic acid and dilute to i litre. 2. A portion must show no change when heated in a boiling water bath for 10 minutes. 3. 0'0005 gram, glucose generally gives the test, whereas 0-02 gram, lactose or maltose, or 0-03 gram, sucrose fail to give the test. 102. Measure 2 cc. of a i per cent, solution of glucose into a test-tube. Add 3 drops of pure glycerol. Measure 20 drops of a 20 per cent, solution of pure crystalUne copper sulphate into the tube by means of a dropping pipette (see fig. 5). Add 2 cc. of 20 per cent, sodium hydroxide. Boil the mixture and keep it boiling for half a minute, shaking the tube during the boiling to prevent loss by spurting. The addition of a couple of glass beads helps smooth boiling. Filter through a small paper or allow the tube to stand in a rack till the cuprous oxide has settled. Repeat the experi- ment, using 21, 22, etc., drops of the copper sulphate if the filtrate was yellow;- 19, 18, etc., if it was blue, until a point is found at which an extra drop of copper causes a change in the filtrate from yellow to a faint blue. Note. — The experiment gives one a very rough method of determining the concentration of a solution of glucose, which can be apphed for finding the approximate dilution necessary when an accurate estimation has to be made. The reason for the addition of glycerol is explained in Ex. 96, note 3. 103. Measure 2 cc. of the i per cent, solution into a test-tube, add 0-5 cc. of pure concentrated hydrocliloric acid, and boil gently for 2 minutes. Cool imder the tap. Add the number of drops of copper sulphate necessary to give a faint blue, as found in the preceding exercise, and three drops of glycerol. Neutralise by the addition of 20 per cent, sodium hydroxide, the neutral point being shown by the appearance of a grey precipitate. Now add 2 cc. of 20 per cent. sodium hydroxide, boil for i minute, and allow to stand. An increase in the reducing power is not obtained. Note. — Compare the results with those from maltose and lactose, Exs. 120 and 126. 104. To 5 CC. of the solution in a test-tube add a large "Icnife point " (half a gram.) of anhydrous sodium carbonate. Shake, and CH. v.] GLUCOSE. 109 heat to boiling. Maintain active boiling for 50 sees., shaking from side to side to prevent spurting. Immediately add 4 drops of a mixtui-e of equal parts of glycerol and 10 per cent, copper sulphate. Shake for a moment to mix and allow to stand without further heat- ing for I minute. The blue colour is discharged, and a yellowish precipitate of cuprous hydroxide forms. A control test with 5 cc. of distilled water should be performed. Repeat the experiment, using 5 cc. of the solution diluted i in 10 and I in 100. (Cole's test.) Note. — The test was elaborated by the author for the detection of very small quantities of glucose in urine (see Ex. 38 1). It is very sensitive, and it is claimed that i part of glucose in 500,000 parts of distilled water can be detected by this means. The instructions given are to be strictly followed. Many samples of glycerol give a slight reduction when boiled with sodium carbonate and copper sulphate, but they do not give a reduction when treated in the way described. The function of the glycerol is to keep the cupric carbonate in solution. 105. To 2 CC. of Nylander's reagent add 10 cc. of the glucose solution, mix, and boil. Immerse the tube in a beaker of boihng water for five minutes. A black precipitate of metallic bismuth separates out. (Nylander's test.) Notes. — i. Nylander's reagent is prepared by dissolving 50 grams, of Rochelle salt and 20 grams, of bisriiuth subnitrate in i litre of 8 per cent, sodium hydroxide. 2. The test is used for detecting small amounts of glucose in urine. It is superior for this purpose to Fehling's solution since it is not readily reduced by urates, creatinine, etc. The introduction of Benedict's solution and Cole's test have, however, led to the disuse of Nylander's. 106. Treat 2 cc. of a o-i per cent, solution of safranine with 2 cc. of the glucose solution and 2 cc. of 5 per cent, sodium hydroxide. Mix and boil, avoiding any shaking. The opaque red colour gives place to a Hght yellow, owing to the reduction of the safranine to a " leuco-base." 107. To 3 cc. of the 0-2 per cent, glucose add i cc. of a solution of sulphindigotate of soda and a large " knife point " of anhydrous sodium carbonate and boil. The blue colour turns green, purpHsh, red, and finally yellow. Shake with air : the blue colour reappears. (Mulder's test.) Note. — These two experiments illustrate the reducing properties of glu- cose in hot alkaline solution. The avidity of the reduced leuco-bases for oxygen is shown by the reappearance of the colour on cooling and shaking with air. 110 THE CARBOHYDRATES. [CH. V. io8. To 2 cc. of the solution add a large " knife point " of an- hydrous sodium carbonate and a rather smaller amount of soUd picric acid. Boil for about a minute. A deep reddish brown colour is produced. Repeat the experiment with 2 cc. of the solu- tion diluted I in 10. A distinct colouration is produced. ^ (NO,), / (NO,), Note. — Picric acid. CjH,<^ , is reduced to picramic acid, C,H,-^ NH, ^\OH \OH. by various substances in alkaline solution. The reaction serves as a basis for the colorimetric method of estimation of sugar in blood (Ex. 311). Note that glucose only gives the test on heating. Creatinine reduces picric acid to picramic acid in the cold (see Ex. 362). 109. To 10 cc. of the solution add i cc. of strong acetic acid. Add as much sohd phenyl-hydrazine hydrochloride as will he on a sixpenny piece, and at least twice this amount of solid sodixim acetate. Dissolve by warming, mix thoroughly, and filter into a clean tube. Place this in a beaker of boiling water for 30 minutes, keeping the water boiling the whole time. Remove the flame from under the beaker, and edlow the solution to cool slowly. A yellow crystalline precipitate of phenyl-glucosazone appears, often before the solution has been heated for more than 20 minutes. Collect some of this by means of a pipette, transfer to a slide, cover with a shp, and examine under both powers of the microscope. Note the characteristic arrangement of the fine yellow needles in fan- shaped aggregates, sheaves, or crosses. Notes. — i. Glucose is an aldehyde, and, like all aldehydes and ketones> forms a compound with phenyl-hydrazine. But this phenyl-hydrazone of glucose is very soluble, and cannot be readily separated. However, in the presence of an excess of phenyl-hydrazine at 100° C. an insoluble osazcne is formed. CHO CH : N.NH.C,H, CHOH C : N.NH.C,H. I -1- 3 C.H5.NH.NH, = I (CHOH)3 (CHOH), I I CHjOH CHjOH Glucose Phenyl-osazone of glucose (pheny 1-glu cosazone) + 2H.O H- NH, -h CeHs.NH, Aniline 2. Phenyl-hydrazine is a yellow basic Uquid, insoluble in water, but soluble in dilute acids to form salts. If the base itself is used, two or three CH. V.J FRUCTOSE. Ill drops should be dissolved In a few cc. of strong acetic acid, and added to the sugar solution. 3. Phenyl-hydrazine hydrochloride, CjH5.NH.NHj.HCl does not give an osazone when boiled with glucose, unless an excess of sodium acetate be added. This acts on the hydrochloride to form phenyl-hydrazine acetate and sodium chloride. In the author's experience it is advisable to have some free acetic acid present. 4. The osazone can be recrystallised as follows : Filter the cold solution through a small paper. Wash well with cold water. Boil a little strong alcohol in a tube and pour the hot solution on to the paper. Collect the filtrate in a clean tube, boil it, and pass it back through the paper. Repeat the process until a strong alcohoUc solution is obtained. Heat it again, and gradually add boiling water until a faint turbidity is produced. Heat again, add alcohol until the solution is just clear again, and then allow the tube to cool slowly. Or the alcoholic solution can be concentrated slightly on a boiling water bath. The product obtained can be filtered, washed and dried in a steam oven. The melting point is 204° to 205° C. 110. To about 2 cc. of the solution add 6 drops of a i per cent, alcoholic solution of a-naphthol. To this mixture add about 2 cc. of strong sulphuric acid, running it down the side of the tube. A purple ring is formed at the junction of the fluids. Note. — ^This reaction is given by all carbohydrates (see Ex. 26). Furfurol is formed very readily from fructose, sucrose and the pentoses. A modifica- tion of the test that only succeeds with these sugars is given in Ex. 114. Fructose (laevulose or fruit-sugar) is a keto-hexose. It can be prepared by the acid hydrolysis of inulin, a polysaccharide found in the tuberous roots of the dahlia, dandelion, Jerusalem artichoke and Inula Helenium, from which plant the name inulin is derived. It can also be prepared by hydrolysing cane sugar with dilute acid and separating the fructose from the glucose by adding calcium hydroxide to the cooled solution. Calcium fructosate crystallises out, and can be decomposed by oxalic acid. It is rather difficult to obtain crystals of the sugar. For the following reactions use a dilute solution of commercial fructose. Certain of the reactions can be demonstrated by the use of a i per cent, solution of "invert sugar," obtained by boiling 100 cc. of i per cent, cane sugar with I cc. of strong hydrochloric acid for two minutes. III. Repeat Exercises 95 to 97. They are all obtained. 112 THE CARBOHYDRATES. [CH. V. 112. Prepare the osazone as directed in Ex. 109. It is identical with glucosazone. Note. — The reaction between fructose and an excess of phenyl-hydrazine is as follows : — CHjOH CH : N.NH.C.Hb I I C = O C : N.NH.CjHj I + 3 C.H5.NH.NH, = I *(CHOH)3 *(CHOH)3 I I CHjOH CHjOH + 2 HjO + NH, + CeH^NHj The configuration of the three secondary alcoholic groups indicated by • is identical in glucose and fructose (see formula on page 10 1). It follows that the osazones are identical. 113. Repeat Ex. no. It is obtained with great brilliance. 114. To about 15 drops in a test-tube add 6 drops of a i per cent, alcoholic solution of a-napthol and 5 cc. of concentrated HCl. Boil. The solution becomes deep purple as soon as the mixture is vigorously boiling. Notes. — i . This modification of the furf urol test is given only by fructose, sucrose and the pentoses. Glucose, maltose, lactose, and the common poly- saccharides only give an intense colour after being boiled from i to 2 minutes. 2. Fructose, either in the free state or produced from sucrose by acid hydrolysis and the pentoses, readily yield furfurol (furfuraldehyde) by the action of strong HCl. With H2SO1 furfurol is readily produced from all carbohydrates (see Ex. no). 3. Furfurol is HC CH HCx /C.CHO O It reacts with a-napthol, thymol, bile salts (see Ex. 315) in the presence of strong acids to give coloured compounds. 4. Proteins that contain a. carbohydrate group also give a reaction (see Ex. 26). 115. SeliwanofE's test for fructose. To 5 cc. of SeliwanofiE's reagent add a few drops of the sugar and heat the solution to boUing. A red colouration and a red precipitate are formed. The precipitate dissolves in alcohol, to which it imparts a striking red colour. Notes. — The reagent is prepared by dissolving 0-05 gram, of resorcin in 100 cc. of hydrochloric acid, diluted with its own volume of water. The test is also given by glucose after long boiling, but a precipitate is not usually formed. CH. v.] MALTOSE. 113 B. The Disaccharides. Maltose is the disaccharide formed as the final product of the hydrolysis of starch by enzymes, such as ptyalin, diastase, etc. It is hydrolysed by boiling acids, and by the enzyme maltase of the small intestine, to two molecules of glucose. It exhibits well-marked reducing properties towards Fehling's solution, but not towards Barfoed's. It forms an osazone with phenyl-hydrazine acetate, which is more soluble than glucosazone and which nielts at 2o6°C. Constitutionally it is glucose-a-glucoside. ii6. Preparation of Maltose. Weigh out 200 grams, of fine potato starch and divide it into three approximately equal portions. Add 50 cc. of cold water to one portion and stir until a uniform cream is obtained. Pour this slowly into 1200 cc. of boiling water contained in an enamelled iron vessel, stirring well during addition. Boil for a minute, stirring all the time. Cool to 55 C. and add 2 cc. of a fresh malt extract (see note i). The starch paste becomes hquified in a few minutes. Boil the liquid again, and to it add the second portion of starch, which has been stirred up with another 50 cc. of cold water. Cool to 55 C, add 2 cc. of malt extract, and liquify as before. Boil again, add the third portion of starch, and cool to 55 C. Add 40 cc. of malt extract, and digest for 24 hours. Boil, filter, and evaporate in a porcelain dish on a water bath until a fairly thick skim forms on the surface. On another water bath heat 500 cc. of 95 per cent, alcohol in a flask and pour it on to the hot syrupy solution, stirring well. The maltose dissolves and the dextrin is precipitated, carrying down with it a considerable percentage of the maltose. Connect the flask to a reflux condenser (fig. 7) and heat on a boihng water bath for 5 hours, repeatedly agitating the mixture. Allow the mixture to cool thoroughly, and pour off the alcoholic solution of maltose from the gummy residue of dextrine. Transfer the alcoholic solution to a distilling flask connected with a condenser and distil off the alcohol as completely as possible by heating the flask on a water bath. Pour the thin syrupy residue 114 THE CARBOHYDRATES. [CH. V. into a beaker and allow to cool. Add a little crystalline maltose and allow to stand for 24 hours in a cool place. The syrup should set to a semi-soHd crystalline mass. Spread this on a porous earthen- ware plate to dry. Recrystallisation. Weigh the solid, add one-fourth of its volume of water, and heat on the water bath until a syrup is formed. For every cc. of water taken add 10 cc. of hot 88 per cent, alcohol. Filter. Cool, add a little crystalline maltose, and allow to stand for about 2 days. Filter on the pump, wash with a Uttle 95 per cent, alcohol, and dry on a porous plate. Notes. — i. Preparation of malt extract. Mix 40 grams, ot finely ground pale dried malt with 100 cc. of cold water. Shake well, and aUow to stand for four hours. Filter. 2. It is easier to prepare a strong solution of starch by using soluble starch (see p. 395). In this case boil 1200 cc. of water, stir 200 grams, of the soluble starch with 200 cc. of cold water, and pour this slowly into the water, kept hot on a boiling water bath. Cool to 55° C. and add 40 cc. of the malt extract. Use a I per cent, solution for the following exercises : — 117. Repeat Exs. 95, 99 and 100. The reactions are indis- tinguishable from those of glucose. 118. Repeat Exercise 98. A reduction is generally obtained. (Distinction from glucose.) 119. Repeat Exercise 102, using 12 drops of the 20 per cent, copper sulphate for the first trial. It will be seen that the maltose has a reducing power of about 60 per cent, that of a glucose solution of the same strength. 120. Repeat Exercise 103, using 20 drops of the copper sul- phate for the first trial. The reducing power of the solution has been markedly increased, owing to the hydrolysis of the maltose to glucose. (Distinction bom glucose.) 121. Repeat Exercise loi. A reduction is not usually ob- tained. (Distinction from glucose.) Note. — It must be remembered that the glucose solution employed was 0-2 per cent. Two cc. of this can only reduce 4 to 5 drops of 20 per cent, copper sulphate (see Ex. 102). So that, though the i per cent, maltose solu- tion employed exhibits strong reducing powers towards alkaline copper CH. v.] LACTOSE. 115 solutions, it has, relatively, a very feeble reducing power towards the acid Barfoed's reagent. It must be emphasised that useful information can only be derived from Barfoed's test if the reducing power of the solution towards alkaline reagents is known. 122. Prepare the osazone as directed in Exercise 109. Malt- osazone is much more soluble than glucosazone, and only separates on coohng. It is important to allow the solution to cool slowly as directed. It generally crystallises in clusters of broad plates, not in needles. It can be recrystallised by dissolving the precipitate in boiling water, filtering, and allowing the hot solution to cool slowly. It melts at 206° C. Lactose is the sugar found in milk, and often in the urine of women during lactation. It has reactions very similar to those of maltose. It is hydrolysed by boiling acids, and by the ferment lactase into equal parts of glucose and galactose. Constitutionally it is glucose-/3-galactoside. It is not fermented by ordinary yeast. Use a I per cent, solution for the following exercises :-•— 123. Repeat Exs. 95, 97, 99 and 100. The reactions are indistinguishable from those of glucose. 124. Repeat Ex. 98. A reduction is generally obtained. (Distinction from glucose.) 125. Repeat Ex. 102, using 14 drops of the 20 per cent, copper sulphate for the first trial. Lactose has a reducing power about 70 per cent, of that of glucose. 126. Repeat Ex. 103, using 18 drops of the copper sulphate for the first tube. The reducing power of the solution has been markedly increased, owing to the hydrolysis of the lactose to glucose and galactose. (Distinction from glucose.) 127. Repeat Ex. loi. A reduction is not usually obtained. (Distinction from glucose.) 128. Prepare the osazone (see Ex. 108). Lactosazone is much more soluble in hot water than glucosazone. It crystallises in irregular clusters of fine needles (" Hedge-hog crystals "). It can be recrystallised from hot water (see Ex. 122). It melts at 200° C. 116 THE CARBOHYDRATES. [CH. V. Sucrose (cane sugar) is widely distributed in the vegetable kingdom, where it functions as a reserve material. It crystallises well, is very soluble in water, and has a much sweeter taste than glucose. It does not reduce Fehling's solution, does not form an osazone, and does not behave as an aldehyde or ketone. It is hydro lysed very readily by boiling acids to a mixture of glucose and fructose. Cane sugar is dextrorotatory, but since fructose is more laevorotatory than glucose is dextrorotatory, a mixture of the two in equal parts is laevorotatory. So the sign of rotation being inverted by hydrolysis, the process is known as inversion, and the product as " invert sugar." This hydrolysis is also effected by the enzyme invertase (sucrase), which is found in the small intestine and in certain yeasts. The constitution of cane-sugar is not yet definitely established, but in all probability it is formed by the condensation of glucose and fructose in such a way as to destroy both the aldehyde and the ketone groups. H.C CHjOH CHjOH Glucose portion. CHgOH Fructose portion. Use a freshly prepared i per cent, solution of pure white crystalline cane sugar (" cofifee sugar ") for the following reactions. 129. Repeat Exs. 95 and 97. Sucrose is not affected by alkali, and does not reduce alkaline copper solutions. CH. v.] STARCH, 117 130. To 3 cc. of the solution add i drop of concentrated HCl. Boil for a few seconds. Cool under the tap. Add about 10 drops of 20 per cent, copper sulphate, 3 drops of glycerol, and about 3 cc. of 20 per cent, sodium hydroxide. Boil. A marked reduction is obtained. Note. — Sucrose is hydrolysed extremely rapidly by acids into glucose and fructose. Though the polysaccharides yield reducing sugars by acid hydrolysis the above procedure would have very little effect on them. 131. Repeat Exs. 114 and 115. Sucrose behaves like fructose. C. Polysaccharides. These compounds are formed by the condensation of an indefinite number of molecules of monosaccharides. The pentosans (C5H804)n yield pentoses on hydrolysis. The hexosans (CgHio05)n yield hexoses, generally glu- cose, on acid hydrolysis. The polysaccharides described below are hexosans. Starch is widely distributed in the vegetable kingdom as a reserve carbohydrate. It occurs in the form of grains in many roots, tubers, seeds, and leaves. The size and shape of the grains are peculiar to each botanical species. These grains probably consist of at least two substances. A. " Amylopectin, " or " starch cellulose." B. " Amylose," or " granulose." Amylopectin forms about 60 per cent, of the grain. It is a mucilaginous substance, which swells up without dis- solving in boiling water or in cold sodium hydroxide. It is hydrolysed by acids into glucose. By certain enzymes, called amylases, found in malt, saliva, and the pancreas, it is converted into a mixture of maltose and " stable dextrin," that is only very slowly hydrolysed to maltose and glucose. It does not seem to give a blue colour with iodine. Amylose is soluble in cold water. It is rapidly and completely converted to maltose by the amylases without leaving a residuum of dextrin. It gives a blue colour with 118 THE CARBOHYDRATES. [CH. V. iodine. The grains are covered with a film of amylopectin, which prevents the amylose from dissolving out in cold water. " Starch paste " is obtained by pouring a suspension of the grains in cold water into boiling water. It is to be considered as a mixture of amylopectin and amylose, both of the substances being colloids. It is opalescent, due to the amylopectin. It is completely precipitated by half saturation with ammonium sulphate, or by the addition of an equal volume of strong alcohol. It has no reducing properties. "Soluble starch " differs from starch paste in being clear and limpid. It is produced by the action of amylases or acids on starch paste. It is only slowly precipitated by half saturation with ammonium sulphate, but is precipi- tated immediately by full saturation. It has no reducing properties. Dextrins are formed by the partial hydrolysis of starch by amylases, acids or superheated steam. The name arises from the fact that they are strongly dextrorotatory. They differ considerably in complexity. There are two main varieties : erythro-dextrins, giving a reddish colour with iodine, and achroo-dextrins, which give no colour. By fractionation with salt solutions Young has separated three erythro-dextrins, I., II., and III. The first two are precipi- tated by full saturation with ammonium sulphate, and give a purple and a red colour respectively with iodine. Erythro- dextrin III. is not precipitated by salts, and gives a red- brown colour with iodine. The achroo-dextrins also are not precipitated by salts. They are insoluble in strong alcohol and in ether. They reduce Fehhng's solution slightly, but do not form osazones nor ferment with yeast. Stable dextrin is the dextrin obtained from starch by the action of amylases continued until the hydrolysis shows an apparent equilibrium. It is, as its name implies, very resistant to the further action of the enzymes, but is apparently broken down slowly to maltose and glucose. CH. v.] STARCH. 119 It is possible to regard it as being formed by the condensa- tion of forty molecules of glucose with the elimination of thirty-nine molecules of water. On this assumption its formula would be 39 (CgHioOg) CgHijOg. Its reducing power is slight, and has [ajn about +195°. At the stage of apparent equilibrium in the action of amylases on starch, about 85 per cent, of the initial weight is in the form of maltose, and about 19 per cent, of stable dextrin is formed. The increase in weight is due to the addition of the elements of water in the hydrolysis. The following equation has been suggested by Brown and Millar. 200 (CeHioOg) + 81 H2O = 80 Q2H22OU + {C^UJ^XCeiiizO, Starch. Maltose, Stable dextrin. Malto-dextrin is the name given to an achroo-dextrin obtained by the action of malt diastase in starch. It is rapidly hydrolysed to maltose by the amylases. The exact relationships between these various dextrins to one another and to the constituents of the starch grain are so imperfectly understood at present that it is not considered advisable to give a scheme of the stages of hydrolysis. 133. Place a small amount of diy potato-starch on a slide, add a drop of water, cover with a slip, and examine under the microscope. Note the characteristic oval starch grains, the concentric markings and the hilum, usually eccentric. Make a drawing of the grains. Run a drop of iodine under the slip ; note that the grains take on a blue colour. 133. Shake a small amount of potato starch with cold water. The starch does not dissolve. Filter, and add a drop of iodine solution to the filtrate. The characteristic blue reaction is not obtained. 134. Shake some dry starch with a little sodium carbonate solution. No change is effected. Shake another portion of starch with a little sodiumhydroxide. The starch is immediately gelatin- ised. To this jelly add a few drops of iodine solution: a blue 120 THE CARBOHYDRATES. [CH. V. colour is not obtained. Treat with strong acetic acid. A deep blue colour appears. Note. — Free iodine is necessary to give the blue adsorption compound with starch. Sodium hydroxide removes free iodine, converting it into iodide and iodate. The action of the acid on the latter causes the appearance of free iodine and the blue colour. Always neutralise an alkaline sohition before testing for the polysaccharides. The following equations show the effect of sodium hydroxide on iodine, and of acid on a mixture of iodide and iodate : (i.) 3l,+ 6NaOH = sNal + NalO, + 3H,0. (ii.) 5NaI + NalOj + 6Ha = 3lj + 6NaCl + sHjO. 135. Preparation of starch paste. Boil about 75 cc. of distilled water in a beaker. Weigh out i gram, of dry potato starch in another small beaker, add about 10 cc. of cold water, and stir to get a uniform suspension. Pour this into the boiling water and stir well. Wash the small beaker out with another 10 cc. of cold water, adding this to the boiling fluid. Stir again, and keep boiling for i minute. Cool, and make the volume up to 100 cc. Note that the " solution " is distinctly opalescent. It should be quite uniform and free from lumps. 136. To a small amount of the paste add a drop or two of dilute iodine. A deep blue colour is produced. Note. — ^The iodine solution should be about ooi N. (See appendix, P- 390.) 137. Treat 5 cc. of the cold starch paste with an equal bulk of saturated ammonium sulphate. Shake the test-tube and allow it to stand for five minutes. The starch is precipitated. Filter through a dry paper, and add a drop of iodine solution to the filtrate. No blue colour, or only the very shghtest tint is obtained, showing that the whole of the starch paste is precipitated by half-saturation with ammonium sulphate. 138. Boil 5 cc. of the starch paste with two drops of concen- trated sulphuric acid for about 15 seconds. Note that the solution becomes perfectly clear and translucent. Add two drops of strong ammonia to neutralise the acid, cool under the tap, add an exactly equal bulk of saturated ammonium sulphate, shake the tube vigor- ously, and allow it to stand for five minutes. Filter through a dry filter-paper and add two drops of iodine solution to the filtrate. A deep blue colour is obtained. CH. v.] STARCH. 121 Note. — Starch paste is rapidly converted into " soluble starch " on boiling with dilute mineral acids. Soluble starch differs from starch paste in that it is not completely precipitated by half-saturation with {NH4)2S04 in the course of five minutes. If it be allowed to stand for twenty-four hours, however, it iS completely precipitated. 139. Measure 2 cc. of the paste into a test-tube, add 6 drops of concentrated hydrochloric acid by n:ieans of a dropping pipette (sea fig. 5). Heat to boiling and maintain the boiling for one minute by the watch. Cool thoroughly imder the tap. Add first one drop, and then another drop, of the iodine solution. A red or violet colour is produced, indicating the conversion of the starch into erythro-dextrin by acid hydrolysis. 140. Boil 2 cc. of the paste with 6 drops of concentrated hydrochloric acid as before. Cool. Add 3 drops of glycerol and 8 drops of 20 per cent, copper sulphate. Add 20 per cent, sodium hydroxide imtil a grey precipitate of basic copper sulphate is pro- duced. Now add another 2 cc. of the sodium hydroxide and boil for a minute. A slight reduction is usually obtained. 141. Repeat the previous exercise, using 20 drops of the acid, and boiling for two minutes. Cool. Add 3 drops of glycerol and 16 drops of the copper sulphate. Neutralise with soda and then add 2 cc. in excess. Boil for one minute. Complete, or nearly complete, reduction of the copper is obtained, indicating that the starch has been hydrolysed to glucose (see Ex. 102). Note.— If 12 drops of hydrochloric acid be added and the mixture boiled for one minute, it wiU generally be found that only a yellow colour is produced with iodine, and that the amount of glucose formed is not sufficient to reduce 9 drops of copper sulphate. At this stage a considerable proportion of the carbohydrate is in the form of achroo-dextrin. It is important to note that the complete hydrolysis of starch by acids is relatively slow compared to that of sucrose and the other disaccharides (see Ex. 130). The addition of a. couple of glass beads makes it easier to obtain smooth boiling in the above exercises. 142. Shake a little commercial dextrin with some cold water. An opalescent solution is formed. Boil the solution. It becomes perfectly translucent. (Distinction from glycogen.) m Use a 3 per cent, solution of commercial dextrin for the following re- actions : — 143. To about 5 cc. of the dextrin solution add iodine solution, drop by drop, noting the colour at every addition. The colour is at 122 THE CARBOHYDRATES. [CH. V. first almost a pure blue, but it changes through a rich purple-red to a red-brown as the iodine is added. Note. — Some samples of commercial dextrin contain a. considerable amount of soluble starch. 144. Repeat the above experiment, but boil and then cool the tube after each addition. The colour disappears on boiUng, but does not reappear on cooUng until several drops of iodine have been added, unless much soluble starch is present. 145. Add a drop or two of the starch paste prepared in Ex. 135 to about 5 cc. of the dextrin solution. To this mixture add diluted iodine solution, drop by drop. The first additions produce a pure blue colour, and it is not till a considerable amount of iodine has been added that the solution acquires a purpUsh tint. Note. — The afi&nity of starch for iodine is so much greater than that of dextrin that the characteristic colour reactions of erythro-dextrin are not obtained until all the starch has been saturated with iodine. Even then it is sometimes difi&cult to detect, owing to the deep blue starch reaction. 146. Treat 5 cc. of the dextrin solution with about 10 drops of the starch paste : to the mixture add an equal bulk of saturated ammonium sulphate, shake vigorously, and allow to stand for five minutes. The starch is precipitated. Filter through a dry paper, and to a portion of the filtrate add a drop or two of iodine solution. The purple red reaction, of erythro-dextrin is obtained. 147. Saturate 5 cc. of the dextrin solution by boiUng with an excess of finely powdered ammonium sulphate. Note the precipitate of erythro-dextrin produced. Cool under the tap and filter. To the filtrate add a drop of iodine. A red-brown colour is produced. Note. — This colour is due to the fact that erythro-dextrin III. is not precipitated by ammonium sulphate. This is the method employed for the identification of erythro-dextrin in the presence of glycogen, which is com- pletely precipitated by saturation with ammonium sulphate. 148. Boil a few cc. of the dextrin solution with a small amount of Fehling's fluid. A well-marked reduction is usually obtained. Note. — Commercial dextrin is generally prepared by the action of dilute acids on starch (see Ex^ 139), the action being stopped as soon as a portion fails to give a blue colour with iodine, and the products then being precipitated by alcohol. Such preparations contain some glucose, and often a little soluble starch. At the same time it must be noted that the achroo-dextrins have a reducing action themselves even when thoroughly separated from the glucose. CH. V.J GLYCOGEN. 123 149. Take lo cc. of the dextrin solution in a small flask ; add 30 cc. of strong (95 per cent.) alcohol, place the thumb over the mouth of the flask and shake vigorously for some seconds. Note that a portion of the dextrin is precipitated as a gummy mass which sticks to the sides of the flask. Pour off the alcohol, filter it and label the filtrate A. Rinse the flask out with a few cc. of alcohol, shake off as much of this alcohol as possible, and add 10 cc. of hot water. Shake this round the flask till the whole of the gummy precipitate dissolves. Divide the solution into three portions, B, C, and D. To B add a drop of iodine : a purple colour is produced. Boil C with a little Fehhng's solution : only a slight reduction takes place. Boil D with two drops of concentrated sulphuric acid for two minutes, neutralise with sodium hydroxide, and boil with a Uttle Fehling's solution : a well- marked reduction occurs. ^50. To a portion of filtrate A, add a drop of iodine solution. No colour is produced. To another portion of about 5 cc. add an equal bulk of strong alcohol. A white precipitate of achroo-dextrin is formed. Glycogen is a reserve polysaccharide found in the Hver and muscles. It forms a white amorphous powder, soluble in water to form an opalescent solution. It is precipitated from solution by the addition of an equal volume of strong alcohol or by full saturation with am- monium sulphate. It does not reduce Fehling's solution, form an osazone nor ferment with yeast. It gives a reddish colour with iodine. By boiling acids it is hydrolysed to glucose : by most of the diastatic enzymes to maltose, but by the diastase found in the liver to glucose. It is not affected by boiling alkalies. It is dextro-rotatory. Estimation. Pfluger's method is undoubtedly the best. 20 to 100 grams, of the tissue is cut into small pieces and placed in an Erlenmeyer flask of Jena glass. 100 cc. of 60 per cent, potash (" pure by alcohol " — sp. gr. 1. 438) is added, a reflux condenser fitted, and the flask immersed for three hours in a boiling water bath. The alkali destroys the proteins -without attacking the glycogen. After cooling 200 cc. of water and 800 cc. of 96 per cent, alcohol are added, and the mixture left to stand over-night. The glycogen is thus precipitated free from protein. The supernatant fluid is carefully decanted and filtered. The precipitate is washed with ten times its volume of 66 per cent, alcohol, containing i cc. per litre of saturated sodium chloride. After settling, the 124 • THE CARBOHYDRATES. [CH. V. fluid is filtered through the original filter paper. This process is repeated once more, and then the precipitate is shaken with ten times its volume of 96 per cent, alcohol and filtered through the same paper. The precipitate is washed with ether, dissolved in boiUng water and the solution made up to one litre. 200 cc. of this are treated with 10 cc. of concentrated HCl and heated in a flask on a boihng water bath for three hours, to convert the glycogen into glucose. After cooling, the solution is neutralised with 20 per cent, potash and filtered through a small paper into a 250 cc. measuring flask. The wash- ings from the flask used for inversion are filtered through the same paper to remove the last traces of glucose, and the solution brought up to 250 cc. The percentage of glucose in the solution is determined by analysis. This multi- plied by '927 gives the amount of glycogen in the 200 cc. of the solution used for inversion, and so the percentage in the tissue can be readily calculated. Prepaiation. A rabbit, which has had a full meal of carrots some five or six hours previously, is killed by decapitation. The liver is cut out as quickly as possible, and the gall-bladder removed. The liver is rapidly chopped into small pieces, a small portion being reserved for Ex. 156, and the remainder immediately thrown into boihng water. After about two minutes boiling the larger morsels are strained off, pounded to a paste with sand in a mortar, and replaced in the boiUng water. The proteins in solution are then coagulated by making the boiUng fluid just acid with acetic acid. The fluid is filtered through coarse filter paper. In this way a crude solution of glycogen is obtained. 151. Boil 5 CC. in a test-tube. The characteristic opalescence does not disappear. (Distinction from erythro-dextrin.) 152. To a smaU amount of the cooled solution add iodine, drop by drop. A red colour is formed, which disappears on shaking, until with a certain amount of iodine added it is permanent. Now heat the solution. The colour disappears, to reappear on cooling. Note. — If much protein is present in solution the colour will not re- appear on coohng unless a considerable amount of iodine be added. This is due to the fact that proteins combine with iodine to form an iodo-protein. 153. Saturate 10 cc. of the solution by boiling with an excess of finely-powdered ammonium sulphate ; cool thoroughly under the tap. The glycogen is precipitated. Filter, passing the filtrate again through the paper if it comes through cloudy. Add a drop or two of iodine to the filtrate. No red colour is produced. Scrape the precipitate off the paper, boil with a small amount of water. The solution is markedly opalescent. Cool the solution and add iodine. A port-wine red colour is obtained. 154. Boil 5 cc. of the solution with a little FehUng's fluid. A very slight or no reduction is obtained. Note. — If the liver has been rapidly boiled, no sugar will be present. If delay has occurred during the initial stages of the preparation, some of the glycogen will have been converted into glucose. (See Ex. 156.) CH. v.] QUANTITATIVE ESTIMATION. 125 155. To 10 cc. of the solution add 20 cc. of strong alcohol, shake vigorously and filter. To a portion of the filtrate add iodine solution. No colour is obtained, showing that the whole of the glycogen is precipitated. Dissolve the precipitate in a Uttle hot water: note that it is opalescent. Add three drops of strong sulphuric acid and boil for about three minutes : the opalescence disappears. Neutralise with sodium hydroxide and apply FehUng's test. A marked reduction occurs, due to the conversion of the glycogen into glucose by the boiling acid. 156. The portion of rabbit's Uver that was reserved has been kept in a warm place for about six hours and extracted with boiling water as before. (Or a decoction of the hver of a sheep obtained from a butcher may be used.) Note that the solution is almost translucent. To a portion add iodine : only a very slight or no red colour at all is produced. To another portion apply Fehhng's test : a weU-marked reduction occurs. 157. Prepare a solution which contains equal quantities of I per cent, starch paste (freshly prepared), of a strong solution of glycogen and of a 3 per cent, solution of commercial dextrin. Note that the mixture is markedly opalescent. To a small portion add diluted iodine, and note that a pure blue starch reaction is obtained. To another portion of about 5 cc. add an equal bulk of saturated ammonium sulphate, shake vigorously, leave for five minutes, and filter. Note that a portion of the filtrate gives a reddish colour with iodine, and that it is distinctly opalescent. Indication of the presence of glycogen. Saturate the remainder of the fluid by boiling with finely- powdered ammonium sulphate. Cool and filter. The filtrate gives a reddish-brown colour with iodine. Indication of the presence of erythro-dextrin. D. The Quantitative Estimation of (he Carbohydrates. A very large number of methods have been introduced for the estimation of glucose, etc., and considerable 126 THE CARBOHYDRATES. [CH. V. experience is required to select the method best suited for a given purpose. The following scheme indicates the principle of the more important methods, only a few of which are described below or in other sections of this book. A. Direct Volumetric Methods. 1. Fehling's. See p. 141. 2. Ling's modification. See p. 141. 3. Pavy's. Strong ammonia is added to Fehling's solution. A measured amount of this is boiled and the sugar solution run in from a burette. The cuprous oxide formed is kept in solution by the ammonia (see Ex. 97, note 5), forming a colourless compound. The end point is thus much easier to see. The practical difficulty of the method is to regulate the heating and also the rate at which the sugar solution is added. 4. Benedict's. See p. 127. 5. Folin and McEllroy's. See p. 129. B. Indirect Volumetric Methods. 6. Amos Peters'. See p. 134. 7. Bertrand's. The sugar is heated with an excess of Fehling's solution. The cuprous oxide formed is filtered off through asbestos and dissolved in an acid solution of ferric sulphate, some of which is reduced to ferrous sulphate. The amount of the latter is determined by titration with standard permanganate. 8. Wood-Ost's. See p. 131. This is very similar to the above, except that copper bicarbonate is used instead of Fehling's. 9. Cole's micro-method. See p. 253. C. Colorimetiic Methods, JO. Beriedid's. See p. 251, CH. v.] BENEDICT'S METHOD. 127 D. Polaiimetric Method. II. This is of great value. The relationships between reduc- ing and rotatory powers of solutions before and after hydrolysis must be determined for the identification and analysis of mixed carbohydrates. Of the various methods proposed, the Author is of the opinion that for ordinary routine work and for urinary analysis, Benedict's direct volumetric method is the most reliable in the hands of the majority of workers. The recent method of Folin and McEUroy has certain advantages, especially in the cost of materials, and may eventually supersede Benedict's. The Wood-Ost process is worthy of more extended recognition. In spite of the lo minutes' boihng that is necessary, the method is a rapid one, and when completed one is left with a sense of confidence that is somewhat lacking in the direct methods. For accurate research work the method of Amos Peters is extremely satisfactory. When a large series of diabetic urines have to be examined sufficiently approximate results can be obtained by polarisation, after removal of the pigment by blood charcoal in the presence of lo per cent, of acetic acid, a little freshly prepared metaphosphoric acid being added if proteins are present. 158. Benedict's Method. Principle of the Method. — ^An alkaline solution of copper sul- phate, containing thiocyanate is boiled and the sugar solution run in from a burette till the blue colour just disappears. The thio- cyanate forms a white insoluble compound with the cuprous hydroxide formed by the reduction of the copper, and so there is no red cuprous oxide precipitated to obscure the blue tint. A little potassium ferrocyanide is also added to prevent any possibihty of the deposi- tion of the cuprous oxide. Preparation of the Solution. — With the aid of heat dissolve Sodium citrate . . . . . . 200 grams. Sodium carbonate (cryst.) . . 200 grams. (or anhydrous sod. carb. 75 grams.) Potassium thiocyanate (sulphocyanide) 125 grams. in enough distilled water to make about 800 cc. of the mixture and filter, and cool to room temperature. Dissolve 18 grams, of the purest, air-dried crystalline copper sulphate in about 100 cc. of distilled water, and pour it slowly into the other hquid with constant stirring. Add 5 cc. of a 5 per cent, solution of potassium ferrocyanide and then distilled water to make 128 THE CARBOHYDRATES. [CH. V. the total volume looo cc. The solution appears to keep indefinitely, without any special precaution, such as exclusion of Ught, etc. Method of Analysis. — Fit a 150 cc. flask into a ring of a retort stand of such a size that it is fairly firmly held. There is no need to use a wire gauze. Arrange the flask at such a height over a Bunsen burner that the reagent can be kept briskly boiling by means of a small flame. In the flask place 3 to 4 grams, of anhydrous soditun carbonate. This can be roughly measured by taking a depth of i inch in a dry test-tube. Then add 25 cc. of the reagent and heat till most of the carbonate is in solution. Run the sugar solution in from a burette, which is best held in the hand. Run the sugar in slowly, till a bulky chalk- white precipitate is formed and the blue colour lessens perceptibly in intensity. From this point the sugar is added more and more slowly, with constant boiling, imtil the dis- appearance of the last trace of blue colour, which marks the end-point. If the volume of the sugar is less than 5 cc, dilute it accurately with water till about 10 cc. are judged necessary. Repeat the titration with this as before. Notes. — There is a tendency to run in an excess of the sugar, unless special care is exercised throughout the titration, and particularly at the end. The solution must be kept steadily boiling during the entire process, and towards the end the sugar must be added in portions of a drop or two, with an interval of about 30 seconds after each addition. Should the mixture become too concentrated, boiled distilled water may be added to replace that lost by evaporation. The titration can also be carried out in a white porcelain dish of 10 to 15 cm. in diameter, but the risk of reoxidation of the cuprous compound is greater than in a flask. Should the solution bump excessively, two or three small pieces of broken porcelain may be added. The 3 to 4 grams, of anhydrous sodium carbonate are added to produce the necessary alkalinity. This proportion of alkali cannot be added to the bulk of the standard solution, for it would crystallise out at room temperature. Fig. 13 A. Apparatus for Benedict's Method. CH. v.] METHOD OF FOLIN AND McELLROY. 129 Calculation of Results. 25 cc. of Benedict's solution are reduced by 0-05 gram, of glucose. 0-053 gram, fructose. 0-074 gram, maltose. 0-0676 gram, lactose. Example. — First titration required 2-4 cc. Solution diluted i in 4 (10 cc. of sugar diluted with 30 cc. water). Second titration required 9-7 cc. So 9-7 cc. diluted solution contain 0-05 gram, glucose. 100 cc. diluted solution contain ""5 x lo" 9.7 100 cc. original solution contain °'°^ x 100 x 4 ^ ^.^^ ^^^ 9-7 159. The method of Folin and McEUroy.* Principle. Five cc. of a 6 per cent, solution of crystalline copper sulphate are treated in a large test-tube with a mixture of sodium phosphate and sodium carbonate. A deep blue solution is obtained on boiling. 2 cc. of a strong solution of potassium thiocya- nate are now added. The sugar solution is run in from a burette. A white precipitate of cuprous thiocyanate is formed. The sugar is run in very slowly until the blue copper colour is just discharged. Owing to the reduction of the alkahnity of the solution, the amount of •copper reduced by a given amount of sugar is considerably greater than in Fehhng's or Benedict's methods. Also the reoxidation •of the cuprous salts to the cupric condition is relatively very slow. The main objection to the method is the relatively slow rate of reduction. Reagents reauiied : 1. Copper sulphate. Dissolve 60 grams, of the best air-dried crystalline -copper sulphate in distilled water, add 2 or 3 cc. of pure sulphuric acid, and make the volume up to i litre. The acid is to prevent the slow deposition of copper hydroxide and silicate due to the action on the glass. 2. Alkaline phosphate powder. Mix together in a large mortar, 100 grams, of disodium phosphate crystals (HNa2P04i2HjO), and 60 grams, of anhydrous sodium carbonate. 3. To 40 grams, of sodium or potassium thiocyanate add 50 cc. of dis- tilled water. When dissolved make the volume up to 100 cc. * Journ. of Biological Chemistry, xxxiii., p. 516 (1918). 130 THE CARBOHYDRATES. [CH. V. 4. Special Burette. Folin and McEllroy describe an ingenious method of measuring small quantities of fluids by means of an accessory fine tip fitted to an ordinary burette. When the fluid is dropping from the burette very slowly, the size of the drop is constant for a particxilar fluid. So if the number of drops emitted by a given tip for a delivery of I cc. be known, the volume of the number of drops required for the titration can be readily calculated. The author pre- fers to use the micro-burette shewn in fig. 14. This may be of 2 or 5 cc. capacity. A rubber tube and spring chp is preferable to a glass tap and accessory tip, as the grease from the tap gets into the burette and makes it impossible to get an accurate reading. As recommended by FoUn and McEllroy, the burette must be filled by suction. Elaborate washing of the burette in the intervals between successive estimations is thereby rendered unnecessary. Method. Weigh out approximately 5 grams, of the phosphate mixture and transfer it to a clean, dry tube, conveniently 7 inches by |ths inch. Add 5 cc. of the 6 per cent, solution of copper sul- phate, shake and heat to boihng. A deep blue solution is obtained. Add 2 cc. of the strong solution of thiocyanate and heat again. Run in about 0-5 cc. of the sugar solution from the burette and boil very gently for 2 minutes by the watch. The tube should be held at an angle and moved about in a small flame : excessive concentration and loss by spurting can be thus avoided. If there is an appre- ciable amount of sugar present a chalky white precipitate appears. If the blue colour entirely disappears there is more than 5 per cent, of sugar present, and the estimation must be repeated with a diluted solution. If the copper is only sUghtly reduced, yielding only a small amount of cuprous thiocyanate, add a further amount of the sugar solution and boil gently for another minute. If now the greater part of the copper has been reduced, complete the titration by adding a drop at a time, boiling for i minute after each addition. The total period of boihng must not be less than 4 minutes, and should not exceed 8. The copper value has been adjusted to a period of 5 to 6 minutes. A second estimation is often necessary. With a Uttle experi- ence it is easy to judge of the amount that should be added, so that Fig. 14 Micro- burette CH. v.] WOOD-OST METHOD. 131 after a preliminary boiling period of 3 minutes, only a few drops more are required to complete the titration. With pure glucose solutions the end point is very sharp. With lactose, maltose and diabetic urines the end point is the transition from a green to a yellow colour. The special precaution necessary is to ensure that the boiling period is within the stated limits. Calcvdation of results. 5 cc. of the copper are reduced by 25 mg. glucose. „ „ „ 25 mg. fructose. „ „ ,, 40-4 mg. anhydrous lactose. „ „ „ 45 mg. anhydrous maltose. [n tlie case of glu^cse the concentration in grams, per cent, is 2-5 divided by volume of solution required. 160. The Wood-Ost copper carbonate method.* Principle. A solution of copper sulphate in carbonate and bicarbonate of potash is boiled with a given volume of the sugar solution. The cuprous oxide formed is filtered off through asbestos and washed with cold water. It is suspended in acid ferric sulphate and the amount of ferrous sulphate formed determined by titration with standard potassium permanganate. The amount of copper reduced being known, the weight of sugar in the "Volume taken can be determined by reference to a curve or tables. Preparation of solutions. 1. Copper carbonate. Dissolve 250 grams, of potassium carbonate and loo grams, of potassium bicarbonate by the addition of about 600 cc. of warm distilled water. Dissolve 23-5 grams, of pure crystalline copper sulphate in about 200 cc. of water. Gradually add the copper to the carbonate solution, mixing well during the addition. Make the volume up to 1000 cc. and filter. The solution seems to keep indefinitely. 2. Acid ferric sulphate. Gradually add 250 cc. of pure concentrated sulphuric acid to 750 cc. of distilled water. Add 25 grams, of ferric sulphate. Warm till the sulphate has dissolved, and filter if Secessary. The solution must not be used until it lias cooled. 3. Standard potassium permanganate. Dissolve about 6 grams, of potas- sium permanganate in a i loo cc. of cold distilled water. Having made certaiil that the whole has dissolved standardise the solution as follows : — Weigh out between 0-3 and 0-4 gram, of pure crystalline ammonium oxalate, determining * T. B. Wood and A. Berry, Cambridge Philosophical Journal, xlvi., p. 103 (1904). 132 THE CARBOHYDRATES. [CH. V. the exact weight to a milligramme. Add about 50 cc. of distilled water, to which about 3 cc. of pure concentrated sulphuric acid have just previously been added. Warm on a water bath until the solid has completely dissolved. Titrate the warm solution with the permanganate. This must be run in very slowly at first, further additions not being made until the colour has com- pletely faded. The end point is reached when a faint rose colour persists for at least a minute. If A be the weight of ammonium oxalate taken, and P the volume of permanganate required, then i cc. of permanganate corresponds to 805 • I >!. A _ -2J. = T mg. copper. It is convenient to have T = 10. If T be greater than 10, add 100 (r - 10) cc. of water to i Utre of the solution. If the titration has been con- ducted accurately, i cc. of the permanganate corresponds to i mg. copper. The calculation is based on the following equations : — CujO + Fej(SOi)3 + HjSO, = 2 CuSO, -I- 2 FeSOj + HjO. 10 FeSOi + 2 KMnOj + 8 H^SOj = 5 Fej,{S04)3 + KjSOj -1- 2 MnSOj + 8 HjO. 5 CjHjO, + 2 KMnOi + 3 HjSOi = K^SOi + 2 MnSO, -I- 8 HjO -I- 10 COjj. So I mol. of oxalic acid or of ammonium oxalate (CjOiNjHj.HjO) requires the same amount of permanganate as 2 Fe, which' corresponds to 2 Cu. So P cc. of permanganate = = 0-8951 x A gm. Cu. 142-1 Method. Measure 50 cc. of the copper solution into a 150 cc. flask of " Duro " glass. Add two or three small pieces of broken por- celain to prevent subsequent bumping. Boil by heating on a gauze with a Bunsen. As soon as the solution has commenced to boil run in exactly 10 cc. of the sugar solution, which should be between 0-2 and 0-9 per cent, of glucose (see note i). Note the exact time when the boiling recommences. The flame should be moderately high at first, but as soon as the solution recommences boiling after the addition of the glucose, it should be lowered so that it just main- tains gentle boiling. After exactly ten minutes' boiUng, stop the reduction by immersing the flask in cold water. Then cool thoroughly under the tap. Filtration of the cuprous oxide. This is done through asbestos by means of a Gooch crucible of 25 to 50 cc. capacity (see fig. 48), or, better, through an asbestos mat supported on a small (15 mm. diam.) perforated porcelain plate, resting in a conical funnel that passes through a rubber stopper fitting the neck of a filtering flask. The preparation of the mat and the subsequent filtration is much f acihtated by use of the special pump connexions shewn in fig. 9, p. 74. The mat is prepared as follows : the suspension of well-washed CH. V. WOOD-OST METHOD. 133 asbestos (see note 2) is poured on to the plate (or into the Gooch crucible) and allowed to settle down without suction. After a short time the tube E (fig. 9) is connected to the filtering flask, the tap C being turned so that it connects to B (thus practically preventing suction through E), and the water pressure turned on. The tap C is then turned through an angle so as to allow suction through E, but before the water has been completely drained off, the tap is rapidly opened again, it being important not to suck too hard. The amount of asbestos required is such as to form a mat about 2 mm. in depth. A small porous plate may be placed on the top of the mat to prevent the latter from being disturbed too much by pouring on water or the copper solution. The mat is then washed two or three times with distilled water, gentle suction being apphed after each addition. The final suction should be sufficient to make the mat quite firm. The mat should be prepared during the heating process, it only requiring a few minutes, in spite of this long description. The cold copper solution is poured on to the upper porcelain plate and suction then started. The pressure must be released before all the solution has passed through, it being most important to avoid caking the cuprous oxide by too high a pressure. The flask is washed out with about 10 cc. of cold distilled water (which may, with advantage, have .been recently boiled and cooled), and this poured on to the cuprous oxide on the asbestos and filtered through as before. This washing is repeated twice more, care being taken aU the time to prevent caking of the cuprous oxide by too high a filtration pressure. Solution of the precipitate. Measure 25 cc. of the ferric sulphate solution by means of a measuring cylinder. Pour about 5 cc. of this into the boiling flask and shake this round to dissolve any cuprous oxide that is sticking to the walls of the vessel. Transfer the filter- ing mat and the filtering discs to a small beaker by means of a small glass rod that has a pointed hook. Remove the funnel from the filtering flask and wash it down into the beaker with the remainder of the acid ferric sulphate. Wash out the flask two or three times with small quantities of water,transf erring this to the beaker through the funnel. Wash down the small rod and stir weU with a larger glass rod, which should not be guarded with a rubber collar, since permanganate attacks rubber. The cuprous oxide may all 134 THE CARBOHYDRATES. [cH. V. dissolve, but a certain amount may remain in suspension until the permanganate titration is nearing completion. Titration of the reduced iron. Run in the potassium permanga- nate from a burette fitted with a glass stopcock, stirring the mixture well. From time to time examine the beaker by holding it above the head. Any lumps of undissolved cuprous oxide can thus be detected. They must be broken up and brought into solution by rubbing with the rod. It is most important that this should be done before the titration is completed. The end point is reached when a faint pink tinge persists for at least ten seconds. Calculation of results. One cc. of permanganate = lo mg. Cu. The amount of sugar corresponding to various amounts of copper are obtained by plotting the results given below. The amount of sugar corresponding to the exact amount of copper reduced is thus foimd. The number of milligrammes of sugar in lo cc. divided by lo and multiplied by the dilution employed gives the percentage of sugar. mg. Cu. mg. Glucose mg. Maltose mg. Cu. mg. Glucose mg. Maltose anhydride anhydride anhydride anhydride 25 7-3 14-0 175 53 99 50 15 28-2 200 60 -5 113 75 22-4 42-3 225 69-5 130-4 100 30 56-2 250 79-2 147-5 125 37-8 70-5 275 89 164-6 150 45-3 84-5 290 95-4 175 Notes. — i. A rough approximation of the concentration of glucose in the original solution can be made by use of Fehling's solution. The sugar should be so diluted that 3 to 5 cc. reduce 3 cc. of Fehling's solution. 2. Preparation of the asbestos. (See appendix.) 161. The estimation of glucose by the method of Amos Peters. Principle. A known volume of the sugar solution is boiled with a measured amqunt of an alkaline solution of copper sulphate. The cuprous oxide is filtered off and th? copper in the filtrate determined by treatment with potassium iodide and titration of the iodine liberated by CH. v.] METHOD OF AMOS PETERS. 135 means of a solution of sodium thiosulphate. From the amount of copper reduced the amount of glucose in the volume of solution taken can be determined. Solutions required. 1. Copper sulphate. 69-278 grams, of the purest crystalline salt CuSOj, jHjO, is dissolved in water and the volume made up to 1 Utre. 2. AlkaUue tartrate. 346 grams, of Rochelle salt and 250 grams, of pure potassium hydroxide are dissolved in water and the volume made up to i Utre. 3. Sodium thiosulphate. 99-2 grams, of the purest thiosulphate are dissolved in boiled out distilled water and the volume made up to i litre with boiled out distilled water. It should be prepared at least a week before it is standardised. 4. Potassium iodide. Saturated solution. 100 grams, of the solid are treated with 70 cc. of hot distilled water and the solution allowed to cool. 5. Soluble starch. Shake i gram, of soluble starch (seep. 391) with about 10 cc. of distilled water and pour the suspension into 90 cc. of boihng water. Standardisation of the thiosulphate. Measure 20 cc. of the copper sulphate into a 200 cc. Erlenmeyer flask. Add 40 cc. of distilled water and 20 cc. of strong (33 per cent.) acetic acid. Insert a thermometer and cool or warm to 20° C. Run in about 6-5 cc. of the saturated potassium iodide, the thermometer being withdrawn and its stem washed with this solution. The iodine liberated is titrated at once with the thiosulphate. When approach- ing the end point add about i cc. of the soluble starch. The colour changes to a chocolate brown when very near the end point. This is best determined by the," spot test " method. AUow a drop of the thiosulphate to fall on the quiet surface of the liquid." If the end point has not been reached, a very perceptible white area is seen around the drop. This is very readily distinguished from the diminution of the slightly yellowish colour of the suspended cuprous , iodide. The volimie of the drop delivered by the burette must be deducted from the total volume added. The copper value of the thiosulphate is calculated as shewn in the following example : — 23 cc. of the copper sulphate = 352 -93 mg. Cu. This required 27-6 cc. of thiosulphate. So i[cc. of thiosulphate = ?^^^ =1278 mg. Cu. 136 THE CARBOHYDRATES. [CH. V. The heating apparatus. Use the apparatus shewn in fig. 15. In a 200 cc. Erlenmeyer flask of Resistance glass, and of about 6 cm. basal diameter, place 60 cc. of distilled water. The flask is fitted with a 2-hole rubber stopper carrying a thermometer so graduated that the stem above 34° C. is visible above the upper edge of the stopper. The lower end of the thermometer should be about 2 mm. from the bottom of the flask. Fig. 15. Cole's apparatus for maintaining a standard heating power. The manometer tube contains a dilute solution of eosin or other dye. It also contains a globule of mercury which nearly fills the bottom of the tube. This prevents the rapid oscillations of pressure due apparently to the explo- sions of local gas engines. Turn on the tap B to its full extent and Ught the flame of a Bunsen or Maker burner, which is placed under a piece of asbestos gauze carried by an adjustable ring stand. The gauze should be from 4 to 6 cm. above the top of the burner. Tighten the screw A till the pressure is reduced about one-third. Allow the gauze to get thoroughly heated and then place the flask in the centre of the CH. v.] METHOD OF AMOS PETERS. 137 heated gauze. By means of a stop-watch note the time for the temperature to rise from 35° to 95°. If the time is greater or less than 120 sees, loosen or tighten the screw A and repeat the experi- ment with another 60 cc. of distiUed water until the temperature of the water rises from 35" to 95° in 120 + 2 sees. The height of the ring and the thickness of the asbestos should be such that the pressure is well under the minimum supplied to the laboratory and yet sufficient to prevent any risk of the flame striking back. Note the manometer reading. The standard heating power can be rapidly obtained for further experiments by adjusting the screw A so that the manometer shews the requisite pressure. Filtering Apparatus. It is convenient to use the apparatus shown in Fig. 16. A is a " Duro " flask of 200 cc. capacity. Tube B is an ordinary calcium chloride tube. The lower end should reach at least 3 cm. below the lower edge of the stopper to prevent loss by splashing during filtration. The filtering mat is made of glass wool, asbestos fibre, powdered pumice and asbestos fibre added in that order. The mat should be washed with nitric acid and then thoroughly washed with water. After a test the cuprous oxide on the mat is dissolved in nitric acid diluted with an equal volume of water and then thoroughly washed. An ordinary Gooch crucible can be used with a mat pre- pared in the same way. The Fig. 16. Filtering apparatus for reduced copper. arrangement is shewn in fig. 33, p. 259. Method of Analysis. Into a 200 cc. Erlenmeyer flask measure 20 cc. of the standard copper sulphate, 20 cc. of the alkaline tartrate, and 20 cc. of the sugar solution (which must not contain 138 THE CARBOHYDRATES. [CH. V. o o o o o •ssooniS •3ni CH. v.] METHOD OF AMOS PETERS. 139 more than i8o mg. of glucose). Fit the two-holed rubber stopper firmly into the neck of the flask, adjust the thermometer so that its lower end is 2 mm. from the bottom of the flask and place on the heated gauze. Note the time when the mercury indicates a temperature of 95° C. Allow the heating to continue for exactly 20 sees, beyond this. Remove the flask by gripping the rubber stopper and swiU it for a second or two under the tap or in a bowl of water. The reduction of the temperature practically stops the reduction. Filter the hot fluid at once, using the stem of the ther- mometer as a stirring rod. Wash the flask twice with about 7 cc. of distilled water. Cool the filtrate by holding the flask under the tap. Add exactly 4 cc. of strong sulphuric acid, insert a ther- mometer and cool to 20°. Add 6-5 to 7 cc. of the saturated solution of potassium iodide, washing the stem of the thermometer with this solution. Titrate at once with the standardised solution of sodium thiosulphate as described above, using soluble starch as an indicator when near the end point. Calculation of results. From the amoxmt of thiosulphate required the amoimt of copper in the filtrate is determined. Know- ing the amoxmt taken (352-9 mg.), the amount reduced by the sugar can be calculated. The amount of glucose corresponding to this copper can be determined by a reference to the curve in Fig. 17. Example. Th2 coppsr in the filtrate required 14-62 cc. of thiosulphate. I cc. of thiosulphate = 12-86 mg. Cu. So copper in filtrate = 14-62 x 12-86 = 188-0 mg. Cu. So copper reduced by glucose in 20 cc.=352-9— 188-0=164-9 mg. From the curve this is seen to correspond to 86-3 mg. glucose. So 20 cc. contain 86-3 mg. glucose. So 100 cc. contain 431*5 mg. glucose. = 0-431 per cent. Note. — If the amount of reduced copper is between 60 and 200 mg., the amount of glucose corresponding to this can be obtained by multiplying by 0-522. 161A. The estimation ol lactose by the copper-iodide method. The method is exactly similar to that described in the previous exercise. The author is responsible for the 140 THE CARBOHYDRATES. [CH. V. copper values for lactose. They are represented graphically in fig. 1 8. It must be noted that the results are given as anhydrous lactose, and not as the crystalline hydrate. 210 140 70 270 200 130 60 260 190 120 50 250 180 T10 40 240 170 100 30 230 160 90 20 - 220 ISO 80 10 210 140 70 10 20 30 40 50 60 70 80 90 100 100 110 120 130 140 150 160 170 180 190 200 200 210 220 230 240 250 260 270 280 290 SCO 300 310 320 330 34 Q 350 860 370 380 380 400 mg. Cu. IFig. i8. Curve showing amount of copper reduced by lactose anhydride. In the case of lactose as much as 250 mg. may be present in the 20 cc. taken. The copper values above 25 mg. Cu. can be converted to anhydrous lactose by the use of the following formula : mg. anhydrous lactose = 1*25 + mg. Cu. x 0-778. CH. v.] FEHLING'S METHOD. 141 162. Fehling's method. Preparation of solution. See Ex. 97, p. 106. Method. With a pipette measure 10 cc. of freshly prepared Fehling's solution into a small flask. Add 40 cc. of distilled water, heat the mixture till it boils and keep it boiling the whole time. Run in the sugar solution from a burette, 0-5 to i cc. at a time, allowing the mixture to boil for about 15 sees, between each addition. A red precipitate of cuprous oxide forms and the intensity of the blue in the supernatant fluid decreases. Continue to add the sugar till this is completely removed. This is best determined by holding the fl:sk by the rim at the neck and viewing it by transmitted light. If an excess of sugar be added a yellow or brown colour appears due to the formation of caramel by the action of the alkali on the sugar. If less than 5 cc. of the sugar are used, the solution must be diluted till about 10 cc. are necessary. Thus if 2 '5 cc. are used in the first rough titration, the sugar should be diluted i in 4, by taking 25 cc. and adding water till the volume of the solution is JOD cc. The burette is washed out and filled with this diluted solution and the process repeated. But this time run in nearly the whole of the sugar solution judged necessary at such a rate that the mixture does not go off the boil. Then add o-i to 0-2 cc. at a time till the reduction is complete. This titration should be repeated at least once more. Calculation. 10 cc. of Fehling's solution are reduced by 0-05 gram, glucose. Exam-pie. 1-5 cc. of the original solution necessary. Sugar diluted i in 7 {10 cc. sugar made up to 70 cc.) iO'2 diluted sugar solution required for 10 cc. Fehling's. IO-2 cc. dil. sugar = -05 gm. glucose. •OS X 100 ,, 100 cc. ,, „ = — IO-2 . . , -05 X 100 X 7 100 cc. original sugar = — '- IO-2 = 3-43 per cent. 162A. Ling's method. Preparation of the indicator. Dissolve 1-5 gram, ammoniiun "thiocyanate and r gram, ferrous ammonium sulphate in 10 cc. water Sit about 45° C. and cool at once. Add 2-5 cc. of concentrated 142 THE CARBOHYDRATES. [CH. V. hydrochloric acid. The solution thus obtained has invariably a brownish-red colour, due to the presence of some ferric salt. Add zinc dust, in small portions at a time, till the fluid is just colourless. On standing for some time the red colour reappears, and must be removed again by a trace of zinc dust. But the deUcacy of the indicator is impaired by being decolourised several times. When this indicator is treated with a cupric salt, the colourless ferrous thiocyanate is oxidised to the red ferric thiocyanate. Method of analysis. lo cc. of FehHng's solution and about 30 cc. of water are boiled in a flask and the sugar solution is run in from a burette as described above in Fehling's method. The indicator is not used till the blue colour has nearly disappeared. Then place a drop of the indicator on a white slab. Transfer a drop of the mixture from the flask to the middle of the drop of the indicator as rapidly as possible by means of a glass tube. If a red colour appears immediately on touching the drop the reduction is not completed. More sugar must be added and a fresh drop of the indicator used as before till no colour or only a faint tinge of red is obtained. If less than 5 cc. of the sugar solution are necessary to complete the reaction, the solution must be diluted tiU about 10 cc. are required, as described above in Fehling's method. Special precautions. Use a glass tube, not a rod, for transferring the drop. Do not put your finger on the top of the tube. Dip it in the flask and transfer it immediately to the indicator. The flask may be taken off the boil for an instant while this is done. Do not stir the drops on the slab. Wash the tube before using it again. Calculation of results. This is the same as in Fehling's method. 163. The estimation of cane sugar by Benedict's method. Measure 50 cc. of the solution with a pipette into a flask. Add 10 cc. of N. hydrochloric acid. Boil over a free flame and keep the mixture very gently boihng for three minutes. Cool imder the tap, neutralise by the addition of 10 cc. of N. sodium hydroxide. Transfer quantitatively to a 100 cc. volumetric flask and make up CH. v.] POLARIMETER. 143 the volume to the mark with cold distilled water, rinsing the boiUng flask out with small amounts of water. Mix carefully, and estimate the invert sugar by Benedict's method. Calculation of results. 25 cc. of Benedict's solution = 0-0475 gram, hydrolysed cane sugar. The concentration found must be multiphed by 2, owing to the dilution made in preparing the hydrolysed solution. E. The theory and use of the Polarimeter. Waves of ordinary light vibrate simultaneously in all directions perpendicular to its direction of propagation. By means of certain contrivances it is possible to affect the light so that the vibrations proceed in a single plane. Such light is plane polarized. The plane in which the light waves vibrate is called the plane of polarization. This conversion of ordinary light into polarized light is generally brought about by means of a modified prism of Iceland spar known as a Nicol's prism. If a beam of light (fig. 19) falls on the Fig. 19. Crystal of Calc Spar. face of a rhombohedron of Iceland spar it divides on enter- ing into two rays, unequally bent, both of which are polarized, their planes of polarization being at right angles to one another. The extraordinary ray (E) is the lesser refracted ray : the ordinary ray (O) is the more refracted ray. Before the calc spar can be utilised for polariscopic purposes one of the rays must be eliminated. This is best effected by Nicol's method of splitting down a crystal in a certain plane, grind- ing down the natural ends to reduce the acute angles from 71° to 68°, and uniting the faces by Canada balsam (fig. 20)^ 144 THE CARBOHYDRATES. [CH. V. A beam of light entering parallel to the long sides of the prism is resolved into its two component rays. The more refracted (ordinary) ray (O) meets the film of Canada ---E c o Fig. 20. Diagram of refraction in a Nicol's prism. balsam (CB), and is completely reflected and absorbed by the black varnish usually placed on the sides of the prism. The other component (the extraordinary ray) (E) passes through the film of balsam and emerges in a polarized condition from the end surface of the Nicol. ^--' Fig. 21. Plan of arrangement of a simple polarimeter. In a polarimeter (fig. 21) a second Nicol prism called an analyser (A) is used in addition. This is mounted in such a way that it can be rotated around its long axis. The polarized ray that emerges from the polarising Nicol (P) falls on the face of the analyser, and will only pass through unimpeded provided that it can contrive to vibrate in the same plane. In this position the Nicols are said to be parallel. If the analyser be rotated through an angle of 45" the ray is completely absorbed and the Nicols are said to be crossed. On rotating through a further angle of 45° the Nicols are again parallel. Suppose a tube of water be interposed between the two Nicols (fig. 21) and a source of light at L be viewed through the system, the Nicols are crossed when the analyser is rotated so that the minimum CH. V.J POLARIMETER. 145 illumination is obtained. If now, instead of water, the tube be filled with a solution of glucose or of certain other substances, it will be found that the illumination is not minimal. To attain this result the analyser must be rotated through a certain angle. The reason for this is that in passing through the glucose solution the plane of polarisation has been gradually rotated so that on emerging and striking the analyser some, of the light can get through. To get the minimum illumination the analyser has to be rotated to the right through an angle equal to that through which the sugar solution has rotated the plane of polarisation of the ray that entered it. The simple arrangement de- scribed is not sufficiently sensitive. 0(D Fig. 22. Plan of a three- field polarimeter. A is the polarising Nicol ; B and C are the small accessory Nicols that resolve the field into three parts, D, E, and F. Fig. 23. The appearances seen in a and 6 indi- cate that the analyser is not in the correct position. When the analyser is correctly adjusted the three parts of the field have an equal feeble illumination as shewn in c. Modern instruments have two small Nicol 's prisms placed between the polariser and the solution (see fig. 22), the effect being to divide the field into three vertical sections. The zero and end points are obtained when the three fields have an equal feeble illumination (fig. 23, c). The source of illumination must be mono- chromatic, since the angle of rotation varies with the wave length employed. That generally used is sodium light, 146 THE CARBOHYDRATES. [CH. V. obtained by heating sodium chloride or bromide in a plati- num ring. The light emitted has a wave length correspond- ing to the D line of the solar spectrum. A much more briUiant illumination can be obtained by use of the green rays emitted from a mercury lamp. The rotation being greater with the shorter wave length, greater accuracy can be obtained.* The rotation varies for diiferent substances. It is increased by increasing the concentration of the solution or the length of the tube. It also varies with the temperature, nature of the solvent, and the wave-length of the light used. The specific rotatory power is the rotation observed through a tube i decimetre in length of a solution calculated to be ICO per cent. This is generally expressed as \_d\. If the sodium light is employed it is expressed as [a]i,. r -, r X ICO where r = the observed rotation. c = the concentration in grams, per loo cc. I = the length of the tube in decimetres. If the temperature is defined, it is usually expressed by Wd 20°. If [aju be known, the concentration m grammes per 1 00 cc. is given by r X 100 C = [a]o X l' The specific rotatory powers of the more common sugars is shewn below. I am indebted to Dr. Lowry for * The apparatus in the author's laboratory consists of a triple field instru- ment by Hilger, of London, fitted with a horizontal sUt and a direct vision spectroscope. A mercury lamp is used as the source of illumination. An accuracy of o-oi° is easily obtained. CH. V.l SPECIFIC ROTATORY POWER. 147 the information concerning the rotations of the sugars to the mercury green. In all cases the final rotations are given (see p. 103). Wd Mh. Glucose . . . + 52-5 62 Lactose hydrate . + 52-4 61-9 Lactose anhydride . + SS-2 65-2 Maltose . + 138 163 Sucrose . . . + 66-s 78-5 Fructose — 93-8 - IIO-8 <^-Galactose . + 81 957 /-Xylose . . . + 19 22-4 Invert sugar . . . — 20'6 - 24-6 For carbohydrates the [ajng can be obtained by multiplying [aju by i*i8i. This relationship is not necessarily true for substances whose molecular construc- tion differs from that of the carbohydrates. F. Optical Activity and the Asymmetric Carbon Atom. If C be a carbon atom attached to a, b and x, different atoms or groups, it is found that there exists only one modification of the type Ca^bx. But if the structural formula be written in one plane it would appear that two arrangements are possible, viz. X A X- a B In A the groups a are adjacent, whilst in B they apparently are separated. 148 THE CARBOHYDRATES. [CH. V. The accepted explanation of the facts is that the carbon atom possesses four valencies or bonds directed towards the apices of a tetrahedron, the carbon atom being 'at the centre. The student is advised to construct such a model from a piece of plasticine and four matches, the central piece of plasticine representing the carbon atom and the matches the four bonds attached to it. The heads of the matches should be left plain or marked with little balls of variously coloured plasticine to indicate the different groups attached to the central carbon atom. Such a model is represented in fig. 24. A Fig .24- Model of carbon atom attached to 3 different groups, representing the compound Ca^x. Another identical model should now be prepared. It will be found that the two can be superposed, as shewn in fig. 25. Now change the positions of any two matches in one of the models. It will be found that the two models can CH. v.] ASYMMETRIC CARBON ATOMS. 149 still be superposed. In fact, no matter how the matches 4^ ^ Fig. 25. Two identical superposable models of the tjrpe Ca^bx, are changed about only one arrangement is possible. This Fig. 26. Model of an asymmetric carbon atom and its mirror image. is in agreement with the fact that only one modification of the. t5rpe Ca^bx exists. 150 THE CARBOHYDRATES. [CH. V. Now make two exactly similar models in each of which the carbon atom is represented as being united to four different groups. Being exactly similar the two models are naturally superposable. Now change the position of any two matches in one model only. The two models thus formed cannot be superposed. On examination it will be found that the two models have a relationship to one another similar to that of the right to the left hand, or of one object to its image in a mirror. This is represented in fig. 26. If now still another model be constructed it will be found that it can be superposed on one or other of the two previous bnes. That is, there exist two modifications and two only, of compounds of the type Cabvcy. If the model of the type Ca^bx be examined it will be seen that it can be divided into two symmetrical halves. The plane of symmetry and method of division is indicated in fig. 27. It must be understood that though a plane of symmetry exists the atoms or groups are not actually split into halves by it. An examination of the figures shewn in fig. 26 will reveal the fact that they do not possess a plane of sym- metry. It has been ascer- tained that all compounds of the tjrpe Cabxy exist in two modifications. The solutions of one of these rotates the plane of polari- sation to the right : that of the other exactly the same degree to the left. The former is the "dextro- rotatory" or the J-com- pound : the latter is the " laevo-rotatory" or /-com- pound. These are sometimes known as enaniiomorpks. Since the compound has no plane of symmetry a carbon Fig. 27. Plane of symmetry of model shewn in Fig. 24. CH. V.J ASYMMETRIC COMPOUNDS. 151 atom attached to four different groups is known as an asymmetric carbon atom. The possession of an asymmetric carbon atom in a compound is essential to the possession of optical activity by that compound. If equal parts of the d- and /- varieties of a compound be mixed together, the solution of the substance is "opti- cally inactive by external compensation." Such an in- active mixture is known as "racemic," and is designated by dl- or i-. When a compound that contains an asym- metric carbon atom is synthesised, it is always found that equal parts of the d- and /- varieties are formed. These can often be resolved into their active constituents by various methods, the most interesting of which is the biochemical, depending on the property of living organisms of selective assimilation, one of the two components being destroyed more rapidly than the other. A few examples of this are given below. Substance Organism Destroyed Lactic acid peniciUium d. bacteria I. Glyceric acid penicilHum I. bac. ethaceticus I. Amyl alcohol fungus I. Glucose, mannose, galactose, fructose yeast d. Racemic acid penicillium d. schizomycetes I. Leucine yeast I. Alanine yeast d. This power of selective Assimilation finds a parallel in the different physiological action of enantiomorphs on the body, and of the body and also of enzymes on enantio- morphs. For instance, c?/-adrenaline has only slightly more than one-half the physiological activity of the natural 152 THE CARBOHYDRATES. [CH. /-adrenaline : c?-asparagine has a sweet taste, whilst /-aspara- gine is insipid : /-nicotine is far more poisonous than fi?-nicotine. Further, if c?/-phenyl-aminoacetic acid be administered to an animal, only the /- variety is found in the urine, the body having the power to destroy the o o II » K o o o w a o o 2 S 1 a , SI a; a| 03 •£. O ^ •e a o o o q d-^"? o <3a. o u P3 a a o o o a^ q a" o a o •« O O 'S U •-; '^ 1 CH2.NH2 I COOH Cysteine. Taurine. The bile acids are hydrolysed into their constituents by boiling acids and also by the intestinal bacteria. The bile salts are soluble in water and alcohol, in- soluble in ether. Their solutions have a remarkably low surface tension. (See Hay's test.) They have the following functions : — 1 . They have a marked adjuvant action on pancreatic lipase. (See Ex. 167.) 2. They are solvents for the fatty acids and markedly increase the absorption of fats. 3. They thus help to remove the fatty film sur- rounding the protein, and allow the proteolytic ferments to act. In this way, by assisting the absorption of pro- teins, they diminish bacterial decomposition. They are not direct antiseptics. Prepaiation oJ Bile Salts. — Mix 40 cc. of ox gall with enough animal charcoal (about lo grams.) to form a paste. Evaporate to dryness over a water bath, stirring at intervals. Grind the residue in a mortar, transfer it to a flask, add about 70 cc. of 96 per cent, or absolute alcohol and boil on the water bath for 20 minutes. Cool and filter into a dry beaker. Add ether to the filtrate till there is a slight permanent cloudiness. Cover the beaker with a glass plate and allow it to stand in a cool place for 24 hours. A crystaUine mass of bile salts separates out. The crystals are filtered off and allowed to dry in the air. For the following tests use diluted ox or sheep gall :— 315. Pettenkofer's test for bile salts. To 5 cc. of the solution add a small particle of cane-sugar and shake or warm till this has completely dissolved. To the cooled solution add 5 cc. of concentrated sulphuric acid, inclining the test-tube so that the acid settles to the bottom. Gently shake the test-tube from side to side. As the fluids gradually mix a deep purple colour develops. CH. XI.] BILE PIGMENTS. 267 Notes. — i. This reaction depends on the production of furfural from the cane-sugar by the strong acid. (See Ex. 114.) 2. If too much cane-sugar be taken the fluid will turn brown or black, owing to the charring produced. 3. Proteins give a very similar reaction with furfurol in the presence of strong acids. (See Ex. 26.) Proteins also tend to give a. brown char with sulphuric acid. For these reasons it is advisable to remove the proteins from solution before attempting the test. 4. The purple colour obtained is only stable in the presence of strong sulphuric acid. It disappears on dilution with water. 5. If a small portion of the coloured fluid be diluted with 50 per cent, sulphuric acid, and examined with the spectroscope, two absorption bands will be seen, one between the lines C and D, nearer the latter ; the other in the green, overlapping E and B. 6. The test cannot be applied directly to urine, owing to the presence of chromogenic substances that yield intense colours with sulphuric acid. 316. Hay's test for bile salts. Take lo cc. of the solution in a test-tube. Sprinkle the surface with flowers of sulphur and note that they fall through the liquid to the bottom of the tube. Repeat the test with water, noting that the particles remain on the surface. Notes. — i. This test for bile salts depends on the remarkable property that they possess of lowering the surface tension of water, thus enabUng the particles of sulphur to sink through the fluid. 2. The test is of great value for the detection of bile salts in urine. 3. This property of bile salts is utihsed by draughtsmen in preparing tracings on oiled paper, on which ink collects in drops, and does not spread well. If the paper be first treated with a little ox gaU and allowed to dry the difficulty is removed, owing to the reduction in surface tension. 4. A method for estimating bile salts in urine has been described by Griinbaum, depending on this property. The rate of escape of the urine from standard capillary tubes is noted, the rate increasing with the concentration of bile salts. 317. Oliver's test for bile salts. Acidify 5 cc. of the solution with two or three drops of strong acetic acid, filtering if necessary. To the acid solution add an equal quantity of i per cent, solution of peptone. A white milkiness or a decided precipitate is produced, insoluble in excess of acid. Notes. — i. The precipitate formed consists of a compound of protein with bile acids. 2. The test can be applied to urine. (Ex. 379.) The Bile Pigments. Bilirubin, CggHagNjOg, is a reddish-brown pigment most abundant in the bile of carnivora. It is readily oxidised by the oxygen of the air into bihverdin, C3aH36N408, the green pigment found mostly in the bile of herbivora. These compounds are formed in the liver cells from the 268 THE CONSTITUENTS OF BILE, [CH. XI. products of disintegration of haemoglobin. Haematin is C3aH32N404Fe, and haematoporphyrin is isomeric with bilirubin. They are weak acids, forming sodium and calcium salts, the latter being insoluble in water. Free bilirubin is soluble in ether and chloroform : the sodium compound is insoluble, as is free or combined biliverdin. By oxidation bilirubin is converted, through a num- ber of ill-defined bodies, such as bilicyanin, and bilifuscin, into choletelin, the end product of Gmelin's reaction. By further oxidation a compound, haematinic acid (CgHgOj), is formed, identical with the product obtained by the oxidation of haematin or haematoporphyrin. By reduction with sodium amalgam in alcoholic solu- tion the bile pigments are converted into hydrobilirubin, which is also formed by the action of more powerful reduc- ing agents on haematin or haematoporphyrin. These facts all indicate the close relationship between haematin and the bile pigments. In the bowel the bacteria first reduce bilirubin to hydrobilirubin. This is then attacked, two nitrogen atoms being probably removed, the result being the for- mation of urobilin, which is mainly excreted in the faeces, being sometimes called " stercobilin." A certain amount however, is absorbed into the blood, and excreted by the liver into the bile, whilst a small amount is excreted by the kidney in the form of urobilinogen. (See p. 278.) 318. Huppert-Cole test for bile pigments. Boil about 15 cc. of the fluid in a test-tube. Add two drops of a saturated solution of magnesium sulphate, then add a 10 per cent, solution of barium chloride, drop by drop, boiling between each addition. Continue to add the barium chloride until no further precipitate is obtained. Allow the tube to stand for a minute. Pour off the supernatant fluid as cleanly as possible or use a centrifuge To the precipitate add 3 to 5 cc. of 97 per cent, alcohol, two drops of strong sulphuric acid, and two drops of a 5 per cent, aqueous solu- tion of potassium chlorate. Boil for half a minute and allow the CH. XI.] THE PROTEINS OF BILE. 269 barium sulphate to settle. The presence of bile pigments is indi- cated by the alcoholic solution being coloured a greenish blue. Notes. — i. To render the test more delicate, pour off the alcoholic solution from the barium sulphate into a dry tube. Add about one-third its volume of chloroform and mix. To the solution add about an equal volume of water, place the thumb on the tube, invert once or twice and allow the chloroform to separate. It contains the bluish pigment in solution. 2. The bile pigment is adsorbed on to the barium sulphate precipitate, but passes into solution again in acid alcohol. The chlorate acts as a very- weak oxidising reagent, converting bilirubin and biUverdin to the characteristic blue compound. 3. The author claims that it is a very much more delicate test than the one that follows. 319. Gmelin's test for bile pigments. Take a few cc. of fuming yellow nitric acid in a test-tube, and by means of a pipette carefully place on the surface of this an equal amount of bile. Shake the tube very gently from side to side, and note the play of colours, in the bile as it becomes oxidised by the acid. Proceeding from acid to bile the colours are yellow, red, violet, blue, and green. Notes. — This test can be modified in many ways. 1. Add a drop of yellow nitric acid to a thin film of bile on a white porcelain plate. The drop of acid becomes surrounded by rings of the various- colours. 2. Filter some diluted bile repeatedly tlirough an ordinary filter paper, and then place a drop of fuming nitric acid on the paper. Cholesterol has been described on p. i6i, and Lecithin. on p. 163. The Protein of Bile. When bile is treated with acetic acid a precipitate is formed insoluble in excess. This was formerly thought to be mucin. But it has been shown that it is nucleo- protein, the bile salts present preventing the re-solution in strong acetic acid. (See Ex. 3 1 7.) In human bile, how- ever, mucin is present as well as nucleoprotein. The protein is secreted by the cells lining the ducts- and the gall bladder, so that bile from the gall bladder contains a much greater percentage than fistula bile. 320. To a small quantity of undiluted bile add strong acetic acid, drop by drop. A precipitate is formed, insoluble in excess of acid. This precipitate consists of a nucleoprotein, together with a considerable amount of the bile salts and bile pigments CHAPTER XII. URINE AND ITS CHIEF CONSTITUENTS. A. The average composition. The composition of the urine varies with the individual and with the diet. Below we given the figures in grams, for the daily output of A. The average man on the average mixed diet. B. An individual on a liberal diet. C. The same individual on a diet deficient in proteins. B. and C. are taken from a paper by Folin. A. B. C. g bo S s s *i ° a 1 I, -P ° (.0 Urea 3° 14 87-5 31-6 14-7 87-5 4-72 2-2 6i-7 Ammonia 0-6 0-5 3-1 •6 0-49 3-0 •51 0-42 "•3 Creatinine 1-55 0-57 3-6 1-55 o-gS 3-6 l-6i o-6o 17-2 Uric Acid 0-7 o^23 1-4 •54 o-i8 I'l •27 0-09 2-5 Undetermined o^7 4-4 0-85 4-8 0-27 7-3 Total— N i6-o lOO-O i6^8 lOO^O 3.6 lOO'O Inorganic SO, 2-92 88^2 3-27 90-0 0-46 1 60-5 Ethereal SO, •22 6^6 0-I9 5-2 O-IO 13-2 Neutral SO, •17 5-2 o^i8 4-8 0-20 26-3 Total SOa 3-3 lOO-O 3-64 lOO-O 0-76 lOO-O CH. XII.] SPECIFIC GRAVITY. 27! B. The Physical Chemistry of the Urine. /. General Properties. Normal human urine is a clear yellowish fluid, the depth of the tint depending largely on the concentration. On standing, a cloud (nubecula) of mucoid containing epithelial cells separates out. After a heavy meal urine may be passed cloudy, due to earthy phosphates and carbonates. On standing, these settle to the bottom of the vessel as a white deposit, insoluble on warming, but soluble in acids. Also on standing a cloud of urates may settle as a reddish deposit that clears up on warming. Fresh urine has a characteristic odour of the aromatic type, due to the presence of some substance that has not yet been recognised. On standing, an unpleasant ammoniacal odour develops as the result of bacterial decomposition. //. The Specific Gravity. Usually -lies between 1012 and 1024 (water = 1000). With copious drinking it may fall to 1002. After excessive perspiration it may rise to 1040. The determination of the specific gravity for clinical purposes is most conveniently made by means of a urino- meter, a weighted cylinder that floats in the urine. The depth to which it sinks depends on t*he density of the fluid, and this can be read directly by means of a graduated scale on the stem. The instrument is calibrated for a certain temperature, usually 15° C. The urine should be either cooled or warmed to this temperature, or a correction made by adding i unit for every 3 degrees above this, or subtracting i for every 3 degrees below the standard. Thus, if the reading be 1018 at 18° C, the corrected Sp. Gr. is 1019. 272 URINE, [CH. XII. To obtain the best results two separate instruments should be at hand, the one calibrated from looo to 1020 and the other from 1020 to 1040. The total amount of solids in the urine can be roughly- calculated from the specific gravity by Long's coefficient. The last two figures of the specific gravity x 2-6 gives total solids in looo cc. Thus specific gravity at 25° C = 1017. Total soUds in 1000 cc. = 17 x 2-6 = 44-2 grams. Haser's coefficient (2-33) on a similar basis, but calculated for 15° C. is probably inaccurate. 321. Take the specific gravity of normal urine by means of a urinometer. Wipe the instrument clean, and float it in the centre of a cylinder containing the urine. Remove all froth, by means of filter paper or by placing a single drop of ether on the smface of the urine. Take care that the instrument does not touch the sides of the vessel. Place the eye level with the surface of the fluid and read the division of the scale to which the latter reaches. Read the level of the true surface of the urine, not the top of the meniscus aroimd the stem. ///. The Osmotic Pressure (Cryoscopy). The method of taking the freezing point of a fluid is described on p. 8, and the subject has been considered from a theoretical standpoint on p. s. In urine the concentrations of certain sub- stances, such as urea, are much greater than they are in the blood. The work done by the kidney in effecting this concentration can be calculated from a consideration of the osmotic concentration, i.e. A, of each substance in blood and urine. It is quite erroneous to imagine that the work done can be calculated from a knowledge of the total osmotic concentration of the blood and urine respectively. But, at the same time, the determination of A of the blood and of the urine secreted by each kidney in certain renal diseases may Fig. 36. Urinometer. CH. XII.] REACTION. 273 give us valuable information as to the relative activities of the two organs. A of blood is about 0-55° C, the same as that of a 0-9 per cent, solution of sodium chloride. A of urine varies considerably with the diet, volume of fluid taken and other conditions. For the mixed 24 hours urine of an average man it is usually about 1-2° C. The following values are of interest in this connection : — A X volume of urine = molecular diuresis. --— ; is of considerable pathological signi- NaCl per cent. f s s, ficance. It is fairly constant in health, varying between 1-25 and 1-6. It exceeds 1-7 in heart disease or in any condition that causes a retardation of the renal circulation. The only febrile condition in which it is less than 1-7 is malaria. IV. Reaction. Normal human urine is generally acid to litmus, the average Ph of 24 hour specimens being about 6-o. The reaction varies with the diet, being greatest on a meat diet, owing to the oxidation (in the body) of the sulphur and phosphorus to sulphuric and phosphoric acids. On a vegetable diet, however, the urine may become alkaline, as it is in nerbivora, owing to the organic salts being oxidised to alkaline carbonates. During the secretion of the acid gastric juice into the stomach, the urine may become alkaline, the so-called " alkaline tide." The acid reaction of the urine is mainly due to the excretion of acid phosphates and of weak organic acids. A certain amount of the acids produced in the body are neutralised by ammonia and excreted as ammonium salts in the urine. The ingestion of acids or of acid phosphates usually leads to an increase in the excretion of titratable acids (or acid salts) and of non-titratable neutral am- 274 URINE. [CH. XII, monium salts. The sum of the two can be taken as a measure of the total amount of acid eliminated from the body. The titratable acid excreted is usually measured by titrating to phenol phthalein, but it is better to titrate to blood reaction, i.e. Ph = 7-4S- By the use of the compara- tor described below, this is a very simple matter. Palmer and Henderson* have studied the relationships between reaction, volume of urine, etc. The following table gives some of the results obtained in apparently healthy subjects : — Ph 24 hours Volume in cc. Titratable Acid in cc. of o'l N Acid. A NH3 in cc. of o-i N Add. N A+N A N R 5-4 1026 320 367 687 0-87 S'7 6-0 "93 I2S9 263 384 365 687 628 0-79 0-72 6-6 1400 Av 224 erage of a 357 11 the case S81 s. 0-63 S-94 1231 278 370 649 0-7S They note that 1. A increases with the hydrogen-ion concentration. 2. With constancy in the excretion of phosphoric acid the hydrogen-ion concentration varies with A. 3. With constancy of A, the hj'^drogen-ion concentra- tion varies as the phosphoric acid. 4. N is fairly constant. Normally the final regulation of the reaction through excretion falls upon the phosphates. 5. The volume increases as the acidity decreases. The variations in these various factors in renal disease * Journ. of Biol. Chem., xvii., p. 305. CH. XIl.] ACID EXCRETION. 275 have been investigated by Palmer and Henderson,* who find that in certain types of the disease ("High Ratio") there is a remarkable increase in R, mainly due to a deficit in the excretion of ammonia. In other types of renal disease the various factors are nearer to normal, as can be seen from the table below. Ph Vol- ume A N A+N R Type 5-2 1650 S18 16S 483 2-o8 High Ratio S-2 1086 287 276 563 1-03 Medium Ratio S-6 1187 244 341 58s 072 Low Ratio It would seem that an important factor in renal disease is a difficulty in the excretion of ammonium salts, so that to maintain the normal reaction of the blood the kidney is forced to excrete an abnormally acid urine. This, in its turn, may cause a further degeneration of the renal tissues. A further point of interest in connexion with the acidity of the urine is that, according to van Slyke, the alkali reserve of the body can be determined and the condition of "acidosis" diagnosed from the indication given by the analyses described below, f The method of determining the Ph of urine is given on p. 29. 322. The estimation of titratable acid in urine (Cole's method). Principle. The urine is titrated to Ph = 7-45 (the average reaction of normal blood), using a large form of Cole and Onslow's * Journ. of Biol. Chem., xxi., p. 37. t A full account will be found in the foUovring papers : — Fitz and vaa Slyke, Journ. of Biol. Chem., xxx., p. 389. Van Slyke, ibid, xxxiii., p. 271. 276 UEINE, [CH. XII. comparator (see p. 21). The concentrations of the indicator in the urine and the two buffer solutions, and also the intensity of the pigment in the three tubes containing urine are kept constant by the addition of equivalent quantities of water and soda respectively. Solutions and Apparatus required. 1 . A comparator for holding the tubes (see fig. 37). with a ground^glass screen, as shewn in the diagram. 2. Tubes of clear resistance glass (7x1 in.) to fit the comparator. These should have the same internal diameter. They can be calibrated by measuring 25 cc. of water from a pipette into a number and selecting those tubes in which the fluid reaches the same level. A better method is to use a hard This should be fitted Fig. 37. Cole and Onslow's Comparator for large tubes. wood gauge, which is slightly conical and is marked with rings corresponding to every 1-64 in. diameter. The gauge is pressed into the tubes and those selected of the same internal diameter. The method of using this is shewn in fig. 38. It is convenient to choose sets of tubes and to mark each member of a set with a distinguishing letter by means of a diamond. Fig. 38- Gauge. 3. Buffer solution, Ph = 7-42. Prepared by treating 50 cc. of 0-2 M. KH2PO4 with the equivalent of 399 cc. of 0-2 N.NaOH and diluting to make 200 cc. with distilled water (see p. 27). 4. Buffer solution, Ph = 7-47. Prepared equivalent of 402 cc. of 0-2 N.NaOH. as above, but use the 5. Standard alkali, see p. 380. It is convenient to use o-i N. from an ordinary burette or 0-2 N. from a microburette. CH. XII.] ESTIMATION OF TITRATABLE ACID. 277 Method. Measure 20 cc. of the urine into tubes (2), (3) and (6). Measure 20 cc. of the buffer Ph = 7-42 into (i). Measure 20 cc. of the buffer Ph = 7-47 into (5). Place about 20 cc. of water into (4). Add 10 to 15 drops of 0-02 per cent, phenol red to (i), (3) and (5), using a dropping pipette (fig. 5) and adding exactly the same amount to each tube. Mix the contents of the tubes by smartly rotating them be- tween the palms of the hands. Titrate (3) with the standard soda. A precipitate of earthy phosphates may appear and the colour as seen through Y gradually approaches that seen through X. Let the amount of soda added be {a) cc. To (i) and (5) add (a) cc. of distilled water from a burette or pipette. To (2) and (6) add (a) cc. of the standard soda. Read the burette. Complete the titration of (3) until the colour as seen through Y is intermediate between that seen through X and Z. The tubes must be spun just before the observation is made to ensure an equal distribution of any precipitate. Let the amount of soda required for this operation be (b) cc. Calculation. 20 cc. of urine require («) + (b) cc, say (A) .cc. of the soda, which is (c) times Normal. So 100 cc. require (A) x 5 cc. of the soda. So 100 cc. contain (A) x 5 x 10 x (c) cc. of o-i N. acid. Notes. — i. It is important that the examination be made in fresh urine. Should this be impossible a little toluol should be added to the speci- men to prevent the ammoniacal fermentation of the urea. 2. Should the urine be so concentrated that a precipitate of urates separates out, the urine may be diluted with an equal volume of distilled water and the mixture gently warmed till it clears. It is then cooled under the tap and the estimation made as described above, allowance being made for the dilution in the final calculation. 3. By a similar method, enzyme solutions, digestion mixtures, etc., can be brought to any desired hydrogen-ion concentration. Suitable buffer solutions and indicators can be prepared according to the directions given in pages 22 to 28. 278 URINE. [CH. XII. C. The Pigments of Urine. Urochrome is the chief pigment of normal urine. It is a yellow substance which has no definite absorption band. Nothing certain is known as to its constitution or origin, except that it is apparently not derived from the bile pigments. It has marked reducing properties. Urobilin occurs in fresh normal urine as its chromo- gen, urobilinogen. This is converted into urobilin by acids or by the action of light and oxygen. The amount excreted is markedly increased in fevers, in diseases of the liver and bile passages, by destruction of the red corpuscles, especially in pernicious anaemia and malaria, and during the absorption of blood clots. In certain of these cases the urobilin itself is found in the urine, and can be identified by its characteristic absorption band, urobilin- ogen not giving a definite band. Urobilinogen is a pyrrol body and is responsible for Ehrlich's reaction with /)-dimethyI-amino-benzaldehyde. The origin of urobilin from the bile pigments is dis- cussed on page 268. It may be added that the urobilin absorbed from the bowel into the circulation is mostly excreted by the liver into the bile, so that only a small portion reaches the urine. Should the liver cells be injured, or should there be any interference with the circulation through the liver, there is a considerable increase in the excretion of either urobilin or urobilinogen in the urine. If the common bile duct is completely occluded by a gall stone or by a growth the urobilin and urobilinogen are absent from the urine and faeces. Should the obstruction be removed there is often a period during which the amounts of these substances in the urine is exceptionally large. Uroerythrin is found in small amounts in normal urine. It is increased in fever and certain diseases of the liver. , CH. XII. J UROBILIN. 279 It is soluble in amyl alcohol. Solutions have a reddish colour, but are unstable to light. The pigment is usually associated with the urates or uric acid of the urine. Haematoporphyiin is found in traces in normal urine. There is a certain increase in fevers, and some other diseases, but a very marked increase in certain cases of poisoning by sulphonal or trional, especially in women. Urorosein occurs in urine as a chromogen which is converted into the pigment by the action of strong acids, such as hydrochloric and sulphuric. It is insoluble in ether and is thus distinguished from indigo blue formed in the test for indican. (Ex. 318.) The chromogen seems to be an indol body, possibly indol-acetic acid. 323. Note the colour of normal urine and examine some in a beaker by the spectroscope. Note that there are no definite ab- sorption bands, but a general absorption of the violet. Urochrome, the chief urinary pigment, yields no bands. 324. Saturate at least 200 cc. of urine with ammonium sulphate. Filter off the precipitate and let it dry completely in the air. Extract it with a small amount of strong alcohol. A brownish solution containing urobilinogen is obtained. Treat this with a few drops of hydrochloric acid: the urobilinogen is converted to urobilin. Examine with the spectroscope, and note a single absorption band situated at the junction of the blue and the green. Its centre is about X 490. 325. Bogomolow's test for urobilin or urobilinogen. Treat 10 cc. of the urine with 10 drops of 20 per cent, copper sulphate. Add about 4 cc. of chloroform, place the thumb on top of the tube and invert 10 times without shaking. If abnormal amounts of urobilin or urobilinogen are present, the chloroform layer is coloured yeUow. Place the finger on the upper end of a dry 5 cc. pipette and insert the lower end into the chloroform layer. Suck up the chloro- 280 URINE. [CH. XII. form solution and transfer it to a dry tube. The chloroform usually separates as a clear fluid, which is of a faint pink colour if urobiUn is present. A characteristic absorption band, with centre about X 500 can be seen. 326. Schlesinger's test for urobilin. To 10 cc. of urine add 3 drops of a 5 per cent, alcoholic solution of iodine ( to convert urobilinogen to urobilin). Into another test-tube place i gram, of zinc acetate and 10 cc. of absolute alcohol. Mix the two solutions and repeatedly decant until all the zinc acetate has dissolved. Filter. Examine the filtrate in a test-tube, 16 mm. wide, in daylight failing from behind the observer. A green fluorescence is seen if urobilin or urobihnogen are present. Note. — The above method is a modification introduced by Marcusseu and Hansen {Journ. Biol. Chem., xxxvi., p. 381). They state that ammoniacal urines should be acidified with acetic acid. They find that in patients suffering from liver complaints they can detect the fluorescence when the urine has been diluted 40 to 80 times, and are of the opinion that unless it can be detected in a dilution of r in 20 a pathological urobihnuria has not been definitely established. D. The Inorganic Constituents. Kaiions. Sodium and potassium are found to the extent of 3-2 gram. KgO and 5-23 gram. Nafi per diem. The ratio KgO : NagO generally equals i : i-54. During starvation this can rise as high as 3 : i, owing to the excretion of the potassium of the tissues, sodium being found in a much smaller amount than potassium. The same is found in ail wasting diseases. Calcium and Magnesium are mainly excreted by the bowel. The amounts in urine are o-i/ to 0-62 gram. CaO and 0-19 to 0-31 gram, of MgO. The amounts of these alkaline earths in the urine are increased by the administration of organic acids, or in conditions such as diabetes in which the formation of such acids is increased. Iron also is mainly excreted by the bowel. It is found in human urine only in organic combination, and then only to the extent of 0-5 to 10 milligrams per diem. CH. XII.] CHLORIDES AND SULPHATES. 28I Anions. Chlorides form the chief part of the anions of the urine. The amount excreted is often calculated as if it all existed as NaCl, though the amount of sodium in the urine is normalty not sufficient to combine with all the chlorine. The amount in the urine depends largely on the amount in the food, but since an important function of the kidney is to maintain a constant osmotic pressure of the tissue fluids, mainly by variations in the amount of NaCl excreted, it follows that anything tending to cause a change in the osmotic equilibrium in the body is liable to alter the excretion of chlorides in the urine. Thus during starvation and during the formation of exudates in pneumonia the chlorides may disappear from urine. The amount of CI excreted per diem is about 7 grams. Reckoned as NaCl it is 12 grams. For the method of estimation see p. 355. Sulphates. Only a small portion of the sulphate excreted in the urine is taken in as such with the food. The greater portion is derived from the oxidation of sulphur containing substances, chiefly proteins. The amount of sulphates is thus a rough measure of the total amount N . 1; of protein metabolised, the ratio — — - being usually - SO3 I Sulphates are excreted very rapidly after a protein meal, reaching a maximum about the third hour. This seems to indicate that cystine, the sulphur complex of proteins, is split off and absorbed very early in the digestion of proteins. i i Ethereal Sulphates are esters formed by the union of sulphuric acid with phenols. O OH O O.CeH, \/ x/ S + HO.CeH, . > S + H,0 y\ //\ O OH OH Sulphuric acid. Phenol. Phenyl sulphuric acid. 282 UKINE. [CH. XII. The proportion of the sulphur that is present as ethereal sulphate varies considerably. Folin has shewn that in starvation and on diets relatively deficient in proteins the proportion increases, as does that of the -"neutral" sulphur. There is also a marked increase after the administration of certain phenolic substances, or when such compounds are formed in the body by bacterial decomposition, as in intestinal obstruction and severe constipation. In such cases the phenols found conjugated with sulphuric acid are CA.OH phenol n TT .--OH. (if 1 [ formed from tyrosine. ^«^'-^0H(4) P-cresolj ^ CsHeN.OH indoxyl, formed from tryptophane. These bodies are poisonous. They unite with sulphuric acid, probably in the liver, to form the innocuous ethereal sulphates. The ethereal sulphates form soluble barium and benzidine salts, and can be separated from the inorganic sulphates by treatment with barium chloride or benzidine hydrochloride and filtering. They are hydrolysed to the phenol and sulphuric acid by boiling with hydrochloric acid. " Neutral " Sulphur. In urine there is always present a certain amount of sulphur in a form less oxidised than that of a sulphate. The exact nature of the compounds in urine containing sulphur in this form is not yet clear. It is probable that the amount of "neutral" sulphur in the urine is independent of the total amount of sulphur excreted. It probably varies with the amount of tissue protein metabolised, so that its determination is often of considerable interest. For the percentages of sulphur excreted in the three forms under different metabolic conditions see page 270. For the methods of determination of the sulphur see pages 358—361. Phosphates. The phosphates of the urine are present on the one hand as salts of the alkali metals and of CH. XII.] PHOSPHATES. aSj ammonium ; on the other, as salts of the alkahne earths,, calcium and magnesium. About 3-9 grams, of P2O5 are excreted per diem in the urine. Phosphoric acid forms three series of salts. The formulse for the three sodium and calcium salts respectively are Ca3(PO,)2. CaHCPOJ. CaH.CPOJ,. Normal phosphate, Na3P04 Mono-hydrogen phosphate, Na2HP04 Di-hydrogen phosphate, NaH2P04 The three sodium salts and CaH4(P04)2 are soluble in water : the other two calcium salts are insoluble. The normal and mono-hydrogen phosphates are alkaline in reaction to litmus : the di-hydrogen phosphates are acid. The phosphates of the urine are derived partly from the inorganic phosphates of the food, partly from the oxidation of phosphorus-containing substances of the food and tissues, such as nucleo-proteins, lecithins and' phospho-proteins, and partly also from the phosphates of bone. The exact share played by these various compounds- in forming the urinary phosphates is difficult to deter- mine owing to the fact that a proportion of the phosphates,, varying between 12 and 50 per cent., are excreted by the bowel. In this connection it may be noted that alkaline phosphates of the food are more likely to be excreted in the urine than are earthy phosphates. The excretion of varying amounts of phosphates by the kidney is one of the methods by means of which the reaction of the body fluids is maintained in equilibrium.- An increased excretion is always seen in cases of acid poisoning and in the acidosis associated with diabetes. As soon as the urine shews a certain grade of alka- hnity, precipitation of earthy phosphates takes place. This is sometimes known as phosphaturia, but it is not necessarily associated with an increase of phosphates in the urine. In the phosphaturia of juveniles it is probable- that there is an excessive amount of calcium in the urine,, due to a defective excretion of the large intestine. 284 URINE. [CH. XII. A certain amount of phosphorus is found in the urine in an organic form, not as a phosphate. It may be present as glycero-phosphoric acid. The average daily amount is about 50 mgms. For method of estimation see Ex. 414. 327. Test for chlorides by adding to about 3 cc. of urine a few drops of pure nitric acid and 3 cc. of a 3 per cent, solution of silver nitrate. An abundant curdy precipitate of silver chloride appears at once. If the chlorides are less in quantity, the solution merely becomes milky or opalescent. Note. — If nitric acid is not added, urates might be precipitated by silver nitrate, especially if the urine be ammoniacal. 328. To a test-tube nearly full of urine add a little strong ammonia and boil. A white flaky precipitate of the phosphates of calcium and magnesium is formed. Filter off the precipitate, wash with water, and dissolve in 5 cc. of dilute acetic acid. Divide the solution into two parts. To one part add a solution of potassium oxalate. A white precipitate is produced, showing the presence of calcium in the urine. 329. To the other portion of the solution add an equal bulk of strong nitric acid and about 5 cc. of ammonium molybdate. Boil: a yellow crystalline precipitate is produced, showing the presence of phosphates. Note. — Neutral urine is very apt to yield a precipitate of earthy phos- phates on boiling, owing to the change of reaction due to the evolution of CO, (see notes to Ex. 28). 330. To demonstrate the presence of acid-phosphates in urine. Treat 5 cc. of urine with an equal volimie of 5 per cent, solution of barium chloride. Filter repeatedly through a small filter paper till the filtrate is clear. Treat the filtrate with a little baryta mixture and boil. Filter; dissolve the precipitate in nitric acid and boil the solution obtained with ammonium molybdate. The yellow precipitate shows the presence in the urine of acid phosphates, such as NaHjPO^. CH. XII.] ETHEREAL SULPHATES. 285 Note. — Any alkaline phosphate, Na^HPOj, present in the urine is precipi- tated by BaCL, as BaHP04. The acid phosphates remain in solution as Ba(H2POj)2. On the addition of the alkaline baryta mixture, the acid phos- phate is converted into the insoluble alkaline phosphates of barium. If no precipitate is produced when the baryta-mixture is added, there are no acid phosphates present in the sample of urine. Since the acidity of a sample of urine varies almost directly with the amount of acid phosphates present, as determined by the above method, it is generally held that the acidity of urine is mainly due to the presence of these acid phosphates. 331. Treat lo cc. of urine with a few drops of strong hydro- chloric acid, and about 3 cc. of a solution of barium chloride. A precipitate of barium sulphate is produced as an opaque railkiness. If the precipitate is thick the sulphates are in excess. (The hydro- chloric acid is added to prevent the precipitation of phosphates.) 332. To demonstrate the presence of ethereal sulphates. To urine add an equal bulk of baryta mixture (two parts of baryta water to one part of a 10 per cent, solution of barium nitrate). A precipitate is formed consisting of the phosphates and the ordinary inorganic sulphates. FUter till quite clear. To the filtrate add a third of its volume of strong hydrochloric acid, boil in a beaker for five minutes, and allow to stand. A faint white cloud of barium sulphate is formed, indicating the presence of ethereal sulphates in the urine. Notes. — i. The ethereal sulphates form soluble barium salts, but are hydrolysed to sulphuric acid by heating with an acid. CsHs - O^,,,^^ ^>S02 + H20=C6H5.0H + HaSOi. HO Phenol. Phenyl-sulphuric acid. The sulphuric acid thus formed is converted into barium sulphate by the excess of barium present. 2. The solution becomes very dark in colour on boiling with the strong acid, owing to the action of the latter on the aromatic chromogenic substances in the urine. 3. Ethereal sulphates can be prepared as follows: warm 10 drops of absolute alcohol with 5 drops of concentrated sulphuric acid in a test-tube. Cool and make alkaline with 5 per cent. soda. Add 10 per cent, barium chloride as long as a precipitate continues to be formed. Boil and filter. The filtrate contains barium ethyl sulphate. Add one-half volume of concen- trated hydrochloric -.acid and boil. A precipitate of barium sulphate is formed. 286 URINE. [CH. XII. E. Urea. Urea is the compound in which the greater part of the nitrogen is normally excreted in man. The percentage of the urinary nitrogen in the form of urea varies. Normally it is about 86 per cent., but in starvation, or on a diet deficient in proteins, it is only about 60 per cent. It is also low in cases of diabetes accompanied by acidosis (owing to the relatively high percentage of ammonia), and also in certain cases of hepatic disorder, notably acute yellow atrophy of the liver, owing to the non-formation of urea by the disordered liver, its seat of formation in the body. The total amount excreted per diem by a normal man on an average diet containing 100 grams, of protein is 30 grams. Urea is also known as carbamide, since it is the diamide of carbonic acid. Carbonic acid. Urea. Urea crystallises in water-free, colourless, long needles, or in four-sided prisms of the rhombic system, which melt and decompose at 130° — 132° C. It is soluble in all proportions in hot water, and to the extent i : i in cold water. In cold alcohol it is soluble to the extent of i : 5. It is also soluble in acetone. In- soluble in pure ether and chloroform. The solutions are neutral in reaction. It forms crystalline compounds with acids. The two most important are urea nitrate CH4N2O.HNO3, insoluble in strong nitric acid, and urea oxalate (CH4N20)2, QHgOj, insoluble in oxalic acid. It forms compounds with the salts of the heavy metals, especially with mercuric nitrate (see below, Ex. 341). With reducing sugars relatively stable compounds are formed, called ureides. They are of importance in connection with the estimation of urea in diabetic urine. CH. XII.] UREA. 287 On heating dry urea to 140° C, ammonia is evolved and biuret formed. \NH3 NHj A. B. The A form exists in strongly acid solutions. It is decomposed by nitrous acid, like all compounds with the NH2 group. The B form exists in neutral or alkaline solutions and is not decomposed by nitrous acid. For the convincing evidence on which this view is based the original papers should be consulted. 333. To a watch-glass half full of distilled water add as much solid urea as will lie on a sixpenny-piece. Note the solubility of urea in water. 334. Place a drop of the urea solution on a slide, add a single drop of a saturated solution of oxalic acid, mix by stirring with a needle or fine glass rod, cover with a slip and examine the crystals of oxalate of urea that separate out. They vary considerably, containing long, thin, flat crystals, often in bundles and rhombic prisms. Draw the crystals. * Journal Chem. Soc, cix., p. 1120. CH. XII.] UREA. 289 333. Dilute the urea solution with twice its volume of water. Place a drop on a slide, add a drop of pure nitric acid, cover with a slip, and examine the crystals of urea nitrate that separate out. They form octahedral, lozenge-shaped, or hexagonal plates, often striated and imbricated. Draw the crystals. 336. Powder two or three crystals of urea in a watch-glass : rub with a small amount of acetone and warm gently on a water bath. The urea dissolves. Allow most of the acetone to evaporate away, and then place a drop of the remaining solution on a watch- glass. Urea crystallises out as the acetone passes off. Draw the crystals. 337. Repeat the above exercise, using strong alcohol instead of acetone. Draw the crystals of urea, which are usually very irregidar. 338. Dilute the remainder of the aqueous solution left from Ex. 335 with an equal quantity of water, and to a portion of this in a test-tube add some yellow nitric acid (or nitric acid to which a little potassium nitrite has been added). An effervescence and evolution of gas take place. CO(NH2)2 -F 2HNO2 = CO2 + 2N2 + 3H2O. Note. — All compounds containing the amino group (NHj) react in a similar manner when treated with nitrous acid (see Ex. 81). The decompo- sition of urea with nitric acid is relatively very slow. 339. To another portion of the solution add sodium hypo- bromite. A marked effervescence and evolution of gas take place. CO(NH2)2 -f- 3NaBrO -F 2NaH0 = 3NaBr -F Na^COs -F 3H2O + Ng. 340. To a few cc. of saturated ammonium sulphate add sodium hypobromite. A marked effervescence and evolution of gas take place. (NH4)2S04 -f- 3NaBrO + 2NaH0 = Na2S04 -F 5H2O -h sNaBr -V N^. Notes. — i. AU ammonium salts and all compounds with the amino group give ofi nitrogen^when treated with an alkaline solution of sodium hypo- bromite. 290 URINE. [CH. XII. 2. The sodium hypobromite is prepared as follows : dissolve 100 grams. c{ caustic soda in 250 cc. of water. Cool, and slowly add 25 cc. of bromine, cooling under the tap as the bromine is added. The reaction is as follows : 2 NaHO + Brj, = NaBrO + NaBr + H,0. It must be freshly prepared before use as it undergoes the following decomposition : 3 NaBrO = 2 NaBr + NaBrO,. 3. As a test for urea the reaction with hypobromite is only useful in a negative sense ; that is to say, if an effervescence is not obtained urea is absent, but if an efEervescence is obtained it does not necessarily follow that urea is present. 341. To some of the urea solution add a solution of mercuric nitrate. A white precipitate of mercuric oxide combined with urea and mercuric nitrate takes place. To the mixture thus obtained add a saturated solution of sodium chloride, drop by drop. The precipitate dissolves, to reappear on a further addition of mercuric nitrate. Notes. — i. The precipitate consists of urea and mercuric nitrate and one, two or three molecules of mercuric oxide, depending on the concentration of the two solutions. 2. The solubility in NaCl is due to the formation of mercuric chloride, which is only very feebly ionised in neutral solutions. 342. Treat a solution of urea with Millon's reagent, and heat. A white precipitate is formed, owing to the presence of mercuric nitrate in the reagent. There is also an evolution of gas due to the action of the nitrous acid on the urea. 343. Specific urease test for urea. To 4 or 5 cc. of a dilute solution of urea add 4 or 5 drops of phenol red. The colour obtained is generally sUghtly pinkish. Add traces of very dilute acetic acid by means of a glass rod until the reaction is very faintly acid to the indicator. Warm to about 45° C. Add a large " knife point " of finely ground Soya bean meal, shake and keep the solution warm. The colour changes to a reddish purple, owing to the enzyme converting neutral urea to alkaline ammonium carbonate. Notes. — i. In applying the test it is important to see that the reaction is only faintly acid to the indicator. For if a considerable amount of acid and only a small amount of urea be present, the amount of ammonium carbon- ate formed may not be sufficient to bring the reaction to the point where a pink colour is given with the indicator. 2. Proteins only interfere by acting as buffers. It is not usually neces- sary to remove them. CH. XII.] UREA. 291 3. The test will not succeed in the presence of the salts of the heavy metals, which inhibit the action of the enzyme. A high concentration of buffer salts, such as phosphates or acetates, decreases the delicacy of the test, by preventing considerable changes in hydrogen-ion concentration. 344. To about 4 cc. of a i per cent, solution of urea add about 2 cc. of strong soda, mix and divide into two portions, A and B. Boil B for 3 to 5 minutes, adding a little water from time to time to replace that lost by evaporation. Cool under the tap. Add phenol red to each and neutralise by the addition of hydrochloric acid, using concentrated acid at first and finish by dilute hydrochloric. Apply the urease test as described in the previous exercise to the two solutions. A gives a strong test, whilst B gives none, or only a slight one, owing to the destruction of the urea by boiling alkali. 345. Place a little urea in a dry test-tube. Heat carefully over a flame, keeping the upper part of the tube cool. The urea melts and evolves ammonia, whilst a white sublimate condenses on the cooler parts of the tube. Cool the tube, add a little water and shake. Pour the solution into another tube and treat it with an equal bulk of sodium hydroxide and a drop of copper sulphate. A pink colour is produced, due to the biuret formed from the urea. 346. Repeat the experiment, but heat more strongly till the melt solidifies and becomes opaque. Cool, add two or three cc. of water, boil and filter wWlst still hot. Divide the solution into two portions, A and B. To A add a few drops of a solution of barium chloride and a single drop of diluted ammonia. A white mass of barium cyanurate is formed on cooling. To B add some ammoniacal copper sulphate solution and boil. On cooling an amethyst precipitate of copper ammonium cyanurate is deposited. Note. — Preparation of ammoniacal copper sulphate, i per cent, copper sulphate is treated with very dilute ammonia till the precipitate that first forms just redissolves. 347. Isolation ol urea from urine. Evaporate about 30 cc. of urine to complete dryness, finishing the evaporation on the water bath (to prevent the destruction of the urea). Turn out the flame and rub the residue with about 10 cc. of acetone till it is boiling Allow the acetone to boil, stirring all the time, till about half of it 292 UKINE. [CH. XII. has evaporated away. Pour off the acetone into a dry watch glass and allow it to cool. Crystals of urea separate out as silky needles. Demonstrate that they are urea crystals by evaporating to dryness, taking up in a small amount of water and applying the urease test (Ex. 343). F. Uric Acid. Uric Acid, C5H4N4O3, is 2-6-8-tri-oxy-purine. NH-CO i C - NH^ I II >co NH - C - NH-^ Its relationship to certain of the other purines is indicated on page 63. When pure it crystallises in microscopic rhombic plates, but when impure it assumes a variety of forms, such as whetstones, dumb-bells, sheaves, rosettes, butchers' trays, etc. It dissolves to the extent of i part in 16,000 parts of cold water and i ,600 parts of hot water. It dissolves in alkalies, and the alkali salts of carbonic, phosphoric, boric, lactic and acetic acids, but not in the ammonium salts of these acids. It dissolves in warm concentrated sulphuric acid to form a sulphate, which is decomposed by the addi- tion of water. It is precipitated by phosphotungstic acid in the presence of hydrochloric acid, slowly b}'^ lead acetate, and completely by picric acid, mercuric chloride and ammonia- cal silver nitrate. By oxidation, allantoin, alloxan, parabanic acid and urea are formed,depending on the reaction and the reagent employed. NH, NH - CO NH - CO CO CO-NH. CO CO CO NH-CH-NH'"^ NH-CO NH - CO Allantoin. Alloxan. Parabanic acid. CH. XII.] URIC ACID. 293 Although the aqueous solutions of uric acid react neutral, it behaves like a disbasic acid C5H2N4O3.H2 and can form two series of salts, C5H2N403.Na2 (neutral, normal, or di-sodium urate) and C5H2N403.HNa (biurate, acid urate or mono-sodium urate). It is also possible that there is a third form of salt, C5H2N403.HNa.C5H4N403 (quadriurate or hemi-sodium urate), though this may be merely a mixture of its two constituents. The di-sodium salts are more soluble than the mono-sodium, but are only stable in markedly alkaline solutions. In the blood and urine urates exist as mono-sodium salts, which react neutral. It is interesting to note that there are two modifica- tions of the mono-sodium salt, called the a- and /3-form. The a-form is more soluble than the /8-form, but is un- stable, and slowly passes over into the other form. They are probably the salts of the two tautomeric modifications of uric acid described by Fischer : NH - CO N = C.OH CO O-NH^ HO.O C-NH. I II \C0 > II II >co NH - C - NH^^ N - C - NH^ Lactam modification forming Lactim modification forming unstable a-urate. stable y8-urate. It is of great interest to observe that in gout the amount of urate in solution in the blood is in excess of the amount of the /3-urate that can be held by normal blood. So that in gout it must be present at least, partly, in the unstable a-form. The deposition of urates in the tissues during an acute attack may be due to the conversion of the unstable a- into the stable, less soluble /3-modification. Urates are completely precipitated as amorphous ammonium urate by saturation with ammonium chloride. They exert a reducing reaction on Fehling's solution and towards alkaline silver solutions, this being the basis of Schiff's test. They yield a characteristic colour reaction when evaporated with nitric acid, the so-called murexide test. 294 URINE. [CH. XII. Uric acid occurs to the extent of about 0-7 gram, in the 24 hours' urine, but the amount excreted varies with the diet and the individual. From its close chemical relationship to the purine bases formed by the hydrolysis of the nucleins of the food and tissues (see p. 63), the view is commonly held that uric acid has its origin in the cellular organs of the body from the oxidation of such substances. Thus we can have uric acid arising exogenously from the free or combined purines of the food and also endogenously from those of the tissues. This view is apparently supported by the fact that the administration of foods rich in nucleoproteins, as sweetbreads, or of certain of the pure purine bases, does cause an increased excretion of uric acid. It is possible that a certain proportion of the uric acid formed in the body is destroyed by the liver, so that the amount excreted is a balance between that formed and that destroyed. In gout, in which there is a deposition of uric acid in the tissues, the excretion is decreased before an acute attack, is increased during the attack, and then falls again. In this condition there is a recognisable amount of uric acid in the blood (see above). For the method of estima- tion in urine see p. 343. 348. Treat a small amount of uric acid with 10 cc. of 2 per cent, sodium carbonate. Heat nearly to boiling and cool. Note that a considerable portion of the uric acid has dissolved in the form of a urate. 349. Filter the solution and treat a portion with a drop or two of strong hydrochloric acid and shake. A white crystalline pre- cipitate of uric acid separates out, showing that uric acid is very insoluble in water. Allow the crystals to settle, remove a few by means of a pipette, and examine them microscopically. They usually form rhombic plates. Draw the crystals. Note. — If the solution is very strong, the uric acid may separate out in an amorphous form. Should this be the case, make the solution alkaline and heat to dissolve. Whilst still hot add some HCl and aUow the tube to cool slowly. CH, XII.] URIC ACID. 295 Uric acid can assume a great variety of crystalline forms, resembling dumb-bells, whetstones, butcher-trays, stars, and sheaves. 350. To another portion of the solution add two drops of ammonia and saturate with ammonium chloride. A white amor- phous precipitate of ammonium urate is formed. Note. — ^This is the basis of Hopkins' original method for the estimation of urates in urine. It is an important reaction for separating urates from physio- logical fluids, such as urine (see Ex. 359), since no other organic substance, likely to be met -with in physiological analysis, is precipitated by saturation with ammonium chloride. The murexide reaction can be appUed to the precipitate obtained. 351. Treat a little uric acid with a little strong sulphuric acid : it dissolves. Pour the solution into water: the uric acid may separate out. 352. Murexide test. Treat a little uric acid in a porcelain dish with two or three drops of strong nitric acid. Heat on the water bath till every trace of nitric acid and water has been re- moved. A reddish deposit remains. Treat this with a dilute solution of ammonia (five drops of ammonia to about a test-tube full of water). The residue turns reddish-violet in colour. Add a little caustic soda. The colour turns to a blue-violet. Notes. — i. This important test needs a certain amount of care. The heating must be performed on the water-bath, and should be continued as long as is necessary to ensure the complete removal of every trace of nitric acid . 2. Xanthine and guanine give a yellow substance (nitro-xanthine) when treated with nitric acid. On evaporation the colour goes to a \'iolet shade, which turns yellow with dilute ammonia. Adenine and hypoxanthine give no colour reaction. 3. The chemistry of the reaction is as follows : From uric acid arises by oxidation dialuric acid and alloxan. They condense together to form aUoxantin. By the action of ammonia on alloxantin, purpuric acid is formed. Murexide is ammonium purpurate. HN-CO HN-CO H OC C-^ + OC CO ! I^OH I I HN-CO HN-CO Dialuric acid Alloxan. Alloxantin + NHs = HN-CO 1 1 OH OC c^ 1 1 HO HN-CO OC 1 c OC -NH 1 CO 1 -NH Alloxantin. HN-CO NH oc- -NH nr. r^^"^^"" 1 1 "^c 1 1 CO 1 1 1 HN-CO Purpuric 1 1 oc- icid. •NH 296 URINE. [CH. XII. 353. SchifE's test. Treat a very small amount of uric acid with a few cc. of sodium carbonate. Pour the solution on to filter paper moistened with silver nitrate. A black stain of reduced silver immediately results. Note. — This useful test cannot be applied in the presence of chlorides. It is important to note that the uric acid is dissolved in sodium carbonate, not the hydroxide, as the latter gives a precipitate of the brown silver hydroxide, which completely obscures the reduction. An amount of sodium carbonate in excess of that required to dissolve the uric acid must be added, as the reduction only takes place in the alkaUne condition. 354. Folin's test. To a very small pinch of uric acid in a beaker add 20 cc. of a saturated solution of sodium carbonate. Stir till the uric acid has completely dissolved, add i cc. of Folin's uric acid reagent. A blue colour is obtained. Notes. — i. Preparation ol Folin's solution. 100 grams, of pure sodium tungstate, 102 cc. of pure ortho-phosphoric acid (B.P. 66-3%) and 750 cc. of distilled water in a flask fitted with a reflux condenser are boiled for 2 hours. On cooling the solution is diluted to i litre. 2. The solution also gives a blue colour with poljrphenols. It is used for the microchemical estimation of uiic acid in urine. 355. Dissolve a little uric acid in sodium carbonate by boiling. .\dd 5 cc. of Fehling's solution and boil for a considerable time. Note the peculiar reduction of the copper, and compare it with the reduction obtained with glucose. 356. Similarly try the effect of uric acid on Nylander's (Ex. 105) and Benedict's (Ex. 100) solutions. A reduction is not obtained. 357. Dissolve some uric acid in sodium carbonate, add an excess of ammonia and treat with silver nitrate. A white amorphous precipitate of a silver compound of uric acid is formed. Note. — Xanthine, hypoxanthine and other substances in urine closely related to uiic acid are similarly precipitated by ammoniacal silver nitrate. 358. A solution of sodium urate and urea is provided. To prepare crystals of uric acid and of urea. Heat a test-tube nearly full of the solution to boiling point and add strong hydrochloric acid till the reaction is distinctly acid. Allow the tube to cool slowly ; the uric add crystals separate out. CH. XIl.] URIC ACID. 297 Cool thoroughly under the tap. Filter off the uric acid. Neutralise the filtrate with sodium carbonate and evaporate to dryness, finish- ing the process on the wdter-bath, to prevent the conversion of the urea to biuret (see Ex. 345). Extract the residue with strong alcohol or acetone. The alcohol or acetone solution is carefully evaporated to dryness, and the urea crystalUses out. 359. To demonstrate the presence of uric acid in urine. To 50 cc. of urine add powdered ammonium chloride and stir tiU the solution is saturated. Add three drops of strong ammonia and stir again. Allow the excess of ammonium chloride to settle for 15 sees, and pour off into another beaker. Note the gelatinous precipi- tate of ammonium urate. Filter : scrape the precipitate off the paper and transfer it to an evaporating dish. Add three or four drops of strong nitric acid and place the dish on the water-bath till a pink, dry residue is obtained. Treat this with a httle dilute ammonia : the purple colour produced indicates the presence of urates in urine (see Exs. 350 and 352). 360. Folin's method of demonstrating the presence of uric acid in urine. To i to 2 cc. (20 drops) of urine in an evaporating dish add one drop of a saturated solution of oxalic acid and evaporate to complete dryness on a water-bath. Allow to cool, add 10 cc. of strong alcohol and allow to stand for five minutes to extract the polyphenols. CarefuUy pour off the alcohol. To the residue add 10 cc. of water and a drop or two of saturated sodium carbonate. Stir to secure complete solution of the uric acid and transfer to a beaker. Add i cc. of FoUn's uric acid reagent (Ex. 354) and 20 cc. of saturated sodium carbonate solution. The blue colour that results indicates the presence of uric acid. 361. Urine has been treated with about one-fiftieth its bulk of strong hydrochloric acid, and allowed to stand from twelve to twenty-four hours. Note the brown crystals of uric acid that have formed on the sides of the vessel. Examine them microscopically : they form very irregular crystals, usually arranged in sheaves. Draw the crystals. Note. — The chief pigment that associates itself with uric add and urate s is known as uroerythrin (see p. 278). 298 URINE. [CH. XII, G. Purine bases, other than uric acid. The most important of these found in normal urine are hypoxanthine, xanthine and adenine (see p. 62), derived from the metabolism of food and tissue nucleins : heteroxanthine (7-methyl-xan thine) and paraxanthine (i, 7-dimethyl-xanthine) derived from the breakdown of caffeine (i, 3, 7-trimethyl-xanthine) and theobromine (3, 7-dimethyl-xanthine) of the coffee, tea and cocoa ingested. In man the methylated xanthines constitute the greater part of these purine bases. But it is interesting to note that the non-methylated ones are much increased in fever. Also during severe muscular exercise there is an increase, accompanied by a decrease of uric acid. After the exercise there is an increase of uric acid, and a decrease of the other purines. The simplest method of estimation is to determine uric acid nitrogen by the method in Exs. 394-397, and the total purine nitrogen by applying Kjeldahl's method to the total purines precipitated by ammoniacal silver nitrate (Ex. 357). The difference is the nitrogen of the purine bases. H. Creatinine and Creatine. The chemical relationships of these bodies are de- scribed on p. 178. In normal human urine creatinine is always present, but creatine only after a meat diet, being derived from that of the food. Creatine, however, is a normal constituent of the urine of children. Creatinine seems to be a product of tissue metabolism, and the amount excreted is regarded by Folin as a measure of endogenous metabolism. (See tables B and C, p. 270.) There is an increase in complete starvation and in fevers, due to the increased tissue breakdown. E. Mellanby has drawn attention to the fact that the liver is probably the seat of formation of creatinine. Thus in most diseases of the liver there is a decreased excretion, an important CH. XII.] CREATININE. 299 exception being hepatic carcinoma, in which condition the urinary-creatinine is increased and is accompanied by creatine. Creatine is excreted when the muscles of the body are broken down. This explains the presence of creatine in urine during starvation and in fevers. When creatinine is given by the mouth it is mainly excreted unchanged, but a small portion is broken down into unknown products. When creatine is administered it also is chiefly excreted unchanged, but a certain per- centage is destroyed in the body. The amount excreted unchanged is considerably increased with diets rich in proteins. Properties. Creatinine dissolves in 1 1 parts of water and 102 parts of alcohol at i6° C. It is insoluble in ether. Its solutions are neutral or very slightly alkaline in reaction. Creatinine is precipitated by phosphotungstic acid, by picric acid, and by the salts of the heavy metals. It forms a characteristic compound with zinc chloride, which is used for the preparation of standard solutions. Alkalies convert it slowly into creatine. On boiling with barium hydroxide it is converted into urea and sarcosine (see p. 178). Creatinine reduces Fehling's solution, but not Bene- dict's or Nylander's solutions. Creatine is converted to creatinine by heating with acids (see Ex. 226). It can be estimated by making determinations of creatinine before and after heating the urine with acid. If aceto-acetic acid is present Graham has found that both results are liable to considerable error (see Graham and Poulton, Proc. Roy. Soc, lxxxvii., B., p. 205). For the method of estimation see p. 338. Preparation of Creatinine from urine. "^ (i.) Preparation of creaiinine picrate. It is best to work on i o Ktres of urine at least. Dissolve 40 grams, of picric acid in 100 cc. of boiling alcohol and use 1 8 grams, of picric acid per litre of urine. Pour the hot solution directly into the urine, stirring well during the addition. * (Benedict, Journal of Biological Chemistry, xviii., p. 183.) 300 URINE. [CH. XII Allow to stand over-riight and syphon off the supernatant fluid. Drain the residue on a Buchner funnel and wash with cold saturated picric acid and then drain dry. (ii.) Decomposition of the pier ate. Treat the dry creatinine picrate with concentrated hydrochloric add in a mortar, using 60 cc. of acid for every 100 grams, of the picrate. Stir thoroughly with the pestle for 5 minutes. Filter by suction, using a hardened filter paper. Wash residue twice with enough water to cover it, sucking dry each time. Transfer the filtrate at once to a large flask and neutrahse with solid heavy magnesium oxide. Add it in small amounts at a time, coohng under the tap after each addition. The solution turns light yellow when aU the acid has been neutralised. Filter with suction and wash the residue twice with water. At once add a few cc. of glacial acetic acid to the filtrate and pour it into 4 volumes of 95 per cent, alcohol. Allow to stand at least 15 minutes and filter under suction. (iii.) Preparation of creatinine zinc chloride (CfH^N^0)2-ZnCl^. Treat filtrate with a 30 per cent, solution of zinc chloride, using 3-5 cc. for each litre of urine taken. Allow to stand over-night in a cool place. Pour off the supernatant fluid and then collect the creatinine zinc chloride in a Buchner. Wash once with water, then thoroughly with 50 per cent, alcohol, then 95 per cent, alcohol and dry. The product should be a nearly white, light crystalline powder. Yield: 1-2 to 1-5 grams, per litre of urine. (iv.) RecrystalHsation of creatinine zinc chloride. 10 grams, are treated with 100 cc. of distilled water and then with 6occ. of N. sulphuric acid. The solution is heated to boiling till a clear solution is obtained. About 4 grams, of pure decolourising charcoal are added and the boiUng continued for about i minute. The solution is filtered ofi through a small Buchner, the filtrate being refiltered through the same funnel two or three times till it is quite clear. The residue is washed with a Httle hot water and the total filtrate transferred to a beaker. It is then treated hot with 3 cc. of a strong solution of zinc chloride and about 7 grams, of potassium acetate dis- solved in a httle hot water. After 10 minutes the solution is treated with an equal volume of strong alcohol and allowed to stand for some hours in a cool place. The crystals are filtered off and stirred up with about twice their weight of cold water, filtered, washed with a little water, and then with alcohol. Yield : 85 to 90 per cent, of the crude material. (v.) Conversion of the zinc chloride compound to creatinine. The powdered, recrj/stallised compound is placed in a dry flask and treated with 7 cc. of concentrated aqueous ammonia for every gram, taken. It is sUghtly warmed and gently agitated until a clear solution is obtained, care being taken to drive ofi as little ammonia as possible. The flask is then stoppered and placed in an ice box for an hour or two. Pure creatinine crystallises out. Yield : 60 to 80 per cent, of the theoretical. 362. JafEe's test for creatinine. To 5 cc. of urine add a few drops of a saturated aqueous solution of picric acid and of a 10 per cent, solution of sodium hydroxide. A red colouration is produced owing to the formation of picramic acid (see Ex. 108). CH. XII.] AMMONIA AND HIPPURIC ACID. 3OI 363. Weyl's test for creatinine. To 5 cc. of urine add a few drops of a freshly prepared 5 per cent, solution of sodium nitro- prusside. Add a 5 per cent, solution of sodium hydroxide, drop by drop. A ruby-red colour appears. Boil. The solution turns yellow. Acidify with strong acetic acid and heat. A green tint appears and a precipitate of Prussian blue may separate. Note. — It is essential to get the ruby-red colour. The formation of Prussian blue is apt to occur with a variety of other substances. I. Ammonia. Ammonia is a constituent of normal urinCj being present to the extent of about 0-7 grams, per diem. There is an increased excretion following the administration of ammonium salts of inorganic acids, in certain cases of hepatic disease, and as a result of acid poisoning. This last condition ("acidosis") can be produced by the adminis- tration of inorganic acids or by the excessive formation of acids in the body, especially if this is not accompanied by an increased intake of alkalies. Thus it is seen in severe diabetes, in starvation, and in delayed chloroform poison- ing, the acids formed being aceto-acetic and y8-oxy-butyric acids. In certain forms of renal disease there is a decreased excretion (see p. 275). For methods of estimation see Exs. 398 to 400. J. Hippuric Acid. Hippuric acid is formed in the kidney by the con- densation of benzoic acid with glycine. CjHs.COOH + HjN.CH^.COOH = CeHj.CO.NH.CHaCOOH + HjO Benzoic acid. Glycine. Hippuric acid. The amount excreted by a normal individual on a mixed diet is about 0-7 grams, per diem. It is increased by a vegetable diet, owing to the presence in most plant foods of an aromatic complex that is oxidised to benzoic acid in the body. Hippuric acid crystallises in 4-sided prisms, somewhat resembling triple phosphate. It melts at 187-5° C. : above 302 URINE. [CH. XII. this temperature the melt becomes red and is decomposed into benzoic acid, benzonitrile and prussic acid. It is soluble in hot water, alcohol and ethyl acetate : insoluble in benzene and petroleum ether : only slightly soluble in cold water, alcohol, ether and chloroform. It forms an insoluble ferric salt. By hot acids or alkalies it is hydro- lysed to benzoic acid and glycine. When evaporated with strong nitric acid, nitrobenzene is formed. 364. Isolation from urine by Boal's method. 500 cc. of the urine of a horse or cow are treated with 125 grams, of ammonimn sulphate and 7-5 cc. of concentrated sulphuric acid. On standing for 24 hours the hippuric acid crystallises out. Filter off the crj'stals, and wash with a httle cold water. Dissolve in a small amount of hot water, boil with a little adsorbent charcoal, filter, concentrate if necessary, and allow to stand for 24 hours. 365. To a little hippuric acid in a small evaporating dish add I to 2 cc. of concentrated nitric acid and evaporate to dryness in a water-bath in the fume chamber. Transfer the residue to a dry test-tube, apply heat, and note the odour of nitrobenzene (artificial oil of bitter almonds). 366. Neutralise a solution of hippuric acid with dilute caustic soda. Add a few drops of ferric chloride. A cream-coloured precipitate of the ferric salt of hippuric acid is formed. E. Certain Constituents of Abnormal Urine. I. Albumin and Globulin. "Albuminuria" is the name given to the condition in which a heat-coagulable protein is found in the urine, no matter whether the protein present is albumin or globulin. As a rule both proteins are present, but albumin is gener- ally greatly in excess of the globulin. Albuminuria can be renal ("true") or accidental ("false"). Renal albuminuria can be brought about by an alteration in the blood pressure in the kidney, by a change in the composition of the blood, or by an alteration in the CH. XII.] ALBUMIN. 303 structure of the kidney. In accidental albuminuria, the protein is not passed by the kidney, but gains access to it lower down in the urinary tract. It is generally accom- panied by haemoglobinuria. For routine work the author now uses the sulpho- salicylic test. It is very rapid and conclusive. For the method of estimating the albumin see Exs. 420, 421. 367. Boiling test. Filter the urine till it is clear. If it wil] not filter clear, as when infected with bacteria, shake with kieselguhr and filter again. If the urine be alkaline to litmus, make it faintly acid by the cautious addition of i per cent, acetic acid. Fill a narrow test-tube three parts full with the clear urine, incline it at an angle and boil the upper layer by means of a very small flame. A turbidity indicates either albumin or earthy phosphates (see note 2 to Ex. 28). Add one or two drops of strong acetic acid, boiling after the addition of each drop. Any remaining turbidity indicates the presence of albumin. 368. Heller's test. Place about 3 cc. of pure nitric acid in a narrow test-tube. Float about 3 cc. of filtered urine on the surface of this, using a pipette to avoid mixing. A white ring at the junc- tion of the fluids indicates the presence of albumin. Notes. — i . The white ring is due to the formation of metaprotein by the action of the acid on the albumin, and the insolubility of the metaprotein in the strong nitric acid (see Exs. 2i and 40). 2. A coloured ring is usually produced owing to the oxidation of certain urinary chromogens. 3. In very concentrated urine, a white ring of urea nitrate may form. It usually has very sharply defined borders. 4. If the urine is very rich in urates, a precipitate of uric acid may form at the junction of the fluids, or, more commonly, somewhat above the nitric acid. Urea and uric acid are distinguished from albumin by the previous dilution of the urine with two or three volumes of water. 5. The presence of resinous substances in the urine of patients who have been treated with balsams leads to the development of a white ring or cloud that disappears on treatment with alcohol. 6. Urine rich in albumose may give a white cloud that disappears on warming. 7. Urine that has been preserved by the addition of thymol gives a ring of nitrosothymol or nitrothymol. The thymol can be removed by gentle agitation with petroleum ether. 304 URINE. [CH. XII. 369. Roberts' test. Repeat the previous exercise, iising Roberts' reagent in place of the nitric acid. A white ring at the junction of the fluids indicates albumin. Notes. — i. Roberts' reagent is prepared by adding i volume of pure nitric acid to 5 volumes of a saturated solution of magnesium sulphate. 2. Coloured rings are not formed, and so confusion is avoided. 370. Spiegler's test. Render the urine faintly acid with acetic acid and repeat the above test, using Spiegler's reagent in place of Roberts'. A white ring indicates the presence of albumin. Notes. — i . Spiegler's reagent consists of : Mercuric chloride . . . . 40 grams. Tartaric acid . . . . . . 20 grams. Glycerine . . . . . . . . 100 grams. Sodium chloride . . . . . . 50 grams. Distilled water . . . . . . 1000 cc. 2. The reaction is also given by albumoses and peptones. 3. The test serves to show i part of albumin in 250,000. It is almost too deUcate for ordinary chnical work, as a large number of apparently normal urines give a positive reaction. 371. Sulphosalicylic test. To i or 2 cc. of the clear, filtered urine add a large " knife point " or a few drops of a 20 per cent, solution of sulphosaUcylic acid (see Ex. 18). A cloud or precipitate indicates the presence of albumin. 2. Albumoses. Albumoses are found in the urine in certain cases of degeneration of the intestinal epithelium ("alimentary albumosuria"). Also in a variety of other conditions such as in the absorption of pneumonic exudates, in some cases^ of an increased breakdown of the tissues in certain fevers, in the puerperium and in urine containing semen. The albumose present seems to be a secondary album ose.. 372. Remove any albumin that may be present by heat coagulation. To the filtrate apply Spiegler's test (Ex. 370). A, white ring indicates the presence of albumose. 5. Bence- Jones' Protein. In certain cases of disease of the bone marrow (multiple- myeloma), and possibly in osteomalacia, a protein with. CH. XII.] BENCE-JONES' PROTEIN. 305 peculiar properties is found in the urine. It is named after Bence-Jones, who first described the condition. It has the property of coagulating at temperatures under 55° C, of redissolving to a clear solution on boiling and of reappearing on cooling. It is precipitated by half- saturation with ammonium sulphate. It is not precipi- tated on dialysis. Hopkins has shewn that the solution of the heat coagulum on boiling depends on the presence of neutral salts, those with divalent cations (as CaClg) being most potent in neutral or faintly acid solutions, and those with divalent anions (as K2SO4) in faintly alkaline solutions. Hopkins has also shewn that the protein excreted is formed in the body, either in the marrow or as a result of the influence of the growth on general metabolism. The amount in the urine is independent of the nature or amount of the proteins of the food. The nitrogen of the protein excreted may be as high as one-third of the total urinary nitrogen. 373. If necessary make the suspected urine faintly acid with acetic acid. Heat carefully by immersing in a beaker of warm water. The urine becomes turbid at 40° to 45° C, and shows a fiocculent precipitate at 60° C. On raising the temperature to ioo°C. the precipitate partially or completely disappears. On cooling it reappears. 4. Blood Pigments. Blood pigments may occur in pathological urine in intact corpuscles ("haematuria") or free in solution (" haemoglobinuria ") . Haematuria can be recognised by determining the presence of red corpuscles by a microscopic examination of the sediment obtained by centrifugalising the urine. It occurs with gross lesions of the kidney or any part of the urinary tract, so that blood passes directly into the urine. If the blood comes from the kidney it is well mixed with the urine. If the blood comes from the bladder 306 URINE. [CH. XII. or genital organs it often forms a clot. In haematuria the urine often has a characteristic smoky appearance, and it is always associated with albuminuria. Haemoglobinuria is a result of haemolysis. It therefore follows a variety of infectious diseases, transfusion of blood, the absorption of haemolytic substances, such as many aromatic com- pounds, severe burns and scalds. Methaemoglobin is nearly always present. The simplest method of detecting blood is by means of the benzidine test, provided that the necessary reagents are to hand. 374. Heller's test. Boil 10 cc. of urine with a little 40 per cent, sodium hydroxide, and aUow the tube to stand for a while. A red deposit indicates the presence of blood-pigment in the urine. Pour off the supernatant fluid and acidify with acetic acid. The precipitate dissolves only partially, leaving a red residue. Notes. — i. The alkali converts the pigment into haematin, which is precipitated with the earthy phosphates. 2. Certain substances, such as cascara sagrada, rhubarb, senna and santonin cause the urine to give a similar red precipitate when boiled with alkaU. But in these cases the precipitate dissolves completely in acetic acid. 375. Sehumm's spectroscopic test. Treat 50 cc. of the urine with 5 cc. of glacial acetic acid and 50 cc. of ether. Shake thoroughly in a separating funnel. Allow to stand and add a drop or two of alcohol to obtain a separation of the layers. Rim off the urinary layer. To the ether add 5 cc. of water, shake and run off the water. To the washed ether add ammonia and shake for half a minute, cooling under the tap. The reaction must be markedly alkaline after shaking. Run off the lower coloured layer into a tube, add 5 to 10 drops of ammonium sulphide solution and examine spectro- scopicaUy for the bands of haemochromogen. (Ex. 305.) 376. Benzidine test. To a large " knife point " of benzidine in a perfectly clean, dry test-tube add about 3 cc. of glacial acetic acid and agitate for about a minute. Add an equal volume of " 10 volumes " hydrogen peroxide. Mix and pour one-half into another clean, dry test-tube. To one of the tubes add i cc. of the CH. XII.] BILE PIGMENTS. 307 suspected urine. The fluid rapidly acquires a deep blue tint if blood pigment is present. Should the untreated fluid also develop a blue tint, the test should be repeated, the control tube being treated with I cc. of a normal urine. By following this procedure the test is a very conclusive one. The reaction can be apphed to an acid ether extract prepared by the method given in the preceding exercise. J. Bile. The constituents of the bile are found in urine when the bile duct is obstructed by a calculus or by catajrrh. The bile is absorbed into the lymphatics, passes into the circulation and reaches all parts of the body, the pigments causing a staining of the various tissues. The condition is known as jaundice. The absence of bile salts from the urine does not exclude the possibility of the presence of bile pigments. With continued obstruction of the bile passages the formation of bile salts seems to decrease. Urine contain- ing bile often has a characteristic appearance. 377. Cole's test for bile pigments. Treat lo to 15 cc. of the urine with 2 drops of saturated magnesfum sulphate and proceed as directed in Ex. 318. If a hand centrifuge is available, the test is more sensitive if an excess of barium chloride is added to the unheated urine and the precipitate driven drown by spinning in the machine. The supernatant fluid is poured off as cleanly as po"ssible, the precipitate stirred with the alcohol and sulphuric acid, trans- ferred to a test-tube and boiled with the potassium chlorate. In a certain number of cases the result is obscured by the presence of certain other pigments. In such cases to render the test more delicate, pour off the alcoholic solution from the barium sulphate into a dry tube. Add about one-third its volume of chloroform and mix. To the solution add about an equal volume of water, place the thumb on the tube, invert once or twice and allow the chloroform to separate. It contains the bluish pigment in solution. 308 URINE. [CH. XII. 378. Hay's test for bile salts. Sprinkle the surface of some urine in a test-tube with flowers of sulphur. The particles fall to the bottom of the tube if bile salts are present. (See Ex. 316.) 379. Oliver's test for bile salts. Acidify the urine with acetic acid and filter if necessary. To it add a clear i per cent, solution of Witte's peptone, also acidified with acetic acid. A white precipitate indicates bile salts. (Ex. 317.} 6. Glucose. Glucose is not, strictly speaking, an abnormal con- stituent of urine. The author was finally convinced of this some years ago when working at a method for the detection of small amounts of glucose in urine* (see Ex. 381). Folinf confirmed this, using practically the same method. Recently Benedict and Osterbergt have introduced a new method for the estimation of glucose in normal urine (see Ex. 407), and although it has only been applied to a few individuals, the results obtained are of very great im- portance, and will probably serve as the starting point for a new attack on the problems of diabetes. According to these observers about i gram, of non-nitrogenous reducing substance is excyeted per diem, of which about 55 per cent, is not fermentable by yeast, and has not yet been identified. The effect of diet is interesting. The excretion is increased by carbohydrate intake, especially at breakfast. A similar intake at mid-day, during normal muscular activity, has a much smaller effect. For this reason a normal individual may pass a urine shortly after breakfast which might cause him to be rejected as a diabetic when examined for life insurance. Such cases would probably be passed as normal if a sample of the mixed 24 hours' specimen were examined. The effect of taking glucose varies with the dose and also with the time of administration. Apparently * Cole, Lancet, 1913, ii., p. 859. ■f Folin, Journ. Biol. Chem., xxii., p. 327. t Benedict and Osterberg, Journ. Biol. Chem., xxxiv., p. 195. CH. XII.] GLUCOSE. 309 glucose is tolerated better on an empty stomach than when taken with an ordinary meal. In general, it might be stated that a normal individual should be able to absorb 50 grams, of glucose on an empty stomach without showing any increase in the amount of sugar excreted in a given time. An important result of these researches is that the concentration of sugar in the urine is of much less signifi- cance than the amount passed per hour. Since urine always contains glucose, they suggest that the term "glycuresis" should replace "glycosuria," to indicate conditions characterised by an. increased excretion of glucose in the urine. There are two types of glycuresis, alimentary and persistent. Alimentary glycuresis is the condition in which the amount of sugar absorbed exceeds the amount that the individual is capable of assimilating. The limit varies with the individual, and is affected by a variety of pathological conditions. Persistent glycuresis is the condi- tion when large amounts of sugar are excreted for a con- siderable length of time, and may be quite independent of the administration of carbohydrate food. The condition is known as diabetes mellitus. The urine is generally much increased in amount, of a high specific gravity, and pale in colour. The classical test for sugar in urine is Fehling's (Ex. 97). It is not a reliable test. Not only is Fehling's solution reduced by certain constituents of normal urine, such as urates and creatinine ; but also certain of these bodies, notably creatinine, form soluble compounds with cuprous oxide, and thus markedly interfere with the delicacy of the test. Also the urea of the urine is decomposed to ammonia, which dissolves cuprous oxide (see p. 106, note 5). Further, glucose is destroyed by boiling caustic soda, so that the presence of a small amount of sugar may escape detection. Benedict's test (Ex. 100) is a great improvement. Owing to the substitu- tion of sodium carbonate for sodium hydroxide the solution is not reduced by urates or creatinine. It does not give a positive reaction with the concentra- tion of glucose normally present in urine, but is very sensitive for small in- creases beyond this. The author considers it the most reliable for general use ; but owing to the fact that it is much more delicate than Fehling's, the result of a faintly positive test is not necessarily an alarming indication of abnor- mality. Cole's test (Ex. 381) is more sensitive than Benedict's, and the manipula- tion has been so arraM;ed as to ensure that it does not give a positive result with normal urines. It is of considerable value in detecting small variations 310 URINE. [CH. XII. from the normal, but such cases should be examined by the application of Benedict and Osterberg's quantitative method. (Ex. 407.) The osazone test serves to confirm the presence of glucose in doubtful cases, and especially to distinguish between glucose on the one hand and lactose and pentoses on the other. The fermentation test is helpful in connexion with the recognition of lactose and glycuronic acid. 380. Benedict's test. To 5 cc. of Benedict's reagent (see p. 107) in a test-tube add eight drops of the urine. Boil vigorously for two minutes and allow to cool spontaneously. If glucose is present the entire body of the solution will be filled with a precipitate which may be red, yellow or green in colour, depending on the amount of sugar. Note. — It is essential to add a small volume of urine. If too much be added the results are apt to be ambiguous. Even with the eight drops recommended, a slight precipitate of earthy phosphates may appear and simulate a feeble reduction. 381. Cole's test for small amounts of glucose in uiine. In a dry boiling tube or large test-tube place about i gram, of adsorbent charcoal. Add 10 cc. of the urine, shake, heat to boiUng and then cool under the tap. Shake at intervals for 5 minutes. Filter through a smaU paper into a dry test-tube. To the filtrate add 4 dropsof pure glycerol and 0-5 gram, of anhydrous sodium carbonate. Shake and heat to boiUng. Maintain the boiling for exactly 50 sees. Immediately add 4 drops of a 5 per cent, solution of crystcJUne copper sulphate, shake to mix and allow the tube to stand without further heating for one minute. With normal urine the fluid remains blue. If glucose is present to the extent of 0-03 per cent, above the normal amount in urine the blue colour is discharged and a yellowish precipitate of cuprous hydroxide forms. Notes. — i. Treatment with adsorbent charcoal removes practically the whole of the urates, creatinine and pigments that interfere with Fehling's test. It also adsorbs so much of the normal amount of glucose present that the filtrate from normal urine fails to give a reduction. 2. 0-5 gram, of anhydrous sodium carbonate is carried by about J the length of a large blade well piled up once. 3. Should the specific gravity of the urine exceed 1025 it is advisable to use 5 cc. of the urine + 5 cc. of water. 4. The test is not given by chloroform nor by glycuronates : it is given by pentoses. CH. XII.] GLUCOSE. 3II 5. Should there be any reason to suspect lactose the procedure should be modified as follows : treat 20 cc. of the urine with t gram, of charcoal as de- scribed above. Treat the whole of the filtrate with another gram, of charcoal and repeat the process. To 5 cc. of this filtrate add the glycerol and sodium carbonate and proceed as above directed. A reduction indicates the presence of glucose, the whole of any lactose up to even i per cent, being removed by this double adsorption, whilst 0-04 per cent, of glucose in the original urine still shows in the filtrate. 382. Fehling's test. Boil 3 to 5 cc. of Fehling's solution (see p. 106) to ascertain whether the Rochelle salt has been de- composed into reducing substances. If no reduction occurs boil the same volume of urine in another tube. Reboil the Fehling's solution and mix the two. Allow the tube to stand without further heating. If any appreciable amount of glucose is present a red or yellow precipitate will appear. Note.- — Prolonged boiling of the urine with Fehling's solution is very apt to lead to the formation of a greenish-yellow precipitate owing to the action of the strong alkali on the normal urinary constituents. 383. Phenylhydiazine test. Treat 10 cc. of urine with i cc. of strong acetic acid. Add enough phenylhydrazine hydrochloride to cover a sixpenny piece and twice this bulk of solid sodium acetate. Dissolve by the aid of heat and filter. Place the filtrate in a tube and immerse this in a boihng water-bath for 30 to 60 minutes. Turn out the flame and allow the tube to cool without removing it from the bath. Examine the deposit microscopically for the characteristic crystals of glucosazone (see p. no). Note. — With small amounts of glucose the crystals are apt to separate in small spherical clusters. 384. Fermentation test. Fill a test-tube with urine and then transfer the fluid to a mortar. Add a piece of washed yeast about the size of a bean and pound it up with the urine. Transfer the mixture to the test-tube and invert, placing the open end under mercury or urine contained in a small dish. Clamp the tube in position, and allow it to stand for at least eighteen hours in a warm place. If glucose is present in the urine there is an accumulation of gas (CO2) at the top of the tube. Notes. — i. Lactose, pentoses and glycuronic acids are not fermented by pure yeast. \!.. A special apparatus called Einhorn's saccharometer has been devised to enable the test to be applied conveniently. Also the volume of COj formed, and the percentage of glucose present can be roughly determined by means of it. 3T?. URINE. [CH. XII. 7. Fructose {laevulose). Fructose occasionally occurs in the urine, sometimes being accompanied by glucose. The significance of fructo- suria is not yet clear. 385. SeliwanoS's test (Borchardt's modification). To a few cc. of urine in a test-tube add an equal volume of 25 per cent, hydrochloric acid and a speck or two of resorcin. Heat to boiling, cool under the tap, and transfer to an evaporating dish. Make the reaction alkaUne by means of solid sodium hydroxide and return it to a test-tube. Add 3 cc. of acetic ether (ethyl acetate) and shake. A yellow colouration in the acetic ether indicates the presence of fructose. 8. Pentoses. Pentoses, that is carbohydrates with 5 carbon atoms, appear in the urine in three conditions, alimentary, persistent or true pentosuria, and admixed with glucose in cases of glycuresis. Alimentary pentosuria is sometimes seen after the ingestion of considerable quantities of certain fruits, as prunes, cherries, grapes and plums. The sugar found varies, but is usually fi?-arabinose. In true pentosuria it is ~ — {A)~ X 14 X (A) mg. A good deal of time is saved if the acid and alkali be labelled with the logarithms of the " acid-equivalent " (acid log.) and of the " alkali-acid ratio " (alkali log.) respectively. 324 ANALYSIS OF URINE. [CH-XIIl. The following example should be carefully studied. Acid employed was 0-0476 N. sulphuric. So 1 CO. = 0-0476x14 mgms. nitrogen. log. 0-0476 = 2-6776 add log. 14 = 1-1461 So " acid-log " = 1-8237 Alkali employed was 0-0421 N. soda. 00421 So I cc. soda = cc. acid. 0-0476 Log. 0-0421 Substract log. 0-0476 — 2-6243 2^6776 So " alkali-log." is o'5 cc. of urine taken. Amount of acid taken was 20-12 for back titration. Log. of 13-6 Add the alkali log cc, f-9467 ., and this i-1335. 1-9467 required 13-6 cc. of alkali Anti-log. of 1-0802 is 12-03. So 20-12 - 12-03 = 8-09 cc. of ammonia formed from 0-5 cc. of urine Log. of 8-09 is Add the acid log. acid have -9079 1-8237 been neutralised by the Anti-log. of -7316 is 5-39. So 0-5 cc. of urine contain 5-39 mg. total Nitrogen. So 100 cc. of urine contain 1-078 gram, total Nitrogen. No allowance has been made for the blank determination, but this should not be neglected, especially when using the micro-method. The blank determination is made with all the materials used for an ordinary analjrsis, distilled water being take instead of urine. Unless the reagents are of very poor quality, the amount of nitrogen found should be very small. This must be deducted from the amount found in the volume of urine taken. An example is given on page 263. 394. KjeldaW's method (distillation by boiling). Into a clean, drj', round-bottomed flask of " Duro " glass A (500 cc. capacity, with a narrow neck 8 inches in length) place 5 to 10 grams, potassium sulphate, 0-5 cc. of saturated copper sulphate solution, 5 cc. of urine (accurately measured) and 10 cc. of concen- trated sulphuric acid, free from nitrogen. Place the flask in the fume-chamber (or use the fmne-absorber, desaibed on page 387), and heat by means of a low flame for 10-15 minutes, then boil briskly f o r 45 minutes or longer. The solution must be heated for at least 15 minutes after it has lost every trace of dark colour. Any particles of carbonaceous matter that adhere to the sides of the flask must be CH. XIII.] TOTAL NITROGEN. 325 washed down into the acid by carefully shaking the flask. When cool add 250 cc. of ammonia-free distilled water, 3 or 4 pieces of broken porous pot, and cool under the tap. Into an Erlenmeyer flask, E, of about 400 cc. capacity, place 20 cc. of standard sulphuric acid, about 0-2 N. This flask is then placed on an ad- justable stand, so arranged that the lower end of the tube D dips below the surface of the acid in E. The bulb in D is to decrease the risk of the acid in E being sucked back by a sudden cooling of A during the distillation. D is connected to a condenser C. The best pattern is Davies', which is shewn in fig. 40. To the flask A add 35 to 40 cc. of 40 per cent, sodium hydroxide, pouring it down the neck and waU of the flask so as to form a bottom layer ; loss of ammonia is thus prevented. Fit the glass tube B into the neck of A by means of a well-fitting rubber stopper. The special bulb on B is to prevent any of the alkaline fluid bumping over into the distillate. Mix the contents of A by shaking and immediately connect up B with C by means of another well-fitting rubber stopper. Heat the mixture in A to boil- ing by means of, a free flame from a Bunsen burner provided with a rose-top. Allow the fluid to boil tiU at least half the total volume of fluid has distilled over, lowering E from time to time, so Fig- 4°- Kjeldahl appara- that D does not dip too far under the direct boiling, acid. Finally, lower E so that the tube no longer dips imder the surface and continue the boiling for another minute or two to wash down any of the standard acid 326 ANALYSIS OF URINE. [CH. XIII. that may have been sucked up into the tube or bulb. Wash down the exterior of the lower end of D with a jet of distilled water, allowing the washings to run into E. To the fluid thus obtained add a few drops of a 0-02 per cent, solution of methyl red and titrate with standard COg- free sodium hydroxide, which may be between o-i and 0-15 N. Calculation, see pages 323 and 324. 395. Kjeldahl's method (steam distillation). The incinera- tion is conducted as described in the previous exercise, but a smaller Kjeldahl flask may be used if desired. After the fluid Fig. 41. Kjeldahl apparatus. Steam distillation. has cooled, add 50 cc. of distilled water, shake round well and transfer the solution to D, a round-bottom flask of i or 2 litres, with a neck sufficiently wide to carry a well-fitting rubber stopper that will allow 3 tubes to pass through, as shewn in the figure. Wash out the Kjeldahl flask twice more, using about 25 cc. of distilled water each time. Measure the requisite amount of standard acid into K, and assemble the apparatus. Arrange the wooden blocks, L, so that the end of the delivery tube, H, just dips under CH. XIII.] TOtAL NitfeOGfeN. 327 the surface of the acid. (It is incorrectly drawn in the figure.) E is a tap-funnel containing 40 per cent. soda. G is a condenser, the double-surface variety being the most efficient. The water in the copper vessel A has previously been vigorously boiled for at least ten minutes to ensure the removal of any ammonia. Remove the flame for a moment and connect the exit pipe of the boiler to C by rubber tubing. Replace the burner. Run in the strong soda from E until the solution is definitely alkaline, as can be seen by the fluid turning blue, due to the formation of cupric hydroxide. The distillation must be allowed to proceed for at least 45 minutes. It is safer to allow an extra half-hour. The only attention necessary is to see that there is a sufficient amount of water in the boiler and that the flask K is at the right height. At the end of the operation, remove the blocks from under K, so that H does not dip into the acid. After a few minutes, remove the flame, wash down the interior and exterior of H into K. and titrate as described on p. 323. Calculation, see p. 323. 396. Kjeldahl's method (alcohol distillation). Into a 500 cc. Kjeldahl flask of " Duro " glass measure 2 cc. of the urine, using an Ostwald pipette (fig. 51). Add 3 cc. of pure concentrated sulphuric acid, 2 grams, of potassium sulphate and 2 drops of saturated copper sulphate solution. Heat over a micro-burner, using a Fohn's fume-absorber. The flame should be about half an inch in height, and should play directly on the bottom of the flask to ensure boiling. Any particles of carbonaceous matter that form on the side of the flask must be rinsed down into the acid. The heating must be continued for 5 to 10 minutes after the solution hsis turned blue. Remove the flame and allow the solution to. cool until the flask is only pleasantly warm to the hand. Add 20 cc. of distilled water from a measuring cylinder. This should be added rapidly, and the mixture immediately shaken to prevent the formation of a cake of potassium hydrogen sulphate. Cool under the tap. Add three or four pieces of broken porous pot and 15 cc. of 95 per cent, alcohol. Assemble the apparatus, seeing that the clamps are correctly adjusted, so tfcat the rubber stoppers fit into the flasks without undue strain. G is a piece of glass rod, with the lower end flattened 328 ANAtYSIS OF URINE. [CH. XIII. out and bent up as shewn. It is passed up through the rubber stopper, and the upper end can then be flattened out, if desired, for convenience of manipulation. The tube is drawn up until the flange is about i inch from the exit tube B. This minimises the risk of any alkaU being carried over by spurting. This risk, however, has been found to be so small that it is hardly worth the trouble of fitting. B is joined to the tube to the condenser by a short piece of rubber tubing. TMs allows the operator to shake A, and, by removing the strain, de- creases the risk of breaking the glass parts. The standard acid is measured into E, a 250 cc. Erlen- meyer flask. 20 cc. of o-i N. acid is usually ample. The wooden blocks, F, should be so arranged that the lower end of D only just dips below the surface of the add. Now remove A and run in 13 or 14 cc. of 40 per cent, soda, nmning this gently down the lower part of the neck and sides of the flask, so that the soda sinks to the bottom of the fluid and the risk of the loss of ammonia is minimised. The flask should be held in a nearly horizontal position during this operation; it should be raised to tlie vertical caut- iously, so as to prevent mixing of the two layers. Re-assemble the appa- ratus, seeing that the two rubber Have ready a Bunsen burner, with a fitted to the rubber tubing. See Kjeldahl's method. Fig. 42. Cole's apparatus for alcohol distillation. stoppers are firmly held. rose-top, and a screw clip, H, that the adjustable stand for the burner is at such a height that the top of the rose is about half an inch under the bottom of the flask. Turn on the water supply to the condenser. Unclamp the flask A and mix its contents by shaking, taking care that the rubber stoppers CH. Xin.] MICRO-KJELDAHL. 329 are not loosened. Place the burner in position and gently agitate the flask until the fluid commences to boil. The flask can then be clamped and the distillation allowed to continue for 15 minutes. Remove the wooden blocks F, and then remove the flame. Remove the rubber stopper from the upper end of the condenser and wash the latter down into E with a jet of distilled water. Wash down the exterior of D also, add a few drops of methyl red and titrate with the COj- free soda, as described on page 323. The soda can be between 0-05 and o-i N. Calculation, see p. 323. 397. Micro-Kjeldahl (Cole's method). Into a 300 cc. Kjeldahl flask measure 0*5 or i cc. of the urine, using an Ostwald pipette (fig. 51). Add 2 cc. of pure concentrated sulphuric acid 2 drops of saturated copper sulphate, and i gram, of pure potassium sulphate. Clamp the flask over a micro-burner, having a flame about J inch in height, just touching the bottom of the tube, and insert a Folin's fume absorber (fig. 54) into the mouth of the flask. Continue the gentle boiling for at least 5 minutes after the solution has lost all trace of its dark colour and has turned light blue. Allow to cool, add 20 cc. of distilled water and 12 cc. of alcohol, and proceed exactly as described in the last paragraph of Ex. 311. B. The Estimation of Ammonia. The ammonia of urine normally exists as ammonium salts of weak acids. In cases of cystitis, however, the urine is nearly always alkaline, owing to the conversion of some of the urea to ammonium carbonate by various micro- organisms. This change may occur after the urine has been passed owing to bacterial contamination from the air, etc. For this reason it is essential that a little toluol should be added to the vessel in which the urine of the 24 hours is being collected, that it should be kept in a cool place and that the estimations should be made as soon as possible. A great many methods have been proposed for the estimation of ammonia in urine. Folin has introduced them at a rate which is almost alarming. Nearly all his later methods are colorimetric, a most excellent modification of Nessler's solution having been elaborated. But the author's experience with large classes is that the majority of workers prefer to use a titration method if possible. Mainly for that reason, the only three methods described here are FoHn's original macro-method, Van Slyke-s modification of it, and the formol method. The latter, however, gives the sum of ammonia and the amino-acids, and the results obtained by it are only of appropdmate value for the ammonia figure. It will be described in connexion with the estimation of amino-acids. 330 ANALYSIS OF tJRiNE. [cm. XIII. Of the two methods described below, the author now always employs Van Slyke's, which is much more rapid than Folin's. The author is convinced that failures to obtain correct results are either due to inattention to essential details or to the use of an imperfect suction pump. An apparatus that sup- plies air under a good pressure is a most valuable adjunct to a modern bio- chemical laboratory. 398. The estimation 0! ammonia by Folin's method. ■A B 'i>-c Fig. 43. Fohn's apparatus for estimating ammonia. A. Wash bottle containing acid. B. Tall aerometer cylinder containing urine. C. Bottle containing standard acid to absorb ammonia from the air. D. Calcium chloride tube, loosely packed with cotton wool, to prevent any sodium carbonate being carried over into C. E. Folin's absorption tube, to bring the air into intimate contact with the acid. Use the apparatus shewn above.* * The parts of the apparatus can be obtained from Messrs. Baird and Tatlock (London). CH. xiir.] AMMONIA. 331 Into C measure 20 cc. of standard sulphuric acid (about o-i N.) and a few drops of methyl red. Into B measure 25 cc. of urine, add 5 or 6 drops of caprylic alcohol (to prevent foaming) and 2 grams, of anhydrous sodium carbonate. Connect up the apparatus at once, and draw air through for two hours. Disconnect the apparatus, wash the tube E with distilled water into C, and titrate with COg- free sodium hydroxide (about o-i N.). Calculation. Determine the percentage of nitrogen in the form of ammonia as described for Kjeldahl's method, p. 323. The result thus obtained is the mgms. of ammonia-nitrogen in 25 cc. To convert this to grams, of ammonia per cent., multiply by 4 ^ I^ >< £553 = 0-00486 (log. 3-6864). To find the ammonia in terms of cc. of o-i N. acid per cent., multiply the 14 = 2-86 (log. ■456c). mgms. of ammonia-nitrogen in 25 cc. by 4 399. Van Slyke's method. Principle. 5 cc. of urine are made strongly alkaline with potassium carbonate, which decomposes the ammonium salts. The ammonia liberated is driven over by an air current into a measured amount of standard acid, which is subsequently titrated with standard alkali. The treatment of urine at room temperature with potassium carbonate does not lead to the formation of ammonia from urea, etc., as does boiling with caustic soda. Apparatus. This is shewn in fig. 44.* A is a wash bottle containing To pump Fig, 44. Apparatus for estimation of ammonia and urea by Van Slyke's methods. * This can be obtained from Messrs. Baird and Tatlock (Ltd.), 14, Cross Street, Hatton Garden, London, E.C. 332 ANALYSIS OF URINE. [CH. XIII. sulphuric acid (i in lo) to remove ammonia from the air. B is a large thick- walled tube, 25 to 30 mm. by 200 mm. C is a sheet of rubber, about 2 mm. thick, cut from a rubber stopper. It fits loosely into B, and has a small groove cut at the side. It decreases the risk of an alkali foam being carried over into E, which is a tube similar to B. D can be made from ra broken 5 cc. pipette and may be loosely filled with cotton or glass wool. F is a tube sealed at the lower end with holes bored in it, whilst still hot, with a hot needle. The tubes B and E are conveniently held by means of a heavy wooden block bored with two large holes. In place of the tube E, a 100 cc. flask with a wide neck may be substituted as shewn in fig. 45. The advantage of the flask is that there is very little risk of the standard acid being carried over with the brisk air current necessary. The objection to it is that the depth of the acid layer being decreased there may be a danger of loss of ammonia. If the air current is a moderate one for the first two minutes this risk is very slight, and perfect results are obtained. An efficient suction pump or blast pump is also „. required. Fig- 45- Method, (i.) Into B measure 5 cc. of the urine, and add 2 drops of caprylic alcohol to stop foaming. 4 or 5 drops of kerosene can be used, but it is not an efficient substitute. (ii.) Into E measure 20 cc. of the standard sulphuric acid, which should be between 0-04 and 0-07 N, and then add a couple of drops of caprylic alcohol. If a flask is used it is advisable to add about 20 cc. of distilled water to give a deeper layer for absorption. (iii.) Place 4 to 5 grams, of pure dry potassium carbonate into B, roughly measuring it with a suitable spoon. Immediately connect up the apparatus, taking care that D is joined to F, and not to the connexion for the pump. Turn on the water supply to the pump so that a rather slow air current is drawn through. After 2 to 3 minutes, turn on the water to full pressure and leave it for 12 more minutes. (iv.) Gradually stop the pump and disconnect the tube E. Lift up the rubber stopper so that F does not dip into the acid, and wash down the interior of F with distilled water, using a fine jet. Repeat this twice, allowing time for proper drainage. CarefuUy wash down the exterior of F with distilled water, add 3 or 4 drops of methyl red and titrate with COj- free soda, which may be between 0-03 and o-o6 N., according to the directions given on page 323. Calculation. Determine the percentage of nitrogen in the form of ammonia CH. XIII.J AMMONIA AND AMINO-ACIDS. 333 as described for Kjeldahl's method, p. 323. The result thus obtained is the mgms. of ammonia-nitrogen in 5 cc. = A. Mgms. of ammonia-nitrogen in 100 cc. = 20 A. Grams, of ammonia-nitrogen in 100 cc. = A x 0-02. Grams, of ammonia in 100 cc. = A x 0-02 x — = A x 0-0243 (log. 2-3853). cc. of o-i N. acid neutralised by NH3 of 100 cc. = 20 A x — = A x 14-29 (log. I -1549) 400. C. The estimation of ammonia and amino-acids by formol titration (Cole's method). Principle. Neutral ammonium salts react with an excess of neutral formaldehyde to give hexamethylene tetramine, the acid being liberated. 4 NHjCl -I- 6 CHjO = Nj (CHj)^ -1- 6 H2O + 4 HCl. From the amount of alkali required to again make the solution neutral, the amount of ammonia can be estimated. Neutral amino-acids also react with formol to give methylene amino-acids [see p. 69 (3) ]. The result of the estimation therefore gives the sum of the ammonia and the amino-acids of the urine. The method usually adopted is to neutraUse the urine to phenol-phthalein, to add neutralised formol, which makes the fluid acid, and then to determine how much standard soda is again required to neutralise the mixture. The great difficulty encountered is that of determining the neutral point, and experience with large classes of students has revealed the fact that considerable variations in results are found, due to the indecision about the two end points As explained on p. 215, the author has overcome this difficulty by the use of the comparator shewn in fig. 27. The results obtained by untrained students now agree very closely. Method. Use the comparator for large tubes described on p. 276. Into tubes (2), (3) and (6) measure 20 cc. of the urine. Into tube (i) measure 20 cc. of buffer solution Ph = 8-4 (see p. 28). Into tube (5) measure 20 cc. of buffer solution Ph = 8'5.* Into tube (4) place about 30 cc. of water. To tubes (i), (3) and (5) add 10 to 20 drops of 0-5 per cent, phenol phthalein, adding exactly the same amount to each by the use of a dropping pipette (fig. 5). The amount necessary varies * If only one buffer solution is used, as is done in the exercise on p. 215, it should be Ph = 8-45. 334 ANALYSIS OF URINE. [CH. XIII. with the appearance of the urine, more being required for deeply pigmented urines. Titrate with standard soda, which may be o-i to o-2 N., as described in Ex. 322, until the colour as seen through Y is inter- mediate between that seen through X and Z. During the course of this titration, the standard soda is added to (2) and (6) and distilled water to (i) and (5), as described in Ex. 322. Usually a considerable precipitate of earthy phosphates appears in the three tubes that contain urine. The contents of these tubes must be well mixed by rotation or otherwise immediately before an observation is made. Measure 5 cc. of commercial formaldehyde (40 per cent.) into a test-tube. Add one-third the number of drops of phenol phthalein added to the urine and then the standard soda, drop by drop, until a faint pink tinge is obtained. Add the whole of this solution to tube (3). Note that the pink tinge and the precipitate of earthy phosphates disappear, owing to the acidity developed. To tubes (2) and (6) add 5 cc. of water, to dilute the urinary pigment to the same degree as that in tube (3). Read the burette containing the standard soda. Titrate the contents of tube (3) ^vith the soda, xmtil the appear- ance at Y approaches that seen at X. To tubes (i), (2), (5) and (6) add the same volume of distilled water as the soda added in this last operation. Mix the contents carefully and complete the titration, so that the appearance at Y is intermediate between that seen at X and Z. Calculation. If (3) cc. of soda of normality («) are required to neutralise 20 cc. after the addition of the formol, then 20 cc. urine contain (a) x («) x (14) mgms. of Nitrogen of ammonia and amino-acids. So 100 cc. urine contain (a) X («) X (70) mgms. of (ammonia + amino-acid) Nitrogen. This amount, less 20 A (the mgms. ot ammonia-Nitrogen determined in the previous exercise) is the mgms. of amino-acld Nitrogen in 100 cc. urine. D. The Estimation of Urea. The use of the enzyme urease (see p. 287) has rendered obsolete a large number of methods that had been devised for the estimation of urea in urine. The time required for D. Van Slyke's method is not much greater than that for the old hypobromite method, and the results obtained are accurate, whereas with h3rpobromite they are most unreliable. The old hypobromite method has, however, been included again because of its convenience for the estimation of urea in McLean's " Urea Goncentratioii CH. XIII .J UREA. 335 Test " of renal efificiency. Under the circumstances of the test, in which the urea concentration is determined in the urine collected during the second hour iollowing the administration of 15 gms. of urea, the urea forms at least 90 per cent, of the total nitrogen of the urine. The hypobromite method in such cases is sufficiently accurate for clinical work, and since it demands very little equipment it is more convenient than the urease method for the majority of general practitioners. 401. Van Slyke's method for urea. Principle. A small volume of the urine is treated with Soya bean meal together with a certain amount of acid potassium phosphate to preserve the optimum reaction for the enzyme. The whole of the urea is rapidly converted to ammonium carbonate. An excess of potassium carbonate is added, and the ammonia formed from the urea, together with that from the preformed ammonium salts of the urine are driven over into standard acid and estimated in the way described in Ex. 399. The amount of ammonia being known, the percentage of urea can be found by difference. Apparatus. This is exactly similar- to that required for Ex. 399. A duplicate set should be obtained so that the ammonia and urea determinations can be conducted simultaneously. Soya bean meal. It is cheaper and better to use the natural meal than any of the commercial enzyme preparations. It is not even necessary to pre- pare an extract. The amount required is about 0-3 gm., which can be approxi- mately measured by means of a small spatula, tube or spoon after a few trials. It is important to use a fine meal.* It will be found that the natural beans are rather difficult to grind finely in a coffee mill. Some samples of meal yield a trace of ammonia, when treated with potassium carbonate, but in the author's experience this is usually so small as to be outside the error of experiment and can be neglected. Method. See that the tube B (fig. 44) and the narrow tube that goes into it have been well washed, and are quite free from any of the alkaline carbonate used in a previous experiment. (i.) Measure 0-5 cc. of the urine into B, using an accurate Ostwald pipette (fig. 51). If the urine is known to be a very dilute one, I or even 2 cc. can be taken. (ii.) Add 2 cc. of the acid potassium phosphate, and 3 cc. of water, washing the traces of urine down to the bottom of the tube with these two fluids. Then add 0-3 to 0-4 gm. of the Soya bean meal. Lightly shake to mix. (iii.) Add 2 drops of caprylic alcohol, to prevent subsequent foaming. (iv.) Fit the rubber stopper with the tubes it carries. * Soya bean meal can be obtained from Messrs. Baird and Tatlock, London. 336 ANALYSIS OF URINE. [CH. XIII. (v.) Into E (or the flask shewn in fig. 45) measure 20 cc. of the standard sulphuric acid add 2 drops of caprylic alcohol, fit the stopper and connect up E to the pump and B to the wash bottle A. (vi.) Immerse the tube B in a beaker or can of water at a temperature of about 45° C, and leave it for 12 minutes, a slow current of air should be drawn through the apparatus to ensure a thorough mixing of the contents and extraction of the enz3mie from the meal. (vii.) Remove the tube from the bath and send a strong air current through the apparatus for i minute to sweep over any ammonia that may have escaped from the fluid and be present in the air of B. (viii.) Stop the air current and disconnect the entry and exit tubes of B. Remove the stopper with these tubes and place it on the bench in such a way that the small amount of fluid on the end of the entry tube is not lost. Cool the tube B. in a stream of cold water. (ix.) Add 4 to 5 grams, of pure dry potassium carbonate to B, roughly measuring it with a suitable spoon, immediately replace the stopper and connect up the apparatus. Send a slow air current through for 2 minutes and then a rapid current for 12 minutes. (x.) Proceed as described in Ex. 399 (iv.). Calculation. Determine the percentage of nitrogen in the form of ammonia and urea as described for Kjeldahl's method, p. 323. The result thus obtained is the mgms. of (urea + ammonia) N. in 0-5 cc. urine = Ua. So mgms. of (urea + ammonia) N. in 100 cc. = 200 x Ua. From Ex. 399, the mgms. of ammonia N. in 100 cc. = 20 A. So mgms. of urea N. in 100 cc. = (200 x Ua) - 20 A = U. Since 60 grams, of urea contain 28 grams, of nitrogen, the grams, of urea in 60 I 100 cc. urine = U x -g x ^3^= U x 000214 (log- 3-33io)- 401A. The estimation of urea by the hypobromite method. Principle. Urine is treated with an alkahne solution of sodium hypobromite and the amount of urea calculated from the volume of nitrogen evolved. The reaction that takes place is as follows : — CO(NH2)2+3NaBrO+2 NaOH=Na+3 NaBr+NajiC03+3 HjO. Hence 60 grams urea evolve 28 grams N.=2X 11-2 htres. Therefore I gram urea evolves 373 cc. Nitrogen, measured at N.T.P. CH. XIII.] UREA. 337 Practically it is found that only 357 cc. are evolved, the other 4'4 per cent, of the nitrogen being converted into nitrates, cyanates, etc. Apparatus. See fig. 46. A 50 cc. burette [a) is held by a clamp in a tall cylinder of water (6) . The upper end of the burette is closed by a tightly- fitting rubber stopper, which is pierced by one Umb of a glass T-piece. The upper limb of the T-piece is fitted with a short length of pressure-tubing carrying a screw- clamp (e) . The side limb of the T-piece is connected by about two feet of small rubber tubing to a glass tube piercing the well-fitting rubber stopper of a wide- mouthed bottle (c) of about 60 cc. capa- city. This bottle is placed in a jar of water, supported at such a height that the burette can be Ufted nearly out of the tall cyhnder without stretching the rub- ber connexion. A small glass bottle or short tube of 10 to 15 cc. capacity is also required [d). (For the method of pre- paring the hypobromite solution see P- 395-) Method of Analysis. Place 25 cc. of freshly-prepared hypobromite solution in (c). Put 4 cc. of urine, accurately measured, in the small bottle [d), and place this inside the other by means of a pair of forceps, taking great care not to upset any urine into the hypobromite. Fit the rubber |^cork tightly into the bottle and place this in the jar of water to cool. See that the burette is as low as possible, that the cylinder has sufficient water in it to reach the zero graduation of the burette, and that the screw clamp is open. Leave the apparatus to cool to the temperature of the water ; clamp the burette in such a position that the water is below the zero mark, and then screw the clamp on the rubber tubing as tight as possible. Note down the level of the water in the burette, keeping the eye level with the meniscus. Take the bottle out of the jar, and gently tilt it so that the urine flows into the hypobromite. Fig. 46. Apparatus for deter- mination of urea by hypo- bromite method. 338 ANALYSIS OF UEINE. [CH. XIII. Gently shake the bottle from side to side, keeping the bottle upright to prevent the froth from being forced up into the tube. Tilt the bottle again and repeat the process till the urine and hypobromite are thoroughly mixed. Place the bottle back in the jar of water for about 3 minutes to cool. Raise the burette tiU the level of water in the tube is the same as that outside, the gas being thus under atmospheric pressure. Read the level of the meniscus as before : the difference in the two readings is the volume of nitrogen evolved. Ascertain the temperature of the water and the barometric pressure. Calculation of results. Let the temperature he t° C, the tension of aqueous vapour at this temperature be T mm. (see Appendix), and the barometric pressure be B mm. of mercury. Let v be the volume of nitrogen measured under the conditions : at 0° C. and 760 mm. this wiU become V X 273 X (B— T) (273 + i!) X 760 Now 357 cc. of N are evolved from i gram of urea. .•. v' cc. are evolved from — gram of urea. 357 .". 4 cc. unne contam — gram urea. 357 and 100 cc. urine contain -=— gram urea. 357 Note.— Performing these two calculations in one operation we obtain for the percentage of urea V X {B - T 273 X 25 V X {B - T) (^3T"0" "^ 760 X 357 = "(^3T7r "" '°^^'^ <'°S- ^■4°°^>- E. Creatinine and Creatine. The methods that are universally adopted are based on Jaffe's test for creatinine (Ex. 227) as applied by Folin for colorimetric estimation. Creatine does not reduce picric acid, but is converted to creatinine either by heating with hydrochloric or picric acid. The combined (creatinine + creatine) is then estimated colorimetrically. Folin and Doisy* have recently pointed out that considerable errors may occur if impure picric acid is used, especially in * Folin and Doisy, Journ. Biol. Chem., xxviii., p. 349. CH. Xni.] CREATININE. 339' the case of the estimation of creatine. They give a method of purifying the very doubtful specimens of wet picric acid that are at present on the market. A much less troublesome method is given on p. 251, but for observations on the excretion of creatine in pathological conditions it would be safer to follow FoMn. Graham and Poulton* have shewn that the presence of aceto-acetic acid in the firine inhibits the reaction with creatinine, so that the estimations are too low. This acid is destroyed by the heating required for the estimation of (creatinine + creatine), so that the result of the analysis always makes it appear as if creatine were present. They give a method for the removal of aceto-acetic acid, which must be followed when the urine gives a distinct Rothera's test. They are unable to confirm the statement that creatine is found in the urine as a result of carbohydrate starvation. It only appears to be present if faulty analytical procedures are adopted, aceto-acetic acid always being found in carbohydrate starvation. 402. The estimation of creatinine (Folin).t Principle. A measured amount of the urine is treated with picric acid and caustic soda. The picric acid is reduced to picramic acid in the cold by the creatinine present, glucose having no effect in the cold (see Exs. 108 and 227). A known amount of creatinine is similarly treated and the solutions compared in a colorimeter. Solutions and apparatus required. 1. A colorimeter, see p. 384. 2. Standard solution of creatinine zinc chloride, i '6106 gram, of the pure recrystallised zinc compound (see p. 300) is dissolved in aboTit 500 cc. of distilled water and 100 cc. of Normal hydrochloric acid and the volume made up to I litre with distilled water, i cc. contains i mgm. of creatinine. The solution is quite stable. 3. A saturated aqueous solution oipure picric acid (about 1-2 per cent.) and a 20 cc. pipette for measuring it. 4. 10 per cent, caustic soda, which can be measured by a pipette or burette. 5. Ostwald pipettes (fig. 51) of i cc. 6. Two 100 cc. measuring flasks. Method. Into a loo cc. measuring flask (labelled " U ") measure i cc. of the urine by means of an Ostwald pipette. Add 20 cc. of the picric acid and then 1-5 cc. of the soda. Allow the mixture to stand for 10 minutes with gentle agitation. As soon as the mixture has been made measure i cc. of the standard creatinine solution into the other lOO cc. flask (labelled " S "), add the picric * Graham and Poulton, Proc, Roy. Soc, Ixxxvii., B., p. 205. f Journ. Piol. CAew., Jfvi}., p. 470, 340 ANALYSIS OF URINE. [CH. XIII. acid and soda as before and mix, noting the time. After the flasks have each stood for lo minutes they are separately filled to the mark with distilled water and the contents well mixed. The solutions are then compared in a colorimeter (see p. 388J, the standard being set at 15 mm. Should " U " read below 10 mm., the determination must be repeated, using i cc. of an accurately diluted urine, say i in 2 or I in 3. Should " U " read above 22 mm. the determination must be repeated with 2 cc. or more of the urine. In such cases there is no necessity to make another standard, the colour being quite permanent for hours. Calculation. Mg. in I cc. urine _ Reading of " S " _ 15 Mg. in I cc. standard ~ Reading of " U " ~ Reading of " U " 15 Mg. in I cc. = -^ — t: c ,, -.. „ = Cn. ° Reading of U If more or less than i cc. of urine have been taken, this must be divided by the volume of urine used. Grams, of creatinine in loo cc. = Cn x o-i. Since 113 grams, of creatinine contain 42 grams, of nitrogen, grams, of 42 - creatimne- N m 100 cc. = Cn x o-i x — — = Cn x -0371 (log. 2-5701). 403. The estimation of creatine and creatinine (Folin). (i.) Weigh a 200 cc. Erlenmeyer flask of " Duro " glass. (ii.) Into it measure the amount of urine that contains between 07 and 1-5 mgm. of creatinine, as determined by the previous exercise. (iii.) Add 20 cc. of saturated picric acid and about 130 cc. of water and a few pieces of broken porcelain. (iv.) Boil gently over a micro-burner for i hour. (v.) Increase the heat and boil down to rather less than 20 cc. (vi.) Weigh the flask and add water, if necessary, to make the total weight of the contents equal to 20-25 grams. (vii.) Cool in running water. CH. Xm.j CKfiAtlNfi. 34f (viii.) Add 1-5 cc. of 10 per cent, soda front a burette and allow the mixture to stand for 10 minutes with gentle agitation. (ix.) Transfer to a 100 cc. volumetric flask and wash out with distilled water to make 100 cc. (x.) Estimate colorimetrically as in the previous exercise. Calculation. This is the same as in the previous exercise, proper allow- ance being made for the volume of urine used. The diflference between the two results is the creatine, which is usually expressed in terms of creatinine. To convert this to creatine it should be multiplied by — = 1-141 (log. 0575). 404. The estimation of creatine and creatinine (Benedict).* Principle. The dehydration of creatine to creatinine is very rapidly effected by evaporation to dryness with hydrochloric acid. A little lead is added to inhibit pigment formation, the traces of hydrogen evolved preventing oxidation. It is not applicable to urines containing glucose. Method. Into a small beaker measure that volume of urine that contains 7 to 10 mgm. of creatinine. Add 10 fc 20 cc. of N. hydrochloric acid and a pinch or two of powdered or granulated lead. Boil down over a small free flame till nearly dry and then evaporate to complete dryness on a boiling water bath. Add 10 cc. of hot dis- tilled water and filter through a small plug of cotton wool into a narrow 25 cc. measuring cylinder. Wash out quantitatively with two successive portions of about 4 cc. of hot water. Cool by immersion in cold water and make the volume up to 20 cc. Measure 2 cc. into a 100 cc. volumetric flask, using an Ostwald pipette. Add 20 cc. of saturated picric acid and 1-5 cc. of a 10 per cent, solution of soda that contains 5 per cent, of Rochelle salt (to prevent the formation of a cloud due to traces of dissolved lead) . After standing for 10 minutes with gentle agitation, make up to the mark with distilled water. A standard is simultaneously prepared from i cc. of the standard creatinine solution, 20 cc. of picric acid and 1-5 cc. of the 10 per cent, soda containing 5 per cent, of Rochelle salt, diluted to 100 cc. after standing for 10 minutes. The two solutions are read as described in Ex. 402. * Journ. Biol. Chem., xviii., p. 191. 342 ANALYSIS OF URINE. [CH. XIII. Calculation. If (a) cc. of urine have been taken originally, the amount actually used corresponds to '^. If the standard is set at 15 mm., and the 10 " U " tube reads at U, then mg. of (creatine + creatinine) in I cc. _ ., u" X (a)- The test of the calculation is the same as that of the previous exercise. Note. — The above method is a slight modification of that published by Benedict, btit it does not differ in any essential. 405. The removal of aceto-acetic acid (Graham and Poulton). Principle. Aceto-acetic acid is converted by heat to acetone, which is distiUed off at low pressure. The following account is slightly modified from the original. Solutions and apparatus required. (i.) A 10 per cent, solution of phosphoric acid. (ii.) A solution of soda of such a strength that i cc. of it neutralises i cc. of the phosphoric acid, phenol phthalein being used as the indicator. A 15 per cent, solution of soda is a convenient starting point for the preparation of this. When it is correctly adjusted, i'5 cc. of it wiU neutraUse all three valencies of 1 cc. of the phosphoric acid, only two of w^h are neutralised to phenol phthalein. (iii.) A suction pump and gauge (see fig. 9, p. 74). (iv.) A thick walled tube (25 to 30 mm. by 200 mm.) similar to the tube E of fig. 44, but in place of F is substituted a tulDe drawn out to a fine capillary, which must reach nearly to the bottom of E. (The upper end of the tube may be fitt' d with a piece of pressure tubing and a screw chp similar to that shown in C of fig. 8.) Method. Into the tube measure 10 cc. of the urine and add i cc. of the 10 per cent, phosphoric acid. Fit the stopper carrying the capillary tube and connect the other outlet tube to the tube E of the apparatus shewn in fig. 9 and have C turned to make connexion with A. Turn on the pump and note the pressure obtained, which depends on the size of the capillary, on the size of A and on the water pressure. It is necessary to maintain a pressure of about' 210 mm. of mercury. Immerse the tube containing the acidified urine in a water bath kept between 65° and 70° C, and leave it for about three-quarters of an hour, seeing that the temperature does not rise above 70° C, nor the pressure fall below 210 mm. of mercury. Release the pressure by turning the tap C to connect with B, and then turn off the water. Disconnect and cool the solution under the tap. Add I '5 cc. of the standardised soda to completely neutralise CH. XIII.] URIC ACID. 343 the phosphoric acid and then transfer to a narrow 25 cc. cylinder. Wash out with water to make a total volume of 20 cc. Mix well and estimate the creatinine by the method given in Ex. 402. 2 cc. of the solution correspond to i cc. of the urine. P. Uric Acid. Most of the methods employed at present are based on one of two main principles. The first is on Hopkins' ammonium chloride method ; the other is colorimetric with Folin's reagent. The author's experience with all modifi- cations of the latter has been so unfavourable that it has been reluctantly abandoned. It is possible that the difficulty of obtaining rehable chemicals accounts for many of the troubles, but the greatest care in this respect has not been rewarded with success. Hopkins' is the standard method. It requires skill and practice to get good results, but it is absolutely rehable. The modification of it introduced by Fohn and Schaffer is a concession to the unskilful manipulator, but it has the disadvantage of an allowance of 3 mgms. for the ammonium urate not precipitated by ammonium sulphate. The author humbly suggests that this is an averaging of results, for comparisons with Hopkins' original method and also with the modification described below, seem to indicate that with certain specimens of urine the allowance should be smaller or greater than this. It is always the same for a given specimen of urine, suggesting that some unknown factor affects the solubility of ammonium urate under the conditions of the experiment. It is an objection to Hopkins' method that the result cannot be obtained rapidly, as the solution must be allowed to stand over-night for the whole of the uric acid to crystallise out. This inconvenience is also a feature of the Folin-Schaffer modification. Many attempts have been made to titrate the original precipitate of ammonium urate, but they have not been very suc- cessful owing to the difficulty of removing the chlorides which also titrate with the permanganate in acid solution. It is generally stated that the addition of manganese sulphate prevents the action of the chlorides. By accidentally using magnesium sulphate on one occasion the author was led to investigate carefully the extent to which the presence of chlorides interfere with a correct result. It was found that moderate concentrations have no effect, owing to the great rate at which uric acid is oxidised compared to the low velocity of the reaction between chlorides and permanganate. As a result of a con- siderable amount of work the modification described below was elaborated. The final result can be obtained in ij hours, and in the hands of the author agrees to i mgm. per 100 cc. with that of Hopkins' original method. It has been regularly used in class work for the past 4 years, and presents little difficulty to the average student. But it must be admitted that it has not been tested with a large number of pathological urines, and for that reason it is possible that in certain cases it will only yield approximate results. There is no a priari reason why it should fail more than other methods. 406. Uric acid (Cole's modification of Hopkins' method). Principle. The urine is treated with colloidal iron to remove an unknown substance that is precipitated by ammonium chloride. The filtrate is treated with solid ammonium chloride and made strongly alkaUne with ammonia. 3^4 ANALYSIS OF URINE. [CH. XIII. The uric acid is rapidly and quantitatively precipitated as ammonium urate. This is filtered off, washed with ammoniUm sulphate to remove the greater part of the chlorides, dissolved in hot sulphuric acid and titrated with standard potassium permanganate. The end point is reached when a momentary pink flush is seen over the whole body of the fluid. This marks a stage when the rate of oxidation of the uric acid suddenly decreases. Up to this point i cc. of 0-05 N. permanganate is found empirically to correspond to 3-7 mg. of uric acid. The chemical changes involved in the oxidation have not yet been determined. Solutions and reagents required. (i.) Colloidal (dialysed) iron, o-6 per cent. (ii.) Pure, dry, recrystaUised ammonium chloride. (iii.) Washing fluid. 100 grams, of pure ammonium sulphate are dissolved in about 800 cc. of distilled water, 10 cc. of strong ammonia are added and the volume made up to i litre with water. It is convenient to use this from a wash bottle with a fine jet. (iv.) 45percent. sulphuric acid (by volume). To 500 cc. of distilled water in a large flask cautiously add 450 cc. of pure concentrated sulphuric acid, cooling at intervals. Cool thoroughly under the tap and make up the volume to 1,000 cc. (v.) 0-05 N. potassium permanganate. Dissolve i -58 gram, of the pure salt in distilled water and make the volume up to 1,000 cc. Care must be taken to see that the whole of the soHd has dissolved before the solution is used. It can be titrated against pure ammonium oxalate as described on p. 132. If Ox. be the weight of the oxalate taken in grams, (about 0-15) and P the volume of permanganate Ox. required, then the normality is -js = Pn. ^ ^ P X 0-07I05 I cc. of 0-05 N. permanganate = 3-7 mgms. uric acid. ^ * 7 X Pn I cc. of PnN. permanganate ss — — — — mgms. Method. Measure 1 50 cc. of the urine into a 200 cc. beaker, marking the 100 cc. level by means of a label. Add 30 cc. of the coUoidal iron, stirring well during the addition. Filter through two dry papers into two dry flasks (two being used to save time) . When at least 100 cc. of the filtrate has been collected, carefully measure the amount taken and transfer it to the marked beaker, which has been previously washed and drained. It is convenient to take 100 cc. but with dilute urines it is better to take 120 or 150 cc. For every 10 cc. of the filtrate taken weigh out 2 grams, of the soUd ammonium chloride ; add this to the beaker and stir well. When it has dis- solved add 3 cc. of concentrated ammonia and stir again. Stir at intervals for 20 minutes, then remove the rod and let it rest on the lim of the beaker. When the bulk of the precipitate has settled CH. XIII.] URIC ACID. 345 the urate is filtered off either through a plain paper, or more rapidly by moderate suction through a paper and paper pulp supported on a perforated plate (20 to 25 mm. diameter) resting in a funnel, as shewn in the accompanying figure. The filter is prepared by cutting a piece of filter paper rather larger than the disc, placing this in position, moistening it and applying suction by a pump. Any creases round the edge are flattened out by the point of a pencil. The pressure being released a little paper pulp is poured on to the disc and allowed to settle and then gentle suction applied. The pulp will completely seal the cracks between the disc and the funnel. It is advisable to cut another circle of paper, smaller than the first, and to place it on the centre of the pulp. It prevents the latter being washed away during filtra- tion. Filter the supernatant fluid first, taking care not to disturb the bulk of the precipitate. Do not use too high a pressure, as this drives the amorphous ammonium urate into a cake which renders the subsequent washing very slow. The pressure can be regulated by use of the apparatus shewn on page 74. If this is not available, a T-piece can be used, as shewn in fig. 47. One limb of this is connected to the pump and the other is fitted with a piece of pressure tubing and a spring clip, by means of which the pressure can be instantaneously released. When the main mass of the fluid has passed through, transfer the bulk of the precipitate, but do not suck quite dry. Wash out the precipitate remaining in the beaker with the ammonium sulphate solution on to the filter and start suction again. Do this twice more, finally sucking the precipitate dry. The object of the washing is to remove as much ammonium chloride as possible from the precipitate, paper Fig- 47- 346 ANALYSIS OF URINE. [CH. XIII. and beaker. Now transfer the paper, precipitate and disc to the marked beaker, by means of a glass rod which has a fine curved point. Remove the funnel from the filtering flask and wash it down into the beaker with a jet of hot water. Also wash the pointed glass rod. Add hot water to make a total volume of loo cc, as indicated by the label. Add 20 cc. of the 45 per cent, sulphuric acid and stir with a thermometer. The whole of the precipitate must be dis- solved, if necessary by the aid of heat, before the titration is com- menced. Cool or heat to 65° C. Titrate with the standard perman- ganate, reading the meniscus by the aid of a hghted match held behind the burette. The permanganate must be added rather slowly, with constant stirring. The end point is reached when the addition of a couple of drops causes a faint pink flush through the whole body of the fluid. A very considerable addition has to be made for the pink to be permanent, but the empirical valuation of the permanganate is based on the end point described above. Calculation. Since 150 cc. of the urine were treated with 30 cc. (one- fifth volume) of colloidal iron, 6 cc. of the filtrate from this correspond to 5 cc. of urine. If « cc. of the filtrate have been taken and p cc. of 0-05 N. permanganate used, then « cc. contain p x 3-7 mgms. uric acid, and 100 cc. of urine contain 6 100 I * ^ X 3-7 X -X — X-J333 gram. = - >< 0-444 gram. Note. — Another method of filtering ofi the urate is to use a Hirsch funnel with a fixed perforated plate, and to use asbestos instead of paper pulp. The filtering mat is prepared in the way described for a Gooch crucible (_see p. 393), the drying being omitted. The mat with the layer of urates can be very neatly removed, and the whole process is much facihtated. The time required for the estimation is considerably overstated above. The whole process can be accompUshed in 40 minutes, it being unnecessary to allow the fluid to stand for more than 10 minutes after the addition of the ammonia. G. Glucose. The estimation of sugar in urine when there is a considerable amount present can be carried out by any of the standard methods, as, owing to the dilution necessary or the small amount required, there is little interference by the normal urinary constituents. If the concentration is less than o-8 per cent, the author prefers to use the Wood-Ost method (p. 131), and to dilute the urine at least three times with water. Though the amount of permanga- nate required is small, the results are quite satisfactory. With Benedict's method of direct titration (p. 127) the end point is apt to be very uncertain with low concentrations of sugar. The polarimetric method referred to on CH. XIII.] GLUCOSE. 347 page 127 is of great service when a large number of diabetic urines have to be examined. The results may be rather low owing to the presence of the laevo- rotatory /3-oxy-butyric acid. The method described below is the only one at present available for the estimation of such small amounts of glucose as are present in normal urine. It is included in the hope that the study of slight variations from the normal will extend our knowledge of the pathology of diabetes, and also as a practical method for the detection of lowered tolerance to carbohydrates. 407. The estimation of glucose in normal urine (Benedict and Osterberg).* Principle. The urine is treated with mercuric nitrate and neutralised with sodium bicarbonate. The creatinine, urates, etc., are thus removed. The mercury is removed by means of zinc and the sugar estimated by the colorirrietric method with picric acid. Solutions and apparatus required. '\ 1. Picric-picrate mixture, see p. 251. -.£. Standard solution of glucose. The stock solution is described on p. 251. 5 cc. of this are diluted to make 50 cc. with distilled water, i cc. contains i mg. glucose. An alternative standard can be prepared from pure picramic acid. The stock solution is described on p. 251. 105 cc. of this are treated with 0-5 cc. of 20 per cent, sodium carbonate and 15 cc. of the picric-picrate mixture and diluted to make 300 cc. with distilled water. The colour obtained corresponds to that of I mg. glucose in 4 cc. of water, treated as described for the final urine filtrates and the coloured solution diluted to 25 cc. It is the most convenient standard to use, as time is saved and a possible error of measurement avoided. 3. Sodium carbonate solution, 20 per cent., see p. 252. 4. Test-tubes graduated at 12-5 and 25 cc, see p. 252. 5. Ostwald pipettes, see p. 385. 6. A colorimeter, see p. 388. 7. Mercuric nitrate solution, see A solution, p. 395. On no account must the B solution be used as a substitute. Method,. Into a 50 cc. beaker measure 20 cc. of the urine and then 20 cc. of the mercuric nitrate solution. Mix and add soUd sodium bicarbonate with gentle shaldng. Considerable frothing occurs. The bicarbonate can be added fairly freely until this ceases. Stir well and see that the material on the sides of the beaker is mixed with the main mass, which forms a kind of paste.' Now add the bicarbonate until the fluid reacts just alkaline to litmus paper. Filter at once through a dry paper into a small dry * Journ. Biol. Chem., xxxiv., p. 195. + These can be obtained from Messrs. Baird and Tatlock, London. 348 ANALYSIS OF URINE. [CH. XIII. flask. The filtrate should be quite clear and colourless. Add a pinch of zinc dust and 2 drops of concentrated hydrochloric acid, shake and allow it to stand for 5 to 10 minutes. Filter through a small dry filter into a dry test-tube. Measure i to 4 cc. of this filtrate (so that 0-5 to 2 mg. sugar are taken), into one of the graduated tubes. If less than 4 cc. are taken make the volume up to exactly 4 cc. with distilled water. Add i cc. of the 20 per cent, sodiimi carbonate and 4 cc. of the picric-picrate mixture and plug the tube with cotton wool. If the standard is pre- pared from glucose, measure i cc. (i.e. 1 mg.) into another graduated tube (marked " S "). Add 3 cc. of water, i cc. of the sodium carbonate, 4 cc. of the picric-picrate mixture and plug with cotton wool. Immerse both tubes in a boiling water bath and note the time. After exactly 10 minutes remove the tubes and cool thoroughly rmder the tap. Dilute the " S " tube to 25 cc. If picramic acid is used as a standard, fill a tube with some of the dilute solution. Dilute the other tube to i2"5 cc. or to 25 cc. depending on the colour obtained. If on diluting to 25 cc. the colour is still much darker than the standard, the experiment must be repeated, using less of the final filtrate from the zinc or diluting the urine and starting again from the beginning. When the two colours are roughly the same, compare with the standard in a colorimeter (see p. 388), setting the standard at a height of 15 mm. It may be necessary to filter the solution contain- ing the urine from a slight precipitate that appears on heating with the alkali and picrate. Calculation. This depends on the amount of final filtrate taken and on the dilution. Suppose that 2 cc. of final filtrate were taken and it was diluted to I2-5 CO., and that the reading was 17-4 mm., against the standard at 15. Now 2 cc. of the filtrate contain i cc. of urine, and the standard contains i mg. of glucose (or corresponds to this if picramic acid be used). Since the urinary solution was only diluted to 12-5 cc, whilst the standard was made up to 25 cc, the result must be halved. So In general mg. of glucose in i cc. = mg. of glucose m i cc. 15 i I - 174 " 2- Reading of " S " Volume after heating '='=• ~ Reading of " U " " Volume filtrate used ** 2 25 15 12-5 2 = 17-4 "" 2 '' 25- CH. XIII.] ACETONE BODIES. 349 Note. — The above method gives the total of glucose and non-fermentable carbohydrate. Benedict and Osterberg give the following for the determina- tion of the latter. To 25 cc. of urine (free from preservative) in a cylinder or test-tube add 20 to 25 mg. of glucose and about one-quarter cake of yeast. Mix well and allow to stand in the incubator at 35-38° C. for 18 to 20 hours. Decant 15 to 20 cc. of the urine and determine sugar as before fermentation. The difference between the two gives the fermentable sugar. H. The Acetone Bodies. A very large number of methods have been proposed, and in this case the latest is undoubtedly the best, since Van Slyke's method gives the ;8-oxy- butyric acid as well as the acetone and aceto-acetic acid. Since the ;8-oxy- butyric acid usually forms about 75 per cent, of the total acetone bodies ex- creted, its estimation is of the utmost importance in all studies related to the origin and excretion of these substances. The method given in Ex. 411 is that used by the author for the determination of acetone and aceto-acetic acid when a large number of cases have to be studied. It is the most rapid method of obtaining a good indication of the severity of the ketosis. 408. Total acetone bodies (D. Van Slyke).* Principle. The urine is treated with copper sulphate and lime to remove sugar and other interfering substances. The filtrate is boUed with mercuric sulphate and sulphuric acid under an inverted condenser. Potassium dichro- mate is run in down the condenser to oxidise the oxy-butyric acid to acetone, whilst the aceto-acetic acid is very rapidly converted to acetone by the influence of the hot acid. The acetone forms an insoluble compound with mercury which is filtered ofE and weighed in a Gooch crucible. The precipitate can be titrated if desired by a method described in the original paper. Solutions required. 1. Copper sulphate^. 200 grams, of the pure crystalline salt are dissolved in water and made up to i litre. 2. Mercuric sulphate solution. 73 grams, of pure red mercuric oxide are dissolved in i litre of 4 N. sulphuric acid. 3. Sulphuric acid. To 500 cc. of distilled water in a large flask cautiously add 500 cc. of concentrated sulphuric acid. Cool thoroughly under the tap and make up to i litre with distilled water. Titrate a portion of 2 cc. with N. soda (or titrate 5 cc. with 5 N. soda) and adjust to 17 N. if necessary. 4. Calcium hydroxide suspension. Mix 100 grams, of pure " light " calcium hydroxide with 1 Utre of distilled water. 5. Potassium dichromate. Dissolve 50 grains, in water and make up to I litre. Method, (i.) Measure 25 cc. of the urine into a 250 cc. volumetric flask. Add 100 cc. of distilled water, 50 cc. of the copper sulphate solution and mix. Then add 50 cc. of the calcium hydroxide suspension (previously well shaken) and shake well. Test the * Journ. Biol. Chem., xxxii., p.'455. 350 ANALYSIS OF URINE. [CH. XIII. reaction with litmus. If not alkaline add more of the calciimi hydroxide. Dilute to the mark and allow it to stand for at least 30 minutes. Filter through a dry folded paper into a dry flask. This will remove up to 8 per cent, of glucose. If more than this is present, the urine must be diluted to bring it down to 8 per cent. If glucose is present in the filtrate a yellow (not white) precipitate will appear if a little of it is boiled in a test-tube. (ii.) Connect up a 500 cc. Erlenmeyer flask with a straight reflux condenser, as shewn on p. 72. Into the flask measure 25 cc. of the urine filtrate, 100 cc. of water, 10 cc. of the 17 N. sulphuric acid and 35 cc. of the mercuric sulphate solution. Connect up to the condenser and heat to boiUng over a free flame (not over a sand bath) . When boiUng has begun add 5 cc. of the dichromate solution, nmning this down the condenser tube. Allow the mixture to boil gently for ij hours. (iii.) Cool the solution and filter off the precipitate, using a weighed Gooch crucible (see notes to Ex. 410). Wash out the flask with cold water, of which about 200 cc. in all should be used. Dry the crucible in an hot-air oven at 110° C. for an hour. Allow the crucible to cool down to air temperature and weigh again. Calculation. If 25 cc. of the filtrate (representing 2-5 cc. of the urine) are used, I gram, of precipitate corresponds to 2-48 grams, of total acetone bodies per 100 cc. in terms of acetone. This is on the assumption that the molecular proportion of the acetone bodies in the form of /3-oxy-butyric acid is 75 per cent., the usual figure. 409. ^-oxy-butyric acid. The acetone and aceto-acetic acid are first boiled off and then the estimation conducted as before. Measure 25 cc. of the filtrate into the open flask, add 100 cc. of water and 2 cc. of the sulphuric acid. Boil gently for 10 minutes with a free flame. Cool and transfer to a measuring cyhnder and note the volume. Return it to the flask and add water to the cyhnder to make a total volume of 127 cc. Add 8 cc. of the sulphuric acid and 35 cc. of the mercuric sulphate. Connect up to the condenser and boil. When boiling add 5 cc. of the dichromate and allow the mixture to boil gently for i \ hours. Then proceed as in Ex. 408 (iii) . Calculation. 1 gram, of precipitate corresponds to 2-64 grams, of /3-oxy- butyric acid per 100 cc, reckoned as acetone : to convert to ;8-oxy-butyric 104 acid multiply by — g = i'793. CH. XIII.] ACETONE BODIES. 351 410. Acetone and aceto-acetic acid. This can be found by difference between the two previous exercises, or it can be determined separately by the method adopted for total acetone bodies, except that (i) no dichromate is added, and (2) the boiling is continued for not less than 30 nor more than 45 minutes. Calculation, i gram, of precipitate corresponds to 2-0 grams, of acetone and aceto-acetic acid per cent., reckoned as acetone. Notes. — i. Van Slyke also gives a method of titrating the mercury- precipitate. It is presumably quicker, since it is not necessary to prepare and dry the Gooch crucible. The author has no experience of it. 2. The Gooch crucible should be of 25 to 50 cc. capacity, and fitted as shewn in fig. 48. The asbestos mat can be prepared as described on page 393. It is thoroughly washed and firmly sucked down and dried in a steam oven or a hot- air oven at 1 10° C. It is allowed to cool in the air and weighed. It is then fitted to the rubber cup and some distilled water carefully added and sucked gently through before the mercury precipitate is filtered off. Several precipitates can be collected and weighed one after another. (See p. 393.) 3. The reagents should be tested by performing an experiment with distilled water instead of urine, starting with the copper sulphate treatment. No precipitate whatever should be obtained. Van Slyke gives a caution that this test should not be omitted. 4. A blank determination of precipitate from other substances in urine other than the acetone bodies may be made by following the procedure of Ex. 409, except that 5 cc. of water are substituted for the dichromate and the boiUng period under the condenser is strictly limited to 45 minutes. The weight of precipitate ob- tained is deducted from that found in any estimation of the acetone bodies. It is usually so small that it can be neglected, except in cases where only small amounts of the acetone bodies are present. Fig. 48. Gooch crucible and fil- tering apparatus. 411. The estimation of acetone and aceto-acetic acid by Messinger's method (modified). Principle. The urine is acidified and distilled into a freshly prepared solution of alkaline hypo-iodite, made by adding strong soda to a known volume of standard iodine. The vapours pass through boiling soda which removes any volatile acids that might react with the hypo-iodite. The pre- formed acetone of the urine and that arising from the decomposition of aceto- acetic acid pass over and react with the hypo-iodite to form iodoform. The alkahne solution is then acidified with hydrochloric acid, which hberates any iodine that has not reacted to form iodoform. This excess of iodine is titrated with standard thiosulphate in the usual way. The amount of iodine originally taken being known, and the titration giving the amount that has not reacted with the acetone, the difference is a measure of the amount of acetone distilling over. 352 ANALYSIS OF URINE. [CH. XIII. The following equations indicate the various reactions that take place : (1) 1^+2 NaOH=NaOI + NaI+HjO Hypo-iodite. (2) CH3.CO.CH3+3 Na OI=CHs.CO.Cl3+3 Na OH. Acetone. Tri-iod acetone. (3) CHj.CO.CIa+Na OH=CH3.COO Na+CHIg. Iodoform. (4) NaOI+NaI+2HCl=l2 + 2NaCl+H20. (5) Ij+2NaS. S03 0Na=S.SOjONa+2NaI S.SO3O Na Thiosulphate. Tetra-thionate. The following reaction also takes place slowly in the alkaline hypo-iodlte : (6) 3NaOI=NaIOs+2NaI lodate. Since iodate does not react with acetone, it is essential to have an excess of at least 10 cc. of the iodine. Further, it is important not to add the iodine to the soda until everything is ready for the distillation. Reaction (6) does not cause any loss of iodine that would be reckoned as due to acetone, for on acidification the iodate reacts with the iodide shewn in equations (6) and (i) to liberate iodine. (7) Na IO3+5 NaI+5 HC1=3 I3+6 NaCl+3 H3O. Solutions leauiied. 1. Standard thiosulphate, about o-i N. (See page 254). 2. Standard iodine, about o- 1 N. Dissolve 50 gms. of potasisum iodide in about 300 cc. of distilled water. Weigh out 27 gms. of iodine, transfer to a large mortar and rub it up with successive portions of the iodide solution until all has dissolved. Wash out with distilled water to make a total volume of 2 litres. Mix thoroughly and make sure that all the iodine has dissolved. Determine the exact strength as follows : Measure 25 cc. with a pipette into a 250 cc. conical flask. Titrate with the standardised thiosulphate from a burette until the yellow colour of the iodine has nearly disappeared. Add a few drops of soluble starch and com- plete the titration, as indicated by the complete disappearance of the blue tint. If V be the volume of thiosulphate used, and / its normahty, the normality of the iodine is = x. 25 From equation (2) above it will be seen that i gm. molecule of acetone reacts with 3 gm. molecules of Na OI, which from (i) is formed from 3 I^, or 6 Utres of normal iodine. So 6000 cc. N. Iodine = 58 grms. acetone. I cc. N. Iodine = ■ mgrs. acetone. and I cc. of x N. Iodine = ^— ^ — mgs. acetone. The stock bottle should be labelled with its normality, and the mgms. of acetone equivalent to i cc. CH. XIII.] ACETONE BODIES. 333 Neither the iodine nor the thiosulphate are indefinitely stable. They should both be carefully stored in a cool, dark cupboard, and re- standardised at intervals. 3. Soluble starch, 2 per cent. (See page 254). 4. Strong soda, about 40 per cent. To 2 litres of distilled water in a large porcelain basin, add i lb. of the best powdered caustic soda. Stir well till all has dissolved. Transfer to a flask or Winchester quart bottle, stopper and allow to stand for 24 hours. Syphon off the clear supernatant fluid and store in a bottle stoppered with a rubber cork. 5). Hydrochloric acid, pure concentrated. Method. I. The distillation of the acetone. Use the apparatus shewn in fig. 49. Into flask A measure an amount of urine that jdelds between 5 and 15 mg. acetone (10 cc. may be taken for a preUminary trial). Add water to make the volume up to about 50 cc. and then Fig. 49. Apparatus for the estimation of acetone. A. 300 cc. " Duro " flask. B. SoUd glass rod for seaUng tube. C. "Dure" flask. D. Glass tube connected by rubber to condenser tube. E. 250 cc. Erlenmeyer flask. F. Liebig condenser. I CC. of strong suplhuric acid. Into flask C place 10 cc. of 40 per cent caustic soda and a few glass beads. Into E place 10 or 20 cc. of 40 per cent, soda and then run in 25 cc. of the standard iodine, measuring this with a pipette. See that the water supply to the condenser is turned on. Close the tube with the glass rod B and then light the burners. The soda in C must boil before the fluid in A. The soda is kept just boiling whilst A is allowed to boil briskly. The first appearance of turbidity in E is noted and the distillation 354 ANALYSIS OF URINE. [CH. XIII. allowed to proceed for another lO minutes. Remove plug B and turn out the flames. Detach tube D from the condenser and wash it with a jet of distilled water into E. 2. Titration of the excess iodine. Acidify with strong hydro- chloric acid, adding this cautiously and cooling well under the tap after the addition of an amount equal to that of the strong soda taken. When the solution is acid, there will be a great intensifica- tion of the yellow colour, due to the liberation of iodine. The neutralisation of the alkali makes the solution hot, and if the mixture be acidified before it is cooled a loss of iodine by volatisation will probably occur. An excess of at least 2 or 3 cc. of the acid should be added. Titrate the cold mixture at once with the standard thio- sulphate, running this in until the yellow colour has nearly dis- appeared. Then add a few drops of the soluble starch and cautiously complete the titration. The end-point is well marked. A return of the blue colour may occur if the reagents employed are not pure, but no notice should be taken of this. Calculation and example. Thiosulphate was 0-1065 N. 25 cc. iodine required 23-85 cc. of the thiosulphate. 25 So I cc. of thiosulphate = — /^ cc. iodine (log. 0204). A , • ,■ 23-85 X 0-1065 ^^ ^ .^.^ And iodine is -^-^ N. = 0-1006 N. 25 I- • ,■ —23-85 X 0-1065 X 58 , „ - SO I cc. iodine =:. -^ — y^ nigs, acetone (log. 1-9922). urine taken = 10 cc. iodine taken = 25 cc. Thiosulphate required for back titration = 1 2 - 1 cc. log. of I2-I = 10828 add -0204 anti-log. of 1-1032=12-68 25 - 12-68 = 12-32 cc. of iodine used, log. of 12-32 = 1-0906 add 1-9922 acid anti-log. of 1-0828= 12-1. So 10 cc. urine contain 12-1 mg. acetone. And 100 cc. urine contain 0-121 gm. acetone from acetone + aceto- acetic CH. XIII.] CHLORIDES. 355 I. Chlorides. The usual method for the estimation of chlorides in urine is Volhard's, which is described on page 199 in connexion with the estimation of gastric juice. The method given below is precisely similar. The silver nitrate solution employed is of a different strength and the thiocyanate is not made up to any defined concentration, but standardised against the silver. If preferred the solutions described on page 199 can be employed, i cc. of the o-i N. silver nitrate being equivalent to 0-00355 gram, of chlorine and 0-00585 gram, of sodium chloride. If this weaker silver solution is used, 25 or 30 cc. of it should be taken for 10 cc. of urine. A method that is rapid, convenient and accurate, is that of Larrson, but it is now difficult to obtain satisfactory charcoal. Recently, however, the author has been presented with a specimen of charcoal that was prepared for use in gas masks. It seems to be an extremely good adsorbent, supeiior to the best German products and admirably adapted for all kinds of analy- tical work. The method is described in the hope that a satisfactory product will soon be on the market. In that case Volhard's method would be super- seded by Larrson's. Van Slyke's method (see page 258) is also well adapted to the estimation of urinary chlorides, especially if only small quantities are available. 412. The estimation of chlorides by Volhard's method. Principle. See page 199. Reagents recLuiied. 1. Standard silver nitrate solution prepared by dissolving 29 '061 grams. of pure fused silver nitrate in distilled water and fiUing up accurately to one litre. The solution should be kept in the dark. I cc. corresponds to -oi gram. NaCl (-00606 gram. CI). 2. Solution of potassium thiocyanate made by dissolving 8 grams, of the salt in a Utre of distilled water. 3. Pure nitric acid, quite free from chlorine. 4. A concentrated solution of iron alum. Standardisation of the thiocyanate. In a beaker place 10 cc. of the silver nitrate, accurately measured : add 5 cc. of pure nitric acid, 5 cc. of iron alum and 80 cc. of distilled water. Titrate the whole with the thiocyanate from a burette until a faint permanent red tinge is obtained. Note the amount required for the 10 cc. of silver nitrate. Method. In a loo cc. cylinder or measuring flask place lo cc. of urine, accurately measured by a pipette, 20 cc. of the standard silver solution, also accurately measured, about 4 cc. of pure nitric acid, and 5 cc. of the iron alum. Add distilled water to the 100 cc. mark, and mix thoroughly by pouring into a beaker and stirring well. Filter off the precipitated silver chloride through a dry paper into a dry vessel. Of the filtrate take 50 cc, accurately measured, and titrate it with the potassium thiocyanate solution till a faint per- manent red tinge is obtained. NoTFS, — I. It is very important to remember to add the nitric acid. It 356 ANALYSIS OF URINE. [CH. XIII. renders the silver chloride insoluble and prevents the precipitation of the silver compounds of the purine bases in those cases in which the urine is alkaline. 2. Some workers titrate without filtering off the silver chloride, but the end point is apt to be uncertain owing to the decomposition of the chloride by the thiocyanate. Calculation and Example- 19-6 cc. of the KCNS were required for lo cc. of the AgNO,. lo So I cc. of the KCNS = — ;^ = 0-51 cc. AgNO,. 50 cc. urinary filtrate required 11 -6 cc. KCNS, So 100 cc. urinary filtrate would require 23-2 cc. KCNS and would therefore contain 23-2 x 0-51 = ii-8 cc. of the AgNO,. So 20 - II-8 = 8-2 cc. of the AgNO, have been precipitated. Now I cc of the AgNO, = o-oi gm. NaCl, So NaCl in 10 cc. urine = 8-2 x o-oi gram. So NaCl in 100 cc. urine = 0-82 gram. 413. The estimation of chlorides by Larrson's method.* Principle. The pigments, urates and other interfering substances are removed from the urine by adsorption with charcoal. The chlorides are estimated in a measured amount of the filtrate by direct titration with silver nitrate, using potassium chromate as an indicator. Reagents regaired. 1. Standard silver nitrate (see Ex. 412). 2. A high quality, pure absorbing charcoal (see p. 394). Ordinary animal charcoal is quite useless. 3. A 5 per cent, solution of potassium chromate. Method of analysis. To I'S gram, of the charcoal in a dry 50 cc. flask add 20 cc. of the urine. Shake vigorously and repeat the shaking at intervals for 10 minutes. Filter through a'^small dry paper into a dry tube. Measure 10 cc. of the filtrate by means of a pipette and transfer it to a small beaker. Add 5 or 6 drops of the chromate and titrate with the standard silver nitrate from a burette until the end point is reached, as indicated by the appearance of a reddish-brown colour. Calculation. 1 cc. of silver = o-oi gram. NaCl. Example. 10 cc. of the filtered urine required io-6 cc. of silver. So 10 cc. contain io-6 x O'Oi gram. NaCl. So 100 cc. contain i-o6 gram. NaCl. • Biochcm. Zeitschrift,, xlix, p. 479. CH. Xin.] PHOSPHATES. 357 413A. The estimation of chlorides by van Slyke's method. Principle, reagents, etc. (See page 258.) Method. Measure i cc. of the urine into a 50 cc. volumetric fiask by means of an Ostwald pipette. Add about 25 cc. of dis- tilled water. Then add 10 cc. of the standard silver nitrate by means of a pipette, and then add about 10 cc. of the magnesium sulphate to flock the precipitate. Make up to the mark with dis- tOled water and shake well. Allow to stand for 10 minutes and filter through a dry No. i Whatman paper into a dry flask. Measure 25 cc. of the filtrate into a kjo cc. flask, add 5 cc. of the citrate mixture and titrate with the standardised iodide solution. Calculation. The grams of NaCl per cent, is (10- cc. iodide required) x 2. 10 J. Phosphates. 414. The estimation of phosphates. Principle. Urine is heated to boiling point, and titrated whilst hot with a standard solution of uranium acetate, which gives a precipitate of (U02)HP04 with phosphates in acetic acid solution. Cochineal tincture is used to indicate by a change in colour when the uranium is in excess. Reagents reauiied. 1. Acetate solution. Dissolve 100 grams, of sodium acetate in a litre of distilled water, and add 100 cc. of strong acetic acid. 2. Cochineal tincture, prepared by extracting the insects with 30 per cent, alcohol and filtering after two days. 3. Standard potassium phosphate. Dissolve 7-672 grams, of pure re- crystaUised acid potassium phosphate in distilled water and make the volume up to I litre. 25 cc. = o-i gram. P2O5. This solution can also be prepared by measuring 28-17 cc. of the 0-2 M.KHjPOj described on page 24 into a 100 cc. measuring flask and making the volume up to 100 cc. with distilled water. 4. Standard uranium acetate. Dissolve by the aid of heat 36 grams, of pure uranium acetate in a litre of distilled water. Allow the solution to cool and then filter. Standardise the solution as follows : . Into a beaker measure 25 cc. of the standard potassium phosphate, add about 25 cc. of distilled water, 5. cc. of the acetate solution and about i cc. of the cochineal tincture. Bring the mixture to the boihng point and titrate with the uranium acetate solution from a burette till the red tinge just changes to a green, heating the mixture to boiUng just before the last few drops are added. If x cc. of the uranium solution are used, then i cc. of the uranium corresponds to o-i — gram. PjOj. If desired the solution can be diluted with water so that i cc. = o -005 gram. (20 - x) -K 100 „ , PjOj. To effect this add cc. of distiUed water to every 100 cc. 358 ANALYSIS OF URINE. [CH. XIII. Method. In a beaker of about loo cc. capacity place 50 cc. urine, add 5 cc. of the sodium acetate solution and about i cc. of the cochineal tincture. Have a burette ready containing the stand- ardised uranium acetate solution. Heat the urine to boiling point, remove the flame and run in the uranium acetate as long as a precipitate is formed. Heat the mixture again just to boiling point, and cautiously add uranium acetate, drop by drop, tUl the red colour is converted to a green. Calculation. I cc. of the uranium acetate = 0'005 gram. PjOj. Thus if 50 cc. of urine require i5-2 cc. uranium, the percentage of PjOj is 2 V 15.2 X 0-005 = 0'i52 gram. E. Sulphates. Sulphates can be determined gravimetrically as barium sulphate or voIumetricaUy by means of benzidine. The latter is much more convenient, but both methods are given below. The difficulty encountered with the gravimetric method is that of preventing adsorption of other substances. For that reason the barium must be added very slowly and the method is extremely tedious. 415. The estimation of total sulphates by Folin's method.* Place 25 cc. of urine in a 250 cc. Erlenmeyer flask, add 20 cc. of hydrochloric acid (i volume of concentrated HCl to 4 volumes of water) and boil gently for 30 minutes, covering the mouth o^ the flask with a small watch glass. Cool the flask under the tap and dilute to about 150 cc. with water. Add 10 cc. of 5 per cent, barium chloride solution slowly, drop by drop, to the cold solution, which must not be stirred or shaken during the addition, nor for at least one hour after. Then shake well, filter through a weighed Gooch crucible (see note to Ex. 410), wash with 250 cc. of cold water, dry in an air bath, or over a very low flame. Ignite, cool and weigh. Calculation. Weight of BaSO^ x 1-366 = SO, per cent. Notes. — Instead of using a Gooch crucible a washed " Barium sulphate " filter paper may be used. After washing and drying the ignition may be carried out in a platinum or porcelain crucible, previously weighed. After ignition, the ash should be treated with a drop of 25 per cent, sulphuric acid, cautiously dried and heated again. A correction must be made for the weight of the ash of the paper. * Journ. Biol. Chem., i., p. 150. CH. xin.] SWLPHAtfeS. 359 416. The estimation of inorganic sulphates by Folin's method. Place 25 cc. of urine and 100 cc. of water in a 250 cc. Erienmeyer flask. Acidify with 10 cc. of hydrochloric acid (i volume of con- centrated HCl to 4 volumes of water). Add 10 cc. of 5 per cent, barium chloride, drop by drop, as in the previous exercise, and proceed as there directed. Calculation. The same as for total sulphates. Ethereal Sulphates. This can be found by difference. Total sulphates less inorganic sulphates = ethereal sulphates. 417. The estimation of total sulphur by Benedict's method."^ Place 10 cc. of urine in a small (7-8 cm.) porcelain or silica crucible and add 5 cc. of Benedict's sulphur reagent. Evaporate over a free flame, keeping the solution just below the boiling point, to prevent loss by spattering. When dry, raise the flame shghtly until the entire residue has blackened. Raise the flame still more and heat to redness for ten minutes after the black residue (which first fuses) has become dry. Allow the dish to pool. Add 10 to 20 cc. of I in 4 hydrochloric acid, and heat again till the residue has completely dissolved to a clear solution. Wash the contents quantitatively into an Erienmeyer flask, and dihite with cold water to 100 to 150 cc. Add 10 cc. of 10 per cent, barium chloride, drop by drop, and allow to stand for about an hour. Shake thoroughly and proceed as in Ex. 415. Calculation. Weight of BaSO, from 10 cc. of urine multiplied by 3'413 = SO, per cent. Note. Benedict's sulphur reagent is : Crystallised copper nitrate, 200 grams. Potassium chlorate, 50 grams. Distilled water to i litre. Neutral Sulphur. This can be found by difference. Total sulphates less total sulphur = neutral sulphur. * Journ. Biol. Chem., vi., p. 363. 360 Analysis of ueine. [ch. xiti. 418. Inorganic sulphates by the benzidine method of Rosenheim and Drummond.* Principle. The urine is acidified with hydrochloric acid and treated with an excess of benzidine hydrochloride. The sulphates are precipitated quanti- tatively. The precipitate is filtered off under suction, washed free from acid with water (or better with a saturated solution of benzidine sulphate) and suspended in hot water. Phenol phthalein is added, and the mixture titrated with standard soda. A pink colour does not develop until enough soda has been added to combine with the whole of the benzidine sulphate to form sodium sulphate. Benzidine sulphate, being the salt of a weak base with a strong acid, suffers hydrolytic dissociation into the base and the acid. The base is only very feebly ionised, whilst the strong add is freely ionised, the solution in hot water thus behaving like sulphuric add, which can be titrated with the standard soda. Solutions required. 1. Benzidine hydrochloride. Rub up 4 grams, of pure benzidine with about 10 cc. of distilled water. Transfer with about 500 cc. of water to "a 2 Utre flask. Add 5 cc. of concentrated hydrochloric add and make up to 2 litres with distilled water. 2. Hydrochloric acid. Dilute i volume of pure concentrated hydro- chloric acid with 3 volumes of distilled water. 3. Saturated benzidine sulphate. Prepare some benzidine sulphate by adding a little sodium sulphate to 200 cc. of the benzidine hydrochloride. Collect the precipitate as described below and wash it thoroughly with cold water. Suspend it in a considerable volume of hot water and allow it to stand over-night in a cool place. Filter from the benzidine sulphate till quite clear. 4. o-i N. sodium hydroxide. See appendix. The exact strength is immaterial, so long as it be accurately determined. 5. Phenol phthalein. A saturated solution in alcohol. Method. Measure 25 cc. of the urine (filtered, if necessary) into a 250 cc. Erlenmeyer flask, with a wide neck. Add 2 cc. of the hydrochloric acid and 100 cc. of the benzidine hydrochloride. Mix and allow to stand for 10 minutes. Filter through paper and paper pulp, as described in Ex. 406. The filtrate must be crystal clear. If it is cloudy it must be passed through the filter again. Wash out the beaker with 10 cc. of the saturated benzidine sulphate and wash the precipitate with this. Repeat this at least once more. Transfer the precipitate, filter pulp and disc to the Erlenmeyer flask and wash the funnel into the flask with a jet of boiling water, using about 50 cc. of water. Any lumps of the precipitate must be broken up by use of a glass rod before the titration is commenced, or, if this is * Biochemical Journal, viii., p. 134. Ch. xffi.] ALBUMIN. 361 impossible, before the titration is completed. Add a few drops of the saturated solution of phenol phthalein and titrate the hot solu- tion with the standard soda. The end point is quite sharp. Calculation, i cc. of o-i N. soda = 4-0 mg. SO3. 419. Total sulphates by the benzidine method. Measure 25 cc. of the urine into the Erlenmeyer flask, add 2 to 3-5 cc. of the hydrochloric acid and 20 cc. of distilled water. Place a funnel in the neck of the flask and boil gently for 20 minutes. Cool thoroughly under the tap, add 100 cc. of the benzidine hydrochloride, and proceed as directed above. Ethereal Sulphates. The difference between the result of the analysis in Ex. 419 and that of Ex. 418 is the ethereal sulphate. Total Sulphur and Neutral Sulphur. The urine can be oxidised by Benedict's method (Ex. 417) and the residue dissolved in hydrochloric acid. Before the benzidine method is applied the excess of free hydrochloric acid must be reduced by the addition of soda until the solution is only just acid to congo red paper. The calculation for neutral sulphur is explained in Ex. 417. L. Albumin. 420. The estimation of albumin by Esbach's method. FiU the albuminometer to the mark U with urine. Add Esbach's reagent (Ex. 14) to the mark R. Stopper the tube, and invert it slowly several times to mix the fluids. Allow the tube to stand upright for 24 hours. Calculation. The graduations on the albumino- meter indicate grams, of albumin per litre. Fig. 50. Esbach's albuminometer. 421. The estimation of albumin by Scherer's method. Measure 50 cc. of urine into a beaker. Place it on a water bath and raise the temperature to 50° C. Add i per cent, acetic acid. 362 ANALYSISiOF URINE. [CH. xni. drop by drop, to obtain a complete separation of the protein (care must be taken to avoid an excess) . Raise the temperature to boiling and keep it so for a few minutes. FUter the urine through a small paper that has previously been washed, dried and weighed. Wash the precipitate in turn with hot water, 95 per cent, alcohol and ether. Dry the paper and precipitate in an air bath at 110° C. till the weight is constant. The weight of protein in 50 cc. is obtained by subtract- ing the weight of the paper. M. Diastase. 422. Wohlgemuth's method. Principle. Varying amounts of urine are added to a given amount of soluble starch, and the mixture digested for 30 minutes at 38° C. After cool- ing, a drop of dilute iodine is added to each tube. The tubes that contain considerable amounts of urine have all the starch digested so that no colour i s obtained on adding the iodine. The tube with the smallest amount of urine that completely digests the starch is found and so the diastatic value calcu- lated (see p. 192). Reagents required. 1 . Stock solution of soluble starch. Accurately weigh out 2 grams, of soluble starch (see p. 395) and transfer it to a dry test-tube. Add about 10 cc. of distilled water and shake. Pour the suspension into about 70 cc. of boiling distilled water and stir well. Wash the tube three successive times with 5 cc. of distilled water, transferring the washings to the boihng solution. Now add 10 grams, of pure sodium chloride. Allow to cool and make the volume up to 100 cc. with distilled water. The solution is stable for months. 2. One per miUe soluble starch in 0-5 pep cent, sodium chloride. 5 cc. of the stock are diluted with distilled water to make 100 cc. This solution must be freshly prepared each day. 3. N/50 iodine, prepared, from N/io iodine (see p. 394) by diluting 2 cc. with 8 cc. of distilled water. The diluted iodine must be freshly prepared each day. Method. Label a series of clean dry test-tubes i to 10. Into the tubes measure the volume of urine and of distilled water stated in the table, using guarded pipettes (see Note 7). Tube. cc. of Urine. cc. of Water. 30' Tube. cc of Urine diluted 1 in 10 witli water. cc of Water. 30 1 3 0-5 4 6 0-9 01 22-2 2 0-4 0-6 5 7 0-8 0-2 25 3 0-3 07 6-6 8 07 0-3 28-6 4 0-2 08 10 9 0-6 0-4 33-3 5 01 0-9 20 10 0-5 0-5 40 CH. XIII.] DIASTASE. 363 To each tube add 2 cc. of the one per mille starch, commencing with tube 10. Mix the contents by agitation and place in a thermo- stat or a water bath at 38° C. for exactly 30 minutes. Remove the tubes and place them in a beaker of cold water for 3 minutes to cool. Arrange the tubes in order in a stand. Commencing with tube i add i drop of the N/50 iodine to each tube and carefully note the colour produced. Should a colour be produced and it rapidly fades, add i more drop of iodine to each tube. Note the tube with the lowest number that shows a blue tinge. The next lower tube contains an amount of urine that completely digests 2 cc. of o-i per cent, starch in 30 minutes at 38° C. Calculation. This is explained on page 192. The d values corresponding to the volumes of urine required are given in the table. Thus if tube 4 shows a bluish tint and tube 3 a red, then since 0-3 cc. cf urine have digested 2 cc. of 2 starch, then icc. of urine would digest = 6-6 cc. starch. So d=6-6. Notes. — i. It is customary to use a freshly prepared o-i per cent, solution of starch in water and to make the volume of the urine up to i cc. with I per cent, sodium chloride. The author has determined that 2 per cent, starch in 10 per cent, sodium chloride is quite stable and that the results obtained agree with these found by tlie original method. It is suggested that the present more convenient method be adopted as a standard. 38° 2. The d-—, of normal urine varies between 5 and 20, with an average of 10. In acute pancreatitis and in many cases of carcinoma of the pancreas the value is high and may be over 200. In such cases the urine is stiU further diluted to i in 100 and the d calculated. 2 Thus if o-oo6 cc. of urine is required d = —-7 = 333. It is of considerable pathological importance to note that the kidney in interstitial nephritis has difficulty in passing the diastase. The index of the urine is characteristically low in this condition. , It is high in certain cases of " toxaemia of pregnancy " (eclampsia). 3 . It is important that the same amount of iodine be added to each tube. Very uneven results are obtained if varying amounts of iodine be employed. 4. Samples of the mixed 24 hours' specimen should be used. 5. The diastase in the urine is quite stable if the urine be preserved by the addition of toluol. 3 cc. are ample for an estimation. 364 ANALYSIS OF URINE. [CH XHI. 6. The pipettes for measuring the solutions must be accurate i cc. pipettes graduated to i/ioo cc. 7. The end of the pipettes that are placed in the mouth should be guarded by plugs of cotton wool, to prevent contamination of the fluids with saliva. Bewildering results in class work disappeared after this precaution was rigorously enforced. CHAPTER XIV. DETECTION OF SUBSTANCES OF PHYSIOLOGICAL INTEREST. If no indication as to the origin of the substance is available the scope of the analysis is very considerable. The following account is not intended to be exhaustive, but merely to suggest a few methods of attack. Success demands a sound knowledge of the properties and reactions of a large number of substances. Experience, practice and enterprise count for a good deal. Many substances are not detected by student analysts mainly because they forget to test for them. The hints on page 372 should be carefully studied. The student is urged to perform his tests on the smallest amount of material that is likely to give a conclusive result. With a limited supply of the substance for analysis a much greater variety of tests can thus be applied. A. Fluids. 1. A portion may be neutralised and evaporated to dryness on the water bath. This allows for a subsequent extraction with strong alcohol, wliich serves for the separation of many substances. It should not be started until there is some indication that it may be necessary, as for the separation of sugars from proteins and poly- saccharides, etc. The evaporation must be conducted in neutral solution to obviate any chemical changes produced by hot acids or alkalies. 2. Note any characteristic smell of urine, bile, etc. In such cases apply tests for characteristic constituents. 3. Note the colour and appearance of the fluid : opalescence suggests starch, glycogen, or certain protein solutions; coloured fluids suggest bile, blood or urine. 366 DETECTION OF SUBSTANCES. [CH. XIV. 4. Note the reaction to litmus. An acid reaction excludes the presence of mucin, nucleoproteins, caseinogen, and usually earthy phosphates. 5. If acid test for free HCl by Gunsberg's test. (Ex. 246.) 6. Sprinkle some flowers of sulphur on the surface of a portion of the fluid in a test-tube. If the particles fall through the surface, bile salts are probably present. (Ex. 316.) Confirm by Petten- kofer's test. (Ex. 315.) 7. If the fluid be brown or green, apply the Huppert-Cole test (Ex. 318) for bile pigments. 8. If the fluid be red or brown, examine for blood-pigments or derivatives by Table F, page 370. 9. If there are any reasons for suspecting the presence of ferments, examine as directed on page 371. If none of the colour reactions for proteins are. obtained, ferments are probably absent. 10. Examine for proteins by Millon's and the biuret reactions (Exs. 22 and 24). If they be present, proceed as directed in Table A, B or C, according to the reaction of the fluid. 11. If proteins are absent, proceed to Table E. 12. Test for uric acid if the fluid be alkaUne, neutral or only faintly acid. Acidify with a drop or two of strong hydrochloric add; uric acid may separate out as a crystalUne powder. Make another portion of the solution alkaline with ammonia, saturate with NH4CI and apply the murexide reaction to the precipitate thus obtained. (Ex. 352.) 13. If the fluid be alkaline, treat a Uttle with a solution of calcium chloride. A white curdy precipitate indicates the presence of soaps. (Their presence should be confirmed by the methods given in Ex. 177.) 14. Treat a portion with a little hypobromite solution. If an effervescence is obtained, test for urea by Ex. 343. If this is negative the solution may contain amino-acids or ammonium salts. The bromine test for free tryptophane (p. 217) may give a valuable indication. CH. XIV.] PROTEINS. 367 >>o •Q a ..9 & 2 0- DO « s in •a'5 ■d a- 9 Id 1— 1 -** u n fcPHH 13 zS, « 3 S a a , Q^aSiE S 1 <^§5 3 H 368 DETECTION OF SUBSTANCES. [CH. XIV. o o. bo a 'a o u 13 _o ? 03 a < tn Cll ■a u s Ct. I. o »4-l U o !9 MH 'o a d o o a ■S s i rt bo fl g 'tS) u ■ ~ *j I-l n o rs _g *o en 0) d .2'-^ H ■S 3 0\ o "o ^ .s rt ""^ *« be a •6 g^ o u i^ '§ ft o Ul •S" o ■? . to _g 22 U V o, o O J3 o 3 « a 1 §■§. J3 m 0) .S .So .22 *3 a 1' a o n S o <— s u ll o o. d .2 >, • 13 < e ■a 62 £2 o ^3S '% is rt d f -a H o < ^ rt -w r] 0) w^ Z 3 ° d 3. « la •■2 o . X cS s .g-d u >2 s a :3 d 1 s .Q d "d 1 ° d a § 2 >. M m i 1 C d u 2 -a U <-( O , ^^ .2 H rn •^ O .Q . T3 1 o Is S d U) ■d g= .W ■S 2 5 00 i "S u ■■S ° C g 3 V a >> S JS |.§ s .a e« rt .«=« ^•§•31 ■1-! en ^ 1 !t T3 •3 " S.S S * = a| m n _o b fi 3 .2 S s ^ M-. ,,^ ^"-s /— s ^-^ o a 3 .a a ^^ »^ •a d ,■♦* •a § ■a o •< ^ CH. XIV.] NON-COAGULABLE PROTEINS. 369 Table D. Examination of Filtrate B for albumoses, peptones and gelatin. Treat a portion with caustic soda and a drop of copper sulpliate solution. No biuret Positive biuret reaction. To portions of filtrate B apply reaction. Millon's and glyoxylic tests. Negative Positive reactions. Saturate filtrate B with reactions. ammonium sulphate by heatin g with excess of solid. Cool under tap and filter. | Gelatin present. Precipitate. Mostly stick- Filtrate. Confirm by ing to tube. Wash with Treat 2 cc. obtaining a cold saturated ammonium with 4 cc. of precipitate sulphate. Dissolve in a 40 per cent. by half- little boiling water and cool NaOH and a saturation under tap. To portions drop of copper with amm. . apply biuret test (using 40 sulphate. sulphate. per cent. NaOH) and the Pink colour glyoxylic test. If both are indicates positive — ■ Proteins Albumoses. Peptones. absent. Table E. Examination of a solution for carbohydrates. If proteins be present they must be removed, as far as possible, by neutraUsing, boiUng and filtering. In any case the solution tested must be neutral. {a) To a small portion add diluted iodine drop by drop, until an excess has been added. If a pure blue colour be obtained at any stage of the addition of iodine, starch is present. If a purple or brown colour be produced and the fluid be quite clear, erythro- dextrin is present and glycogen absent. If a blue colour be produced, or if the fluid be opalescent, proceed as follows : AA 370 DETECTION OF SUBSTANCES. [CH. XIV. To a portion of the fluid, prepared as directed above, add an equal bulk of saturated (NH4)2S04, shake vigorously, and filter through a dry paper after about ten minutes. Precipitate. Scrape off the paper, dissolve in a little hot water, cool and add a drop of iodine. A blue colour shows the pres- ence of starch. Filtrate. To a small portion add a drop or two of iodine. If a reddish or purple colour be produced, glycogen or dextrin is present. If the fluid be opalescent after warming, glycogen is present. Saturate the remainder with (NHi)aSOi and filter. Precipitate. Neglect, Filtrate. Add a drop of diluted iodine, a red-brown colour shows the presence of erythro-dextrin. (6) Apply Benedict's (Ex. loo) or Fehling's test (Ex. 97) for reducing sugars. Note that the tests do not succeed in the presence of any considerable amount of ammonium salts. Also that if albumoses, peptones or gelatin are present they should be removed by alcoholic extraction as described in Ex. 58. (c) If a reduction be obtained, apply Barfoed's test (Ex. loi) to distinguish between mono- and di-saccharides. The osazone test (Ex. 109) also can be applied if necessary. (i) Test for cane-sugar and fructose (see Exs. 130 and 131). Table P. Examine the solution spectroscopically : gradually dilute the solution, noting the spectrum at all stages of dilution. Take the reaction of the undiluted iluid to litmus paper, wash- ing the surplus off the paper with a stream of distilled water, if you are unable to note the reaction directly. If the fluid be neutral or alkaline, treat it with Stokes' fluid or warm it with ammonium sulphide, and note whether the spectrum is altered by reduction. This should be done after various dilutions of the original solution. CH. XIV. J PIGMENTS AND ENZYMES. Acid — Acid haematoporphyrin, two bands. (Ex. 307.) 371 Fluid red Neutral Dilute till two bands are well seen and then reduce. Oxyhaemaglobin, the two bands merge into one faint band (Ex 293.) CO-haenioglobin, the two bands are unaltered. (Ex. 296.) Alkaline ■* f Alkaline haematoporphyrin, four bands, converted into acid haematoporphyrin by strong acids. (Exs. 307 and 308.) Haemochromogen, two bands in green, one much more distinct than the other, unaffected by reduc- ing reagents. (Ex. 305.) (Acid — Acid haematin, band in red. Ex. 301. < Neutral. Methaemeglobin, band in red : gives spectrum of oxyhaemoglobin and then of reduced haemoglobin if reduced. (Ex.299.) Alkaline haematoporphyrin — four bands. (Ex. 308.J Alkaline \ Alkaline haematin, faint band in red, converted to haemochromogen by reducing reagents. (Exs. 303, 305.) Enzymes. In testing for enzymes it is important to note the reaction. It is not necessary to determine the exact Ph, but trials should be ijiade with litmus, followed by phenol-red and phenol-phthalein for alkaline solutions, and methyl-red and brom-phenol-blue (or thymol-blue) for acid solutions. In this way certain valuable indications may be obtained. The next point to remember is that all tests must be made in parallel with a control. In the control test, the solution is well boiled to destroy any enzyme that may be present : in other respects it is carried out exactly as the test proper. Without this precaution it is quite impossible to make any safe deduction. To test for proteolytic enzymes see if the solution will clot calcified milk (Exs. 252 and 257). The solution should be nearly neutralised before appljnng the test, but if it is acid it must not be made alkaline (see Ex. 253). If this test is positive, pepsin can be identified by the method given in Ex. 248. Rennin can be distinguished from pepsin by Ex. 256, though it takes a good deal of time. It is unusual to find a rennin solution free from pepsin, but many commercial pepsins are practically free from true rennin. Trypsin can be identified by Ex. 258. If the solution be made faintly alkaline to phenol phthalein, incubated for 10 mins. at 38°C., and then neutralised to litmus, pepsin and rennin are destroyed. If^the solution rigw clots milk;, trypsin is present. 372 DETECTION OF SUBSTANCES. [CH. XIV. Diastatic enzymes are probably absent if the solution is strongly acid or alkaline. The reaction and salt content are factors of importance. The best method of testing for these enzymes is some modification of Ex. 237, adding the buffer and the salt for the reasons given in that exercise. The solution should be carefully neutraUsed to Utmus before making the tests. The enzymes acting on the disaccharides are usually more dif&cult to identify. Sucrase is sometimes found in a very active condition, but the tests for maltase and lactase generally require an incubation period of at least 15 hours. For details see Exs. 266-268. Lipase is rather unstable to acids. For tests see Ex. 168. A few special hints on the examination of physiological fluids. 1. It is impossible to obtain a heat coagulum of albumin or globulin in an acid or alkaline fluid. The reaction must be neutral or only very faintly acid. 2. A little Utmus solution in the fluid does no harm, and often reminds one that the reaction changes after boihng (owing to the evolution of COj). 3. In testing for peptones, after removing the albumoses by saturation with ammonium sulphate, the biuret test succeeds only if at least two volumes of 40 per cent, soda are used. The test wiU not be obtained with the ordinary 5 per cent, soda (see notes to Ex. 57). 4. Gelatin reacts very much Uke the albumoses, except that it does not jdeld the glyoxylic reaction. It can be precipitated by half-saturation with ammonium sulphate. If the precipitate is collected, squeezed and dissolved in a very little hot water, the solu- tion will often set after being thoroughly cooled for some time. 5. It is impossible to obtain Fehhng's or Benedict's test for the reducing sugars in the presence of any considerable amount of ammonia or ammonium salts. 6. The sugars reduce only in an alkaline medium. If the fluid under examination be acid, it must be neutrahsed before boihng with the Fehhng's or Benedict's solution. 7. In testing for cane sugar do not forget that starch and the dextrins are hydrolysed to glucose by boihng acids. But whereas CH. XIV.] ANALYSIS OF FLUIDS. 373 cane sugar is hydrolysed very easily, starch, etc., are only slowly acted on. 8. Starch, glycogen and the erythro-dextrins do not give any colour with iodine solutions, if the reaction of the fluid be alkaline. If this be the case, make the reaction acid with acetic acid. 9. The proteins interfere with the iodine tests for these substances, and should therefore as far as possible be removed before testing for the polysaccharides. 10. Fat is insoluble in water, so do not waste time in testing cin ordinary solution for fats. 11. The only reliable test for urea is the urease test (Ex. 343). In this connection it must be remembered that urea is soluble in alcohol, and can thus be separated from the proteins and other substances likely to interfere owing to their " buffer " action. 12. Ammonium chloride is a very valuable reagent in testing for uric acid or urates. The only other physiological substance precipitated by it is soap. 13. Never omit " control " tests when investigating the ferment action of a solution. 14. Use " carmine fibrin " in testing for pepsin ; never when testing for trypsin. 15. In testing solutions for pigments, examine spectroscopi- cally in various dilutions. Note the reaction of the fluid ; it is no good loolcing for haemochromogen in a markedly acid solution. 16. Creatinine, acetone, aceto-acetic acid and lactic acid can usually be identified by specific colour reaction, though the latter generally involves an extraction with ether. Creatine can only be identified after conversion to creatinine, and then an estimation of total creatinine is necessary. 17. A solution of amino-acids evolves nitrogen gas with nitrous acid and also with alkaUne hypobromites. Ammonia can be removed by gentle boiling in an open dish with a little alkah. 374 DETECTION OF SUBSTANCES. [CH- XIV. B. Solids. 1. Examine a little microscopically, both dry and with the addition of a drop of water. Look for starch grains, crystals of urea, uric acid, urates, leucine, tyrosine, cholesterol, and haemin scales. 2. Heat a small amount of the solid in a dry tube, at first gently and then mare strongly. (a) If sublimation take place and an odour of amylamine be given off, leucine is present. (b) If sublimation take place and a strong smell of ammonia be evolved, urea is indicated. (c) A smell of phenol and nitro-benzol indicates tyrosine. {d) A smell of burning feathers indicates proteins, gelatin, etc. (e) A smell of acrolein indicates fats. 3. Boil some of the soUd \vith a small amount of water in a tube, cool under the tap and leave the test-tube in a beaker of cold water for 10 minutes. If gelatin be present, the solution will set to a jelly. (Starch, if present, may form a thick paste, which may be confused with the clean jelly given by gelatin. If the tube be subsequently placed in boiUng water, gelatin becomes quite limpid, whilst starch remains thick.) 4. If the solid be of a dark brown or red colour, boil a portion with dilute alkali, filter, heat the filtrate with Stokes' fluid or ammonium sulphide, and examine for the spectrum of haemochro- mogen. (Ex. 305.) If this be obtained, the solid contains dried blood or haematin. Confirm by obtaining haemin crystals. (Ex. 309) 5. The table on the next page can be followed, but the method adopted will depend on the indications obtained by preliminary tests. It is advisable to test for starch before deciding on a plan of operation. CH. XIV.] SOLIDS. 375 O J3 CO 2 1 > C! (U •a 4-» rt cd ^ o C3 cS a "OB O a , o O o .a o >> o I) £ n! m > .-H ,2 3 ■a J3 Cll d > ••H a V (S i-n '55 o a £ C lU o 8 « fi s (J +-< 3 •n ti o (1) CO 11 ■qj c o ^ la a u H^ O o u •a 5 «l.3 2 o S rS ;i o ^. « s g^a o ? « O *J O 4J c3 ri a .o ■J;; OJ ^■d O. ^ ■*! L, rt rM ^ to <2 _3 O " o '*^ CO s S rt tn Ho o o ,^ ■"t^-OrtOxZlta I^SH^^^s-a OS CO O W OX! CO Po g h t, a^i S .l = S2"c"l on Eva water ba heat and Q »* +j TJ nJ nJ to ^2§«^^¥^ ^-M U Ih tn CO CO 1 ■« (U - Solut on th water S-fS . §43^.3 ness c. of »• .3 '^ 1 l&o ^ as s .5J "O -o ^ 05 376 lO O < « H a w a. i/j Z o H- ( H PM (/J Pi z O o rn t— f pq H J W o K Crt H H a « C7 Ce! HH O Uh U w O 0:5 O fe H « <; ffi o I T 377 a o o o h •30,0, JJ ■S 2 2 c o o rt d « -s e s 63 § (D Q) , >> •3 ■a a .Q ^ 01 " 0^ 1 OJ Q. OJ a; P:^ « o H &; a »n b ■-4 II II II II II 'J 6 T3 V •d 0} ■^^ "o 1 'S o u 3 S "" f^ 6 tl M 2 S So o 3 B ^ 1 a S a •A c ij ^1 2 g ■a o en qj rt J5 a « in t/i ll u ■5 8 .a 55 1 < 'a c ^ 5 g.i 6 ■3 'S ? 1 a s < o 5 > Hi a B S c B 3 s 5 J3 V4 3 U v (J 3 P < P u u P < < H M z < ti tn Ph 379 APPENDIX. WEIGHTS AND MEASURES. I grain = -0648 gram. 1 ounce = 437-5 grains = 28-3595 grams. I lb. = 16 oz. = 7000 grains = 453-5925 grams. 1 gram. = 15-432 grains. I kilogram = 1000 grams = 2-2046 lbs. I minim = -059 cc. I fluid drachm = 60 minims = 3-55 cc. I fluid ounce = 8 fluid drachms = 28-4 cc. I pint = 20 fluid oz. = 567-9 cc. I cc. = 16-9 minims. I litre = 1000 cc. = 35-2 fluid oz. = 1-76 pints. I gallon = 8 pints = 4-542 litres. I gallon distilled water =10 lbs. I inch = 2-54 cm. 1 inch = 2-54 cm. I foot = 30-48 cm. I yard = 91-44 cm. I cm. = -39 in. I metre = 39-37 in. I litre of hydrogen at 0° C. and 760 mm. Hg. = 0-0896 grams. 38o APPENDIX. TENSION OF AQUEOUS VAPOUR in millimetres of mercury from 8° to 20° C. °c. mm. °C. mm. °C. mm. 8 8-0 14 II-9 20 17-4 8-5 8-3 14-5 12-3 20-5 17-9 9 8-6 15 12-7 21 i8-5 9-5 8-9 15-5 131 21-5 I9-I 10 9-2 16 13-5 22 19-6 IO-5 9-5 16-5 14 22-5 20-2 II 9-8 17 14-4 23 20-9 "•5 lO-I 17-5 149 23-5 21-5 12 IO-5 18 15-3 24 22-2 12-5 IO-8 i8-5 15-8 245 22'9 13 II-2 19 16-3 25 23-5 13-5 "•5 19-5 i6-8 INTERNATIONAL ATOMIC WEIGHTS. Revised Revised 0= 16 0=16 Barium Ba. 137-37 Mercury Hg. 200-6 Bromine Br. 79-92 Nitrogen N. 14-01 Calcium Ca. 40-07 Oxygen 0. 16 Carbon C. 12-005 Phosphorus P. 31-04 Chlorine CI. 35-46 Potassium K. 39-10 Copper Cu. 63-59 Silver Ag. 107-88 Hydrogen H. 1-008 Sodium Na. 23- Iodine I. 126-92 Sulphur S. 32-06 Iron Fe. 55-84 Tungsten W. 184- Lead Pb. 207-2 Uranium u. 238-2 Magnesium Mg. 24-32 Zinc Zn. 65-37 Manganese Mn. 54-93 APPENDIX. 381 SPECIFIC GRAVITIES TABLES. I. SULPHURIC ACID. Sp. Gr. Gtns. of Sp. Gr. Gms. of 15° 4° HjSO, in 100 cc. 15° 4° HjSO, in 100 cc. 1-840 175-9 1-552 100 1-838 173-9 1-54* 98-1 1-835 171-7 1-520 93-6 1-833 170-4 1-492 88-95 1-830 168-5 1-420 74 1-825 166-1 1-380 66-2 1-815 161-8 1-295 50 I -800 156-4 1-200 32-8 II. H-ITOROCHLORIC ACID. Sp. Gr. Gms. of Sp. Gr. Gms. of 15° 4° HCl in 100 cc. 15° 4° HCl in 100 cc. l-i6o 36-6 '•133 30 1-155 35-3 1-113 25 1-152 34-5 1-091 20 1-150 34-0 I -056 12 1-145 32-8 1-047 10 1-140 31-5 1-0375 8 382 APPENDIX, III. SODIUM AND POTASSIUM HYDROXIDES. Sp. Gr. Gms. of Gms. of 15° NaOH KOH 4° in 100 cc. in 100 cc. 1-634 — 94 1-615 — 90-2 1-530 — 75-6 1-438 57-5 — 1-397 50-6 54-3 1-370 46-2 50-6 1-332 40-0 45-1 I'igo 20 25-5 IV. AMMONIA. Sp. Gr. 15° 4° 1 Gms. NH, in 100 cc. Sp. Gr. 15° 4° NH, in 100 cc. •880 3» -896 266 •882 30-83 •898 2605 •884 30-14 -900 25-5 •886 29-46 -902 24-94 •888 28-86 -906 23-83 •890 28-26 -910 22-74 •892 27-70 -920 20-01 •894 27-15 •926 18-42 APPENDIX. V. ALCOHOL. 383 Sp. Gr. Volume Sp. Gr. Volume is-ss" per cent. 15-56° per cent. •79391 100 •83065 91 •79891 99 •83400 90 •80359 98 •86395 So •80800 97 •87740 75 •81217 96 •89010 70 •81616 95 •90214 65 •81997 94 •91358 60 •82365 93 •92439 55 •82721 92 •93445 50 BOILING POINTS. Acetic acid 119 Acetone 565 Alcohol, anyl ... 129-6 „ butyl, normal 117 „ butyl, iso 106 „ butyl, secondary 99-8 „ capryUc 179-5 „ ethyl 78-3 „ methyl 66 Benzene 80 Carbon bisulphide 46-2 Chloroform 63 Ether... 34-18 Toluol III 384 APPENDIX. Sp. Gr. Gms. in 100 cc. Nitric acid 1-42 99 Acetic acid, " glacial " . . I 06 Ili-i Acetic acid, " strong " . . 1-044 33 Sodium carbonate, " saturated " I-I5 l6-2 STANDARD ACIDS AND ALKALIES. A normal solution of a substance contains in 1 000 cc. that weight in grams, which corresponds to i equivalent in grams, of available hydrogen (i-oo8 grams.) or its equivalent. Thus normal hydrochloric acid contains 35-46 + 1-008 = 36-468 grams, of HCl per Utre. Normal sulphuric acid contains 2-016 + 32-07 4-64 . TTO,-^ i-._ = 49-043 grams, of HjSOj per utre. It is customary to employ normal, half-normal, fifth-normal, etc., solu- tions according to circumstances. But it is often much more convenient to determine the exact strength of a solution than to adjust it to some even fraction. For this reason it is better to express the normality as a decimal coefficient rather than as a fraction. Thus, suppose an acid be found by titration against a known standard to be 0-107 N, it can be labelled as suci and used when a solution about one-tenth normal is convenient, the necessary adjustment in the calculation being very simple. The relationship is not N so obvious if it be labelled — —-r. 9-346 In the author's experience the simplest and most reliable starting point for the preparation of standard acids and alkalies is COj - free, sodium hydroxide, made and stored as described on p. 26. From such a stock it is a simple matter to prepare acids or alkaUes of any desired concentration. For further details concerning the preparation and storage of the alkali see p. 322. Thus, suppose that o-i N.HCl be required. Dilute pure concentrated hydrochloric acid about 90 times with distilled water, measure out 25 cc. and titrate it with the standard alkali, using either methyl red or phenol phthalein as the indicator. Suppose that the 25 cc. of dilute acid require 13-8 cc. of alkali which has been found to be 0-1965 N. Then the normality of the 13-8 acid is x 0-1965 = 0-1085 N. The acid can be used as such, or if exactly o-i N. be required, then 8-5 cc. of distilled water is added to every 100 cc. of the acid, thus bringing it to the desired concentration. It is important to note that acids and alkalies act on glass, and thereby suffer a change in concentration. This is practically avoided by storing in bottles that have been coated internally with a fairly thick layer of paraffin wax. APPENDIX. 385 PIPETTES, ETC. Delivery. In using an ordinary single-volume pipette the fluid is drawn by suction just above the mark and closed with the finger. The lower end of the pipette is then allowed to touch the side wall of the bottle or beaker and the fluid run out till the meniscus is exactly at the mark, the eye being level with the meniscus. The fluid is then allowed to run out into the desired vessel and is then drained for 15 seconds with its point touching the wall of the vessel. The majority of pipettes are calibrated for such a delivery, but for certain operations the author prefers to use pipettes which are caUbrated in such a way that they have to be blown out after draining as above for 15 sees. The reason why these are sometimes preferable is that the amount left in the nozzle of the pipette after drainage may alter considerably with variations in the surface tension of the fluid measured. It is suggested that such pipettes caUbrated for dehvery by blowing should be engraved with the letter "B" to distin- guish them from pipettes calibrated for drainage " D." Ostwald pipettes (see fig. 51) are always calibrated for delivery by being completely blown out. The orifice must be so narrow that it takes about 30 seconds for the delivery of I cc. They are filled by suction to above the mark and then closed with the finger. The exterior is wiped with a piece of filter paper and the fluid run to the mark by holding the point on the filter paper. The fluid is then allowed to fall out by its own weight, the delivery being completed by blowing Fig. 5 1 . ont whilst drawing the point of the pipette up the sides of the Ostwald receiving vessel. pipette. Burettes. The chief precautions to be taken are to allow time for proper drainage, and to be sure that the meniscus is read in the same way at every operation. The author prefers to use burettes that have the marks engraved as complete circles, and to read the meniscus by means of a piece of black paper held behind the burette. The black paper is pasted on to a piece of white card, which is sharply folded at the black edge (see fig. 52). The black edge is held against the back of the burette a trifle below the meniscus, the slanting white card reflecting the light. The meniscus appears as a very sharp black hue. In order to avoid parallax it is important that the eye should be exactly at the level of the meniscus. To ensure this for very accurate work the author has constructed the device shewn in fig. 53. Should the fluid be run rapidly out of a burette ample time must be allowed for proper drainage, the meniscus gradually moving upwards as the fluid runs down the side of the burette. Details of the methods employed for the calibration of pipettes and burettes will be found in most standard works on Quantitative Chemical Analysis.* It is essential that pipettes and burettes should be clean and free from grease. Very considerable errors can be caused by variations in * Representative Procedures in Quantitative Chemical Analysis, by F. A. Gooch (Chapman & Hall, London, 1916), can be recommended. 386 APPENDIX. the amount of fluid adhering in the form of drops in a greasy pipette or burette. Should the burette have a glass stopcock this must be greased. Under these circumstances the fluid must always be run out of the burette by the stopcock. If it is emptied by opening the tap and inverting, the inner wall of the vessel is almost certain to become greasy. When this occurs, WaJ Fig. 52. Fig- 53- Author's device for reading burettes. A is a draw tube containing a lens B. E is a paper disc pierced with a small hole. The tube C and D are blackened. The whole is fastened to a wooden block, F. This is firmly held to the burette by a clip and spring. By placing the finger in the groove G and pressing with the thumb on H, the tube can be moved up and down until the meniscus is sighted. L is a piece of paper, the lower half of which is blackened. The device is for reading to one-tenth of the ordinary graduations of the burette. The nearest tenth is best obtained by the method described above. wash the burette out with water. Fill it with strong (40%) soda. Run this out and then wash it out repeatedly with tap water. Now fill the burette with chromic acid cleaning fluid and allow it to stand over-night. Wash out as before. The burette will now keep free from grease for some time if properly used. APPENDLX. 387 FOLIN'S FUME-ABSORBER. Since the fumes arising from the incineration of a substance with boiling sulphuric acid are extremely irritating, that operation should be conducted in a fume chamber or under a hood. But it is preferable to use the very con- venient apparatus devised by Fohn, since the removal of the condensation water materially accelerates the incineration. r=3I> Fig. 54. FoHn's fume-absorber. The apparatus consists of a bulb C (ij inches in diameter) blown into a piece of fths. tubing. The lower end has blown into it an open piece of narrow tubing 2 J inches in length. The bulb rests on the neck of the flask or test-tube in which the incineration is conducted. To the upper end of the tube is fixed a piece of narrow glass tubing which is bent at a convenient angle and connected by a short length of rubber tubing to a glass tube B. This is of such a size that it just slips into one limb (A) of a T-piece. This is fastened to a board or shelf by metal chps. One end of the horizontal Umb of the T-piece is connected to a suction pump, the other end being joined by a piece of pressure tubing (D) and a length of metal tubing (E) to another T-piece. This can be connected to another fume-absorber. One good pump is sufiScient for 3 absorbers. Those not in use should be stoppered with corks. It is sometimes necessary to fit a rubber collar on to B, so that good suction is obtained through C. Owing to the rubber joints the angles of the hmbs A and F can be varied to suit the heights of the vessels in which the incineration is being conducted. The fumes are carried over by the air current into the pump, a wash bottle containing soda being interposed to prevent damage. The condensation water collects in the pocket below C and can be removed by inverting the fume-absorber at the end of the operation. By inverting a funnel over an evaporating basin, and arranging the apparatus so that the end of the funnel fits loosely into the neck of the absorber, the fumes from boihng nitric acid can be carried ofi. 388 APPENDIX. COLORIMETERS. A high grade colorimeter is a necessary adjunct of a Biochemical Labora- tory, a number of important analyses being made by its use. Fig. 55. Duboscq's Colorimeter. Inset shows construction of vernier scale. The best known instrument is that of Duboscq, which is shewn in fig. 52. The standard solution is placed in one of the cups, B, and the unknown solution in the other cup. The plungers, D, are either cylindrical hollow cups, closed at the bottom, or, preferably, are made of a solid piece of optically clear glass. They can be moved up and down by turning the screw E, which works on a rack and pinion. The height of the bottom of the plungers from the bottom of the cups, that is the depth of the solution used, can be read by means of a scale and vernier at the back of the instrument. The standard is set at a given height (say 15 mm.) and the height of the other plunger adjusted until exact equality of tint is obtained. The light is reflected through the solutions from A, which is either a mirror or a piece of opal glass. After passing through the layers of the fluids on the two sides the hght falls on to the prisms shewn in K of fig. 56. These prisms are contained in the case marked J in fig. 55. The light then passes through the eye piece as shewn. APPENDIX. 389 There are several important points of detail that must be attended to before accurate results can be obtained. (i) See that the zero points of the scales are correct, by carefully lowering the plungers until they touch the cups and noting the readings. ^K ■£ ■^ 8 3 (2) See that the prisms and eye piece are ■clean. Specks of dust seen in the field are apt to lead to erroneous judgments. The prisms can be removed and carefully cleaned with a pointed match covered with two or three layers of silk. Great care must be taken to avoid breaking the prisms away from the cement. (3) See that the illumination of the two fields is equal. This is best tested by placing a coloured solution (such as a 2-5 per cent, solution of potassium dichromate) in both cups, placing one plunger at 15 mm. and adjusting the other until the two fields have an identical appearance. The other plunger should also be at 15 mm. Attention must be paid to the point considered below. (4) Folin has suggested that the best place for an instrument is in the middle of the Laboratory, so that the eye is not dazzled by the light from the window. Retinal fatigue will undoubtedly cause very serious errors and inconsistencies, and Folin's recommendation is valuable. (5) A comfortable body position is im- portant. For some reason the best results are obtained when the observer is in an unstrained position. Folin suggests that the best way of using the apparatus is to place it on a stool about the same height as an ordinary chair, and to sit at the side of the instrument. His method of reading the unknown is to place the standard solution into both cups and to set them at the same height. The instrument being adjusted, both fields should look alike, and the eye gets accustomed to the appearance. The standard in one cup is replaced by the unknown, and one very careful observation is taken. When making a series of comparisons, he re-reads the standard against itself after each of two un- tnowns. Kober's Colorimeter* is a great advance on Duboscq's. The manu- facturers (Klett Manufacturing Co., New York, U.S.A.) kindly sent one to Cambridge for trial. It has been found admirable in every way, and is always used now in preference to the various patterns of Duboscq's. Kober's instrument can also be used as a Nephelometer, i.e. for estimating substances Fig. 56. Diagram of path of rays in Duboscq's Colorimeter. Below are representations of the appearance of the field imder different conditions, that on the left with no fluid in B, and that on the right when the tints are matched. * Journ. of Biol. Chem., xxix., p. 155. 390 APPENDrX. iiiiniiiiiiiiiiiiiiiiiiiiiiMiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iiii|iiiiiiii)niriiiiiiiiii,iiiin Fig. 57. Kober's Colorimeter. Inset shews the form of two of the cups. APPENDIX. 391 by the density of a cloudy precipitate. Though this book does not contain an example of this method of analysis, it is of growing importance, being valuable ■when only very small amounts of material are available. It is also supplied, if desired, with an excellent lamp house, so that it can be used at night. In fact, it is more satisfactory to use artificial light, since by means of mirrors the illumination of the two fields can be made exactly equal. The instrument is shewn in fig. 57, and it will be seen that the plungers are fixed and the cups movable. This does away with the space between the top of the cup and the prism, which is apt to allow an indefinite amount of light through in a Duboscq. The prisms are enclosed and so remain free from dust. The use of dark glass for the sides of the cups and the plungers, the absence of cements, improved mechanical arrangements for adjusting the zero and moving the cups, and the reduction in the volume of fluid necessary, all combine to make the instrument nearer perfection than anything yet introduced.* The calculations necessary in colorimetric work are very simple. The assumption is that the depth of colour is proportional to the concentrations of the substance in the standard and in the unknown. Also that the depth of field of the solutions that must be taken to get equality of tint vary in- versely as the intensity of the colour produced, and therefore as the concen- trations in the standard and unknown. So if the standard contains a certain amount of material in a given volume and the unknown is made up to the same volume, then Amount in standard Reading of unkn own Amount in unknown "" Reading of standard * Both instruments can be obtained from Messrs. Baird and Tatlock (London). 392 APPENDIX. TORSION BALANCE. This instrument is of value for the rapid weighing of small amounts of substances, such as blood taken from a finger-prick, etc. The instrument is used as follows for the weighing of blood on a piece of absorbing paper. Remove the clip G and paper from the arm D. Move C until the indicator A is at zero on the scale. See that the lever E is in such a position that F points to " Free." The movable arm B should now be at O. If this is not so, bring B to O by means of an adjusting screw on the back of the instrument. Now set F to " Stop " by means of E. Hang the clip and paper on to D, seeing that the paper hangs freely. By means of C move the lever A to mark about 120 mgm., set F to " Free," and then move C until B is at O. The reading at A is the weight of the paper and the clip. After the blood has been drawn on to the paper, the weight is again taken as before. This should be done as rapidly as possible to avoid errors due to evaporation. The paper and chp should never be put on or taken off D with F at " Free." The indicator should be set at the approximate weight before the spring is released. By taking these precautions the instrument will remain reliable for a very long time. APPENDIX. 393 THE PREPARATION OP CERTAIN REAGENTS AND LABORATORY REQUISITES. Acid potassium phosphate, see p. 24. Acid potassium phthalate, see p. 24. Almin's reagent. 4 grams, of tannic acid in 8 cc. of strong (33 per cent.) acetic acid and 190 cc. of 50 per cent, alcohol. Alpha-naphthol. i per cent, in strong alcohol. A mmonium molybdaie. Rub up 75 grams, of crystaUine ammonium molyb- date with 300 cc. of strong ammonia. When it has dissolved gradually add the solution to a mixture of 900 cc. of concentrated nitric acid (sp. gr. 1-42) and 400 cc. of distilled water, cooling thoroughly during the addition. Add 1600 cc. of distilled water and filter, if necessary. Ammonium oxalate, 0-2 N. 1-42 per cent, of (COO.NHJjHjO. A mmonium sulphate, saturated solution. Boil 800 grams, of pure crystal- line (NHjjjSOj with about i litre of distilled water. Filter when cold. A mmonium sulphide is usually purchased. Can be prepared by saturating ammonia (i part of concentrated to 2 parts of water) with sulphuretted hydrogen and then adding to this one-third of its volume of ammonia of the same dilution. Asbestos pulp for Gooch crucibles, etc. Cut some long-fibred, soft asbestos into pieces about a quarter of an inch long and digest with concen- trated hydrochloric acid in a large flask in a water bath for an hour, shaking thoroughly at intervals. Filter through a plate or on a Buchner under Ught suction and wash thoroughly with water. Transfer to a large wide-neck stoppered bottle and shake thoroughly with water. A good quality asbestos forms a sludge, whi^h allows of rapid filtration, provided that it be not unduly compressed by too great a suction. To prepare a Gooch crucible for gravimetric analysis, set up the apparatus shewn on p. 351. Pour on enough of the asbestos sludge to form a layer I to 2 mm. thick. On this place a perforated porcelain plate, the diameter of which is shghtly less than that of the crucible, and then add another layer of asbestos. Filter under Ught pressure and pass water through until the filtrate is absolutely clear. The crucible can then be dried in a hot air oven at 110° C, cooled and weighed. The drying and weighing should be repeated until the weight is constant. It should then be tested by passing about 500 cc. of water through it, drying and weighing. If constant it is ready for use. The same crucible can be used for a large number of determinations. Barfoed's reagent, see p. 108. Barium chloride, N. 122 grams, of BaCl2.2H20 to i litre. Baryta mixture. One vol. of barium chloride solution is added two vols, of baryta water. Baryta water. One part of crystalline barium hydroxide is dissolved in 15 parts of boihng water and filtered hot. The filtrate on cooling throws down crystals of barium hydroxide. The supernatant fluid is baryta water. It is about 0-25 Normal. Benedict's solution, qualitative, seep. 107. Benedict's solution, quantitative, see p. 127. 394 APPENDIX. Benzidine hydrochloride, see p. 357. Bromine water, saturated. Made by shaking bromine with cold distilled water. BrUcke's reagent, see p. 37. Calciumlchloride, normal. 55-5 grams, pure anhydrous CaClj, dissolved in water, made up to i htre and filtered. One-fifth normal is a convenient strength for certain exercises. Charcoal, adsorbent. The author has found that certain samples of the charcoal prepared by the Chemical Warfare Department for filling protective gasmasks are highly efficient, being superior to Merck's "blood charcoal." It is hoped that carefully selected supplies wiU shortly be obtainable from Messrs. Baird and Tatlock's and other dealers. Chromic acid cleaning fluid. Ten per cent, of chromic acid in water, or 10 per cent, of potassium bichromate dissolved in 10 percent, (by volume) of sulphuric acid. Cochineal tincture, see p. 357. Collodion solution, see p. 3. Congo red paper. White filter paperis thoroughly wetted with a 0-2 per cent, solution of congo red in water. The paperis pinned up tiU dry and cut into strips. It is turned blue by strong acids. Copper sulphate. 200 grams, of pure crystalUne CuSOj.sHjO aredissolved in distilled water by the aid of heat, cooled and made up to i htre. For the biuret reaction a i per cent, solution is prepared by diluting 5 cc. to 100 cc. Ehrlich's reagent for indol. Para-dimethyl-amido-benzaldehyde 4 parts Alcohol {95 to 98 per cent.) . . 380 ,, Concentrated hydrochloric acid 80 „ Eshach's reagent, see p. 36. Fehling's solution, see p. 106. Ferric chloride, 10 per cent. Glyoxylic reagent, see p. 39. Grease paint, for marking beakers, etc. Dilute Brunswick Black to the desired consistency with naphtha or benzene. Apply with a fine brush. Can be scraped off or removed by means of a pad of cotton wool soaked in naphtha. Gunzberg's reagent. Dissolve 2 grams, phloroglucin and i gram, of vanillin in 30 cc. of absolute alcohol. The solution should be freshly prepared, but it can be preserved for a certain time in dark bottles. The most economical way of preparing it is to make 10 per cent, solutions of phloroglucin and of vanillin in absolule alcohol. These keep for a long time when not. mixed. When wanted the reagent is made by taking i cc. of the phloroglucin and 0'5 cc. of the vanillin solutions and mixing. Iodine solution. About o-i N. Dissolve 25 grams, of potassium iodide in about 200 cc. of distilled water in a stoppered flask. Add 12-7 grams, of iodine and shake till dissolved. Make up to i litre with distilled water. For many purposes this can be diluted 10 times or even more with distilled water, but these weak solutions should be prepared as required. Lead acetate (basic). Boil 464 grams, of normal lead acetate and 264 grams, of litharge in 1500 cc. of distilled water for half an hour with constant stirring. Cool and filter. Or use a saturated solution of the commercial basic lead acetate. APPENDIX. 395 Lead acetate {normal). Saturated solution. Litmus solution. Extract the crushed litmus several times with warm distilled water, mix the extracts and filter. Adjust the solution to a neutral tint by means of hydrochloric acid. The sensitiveness of the indicator is much increased by dialysing it against distilled water. A drop or two of chloroform may be added to the solution to prevent the growth of organisms. Mercuric chloride. Saturated solution, about 8 per cent. Mercuric nitrate. A. To i6o cc. of concentrated nitric acid (sp. gr. i -42) m a beaker add, in small portions, 220 grams, of red mercuric oxide. Stir well and then add 160 cc. of distilled water. Heat till the oxide has dissolved. Cool and nearly neutralise by adding 75 cc. of N. soda. Make up to i litre and filter. Preserve in a dark-coloured bottle. This solution is used for removing various nitrogenous substances from urine when estimating small quantities of sugar (see p. 347). B. To 143 grams, of pure mercury in an evaporating basin add 200 cc. of concentrated nitric acid (sp. gr. 1-42). Heat until thick fumes are evolved and then turn out the gas. When the reaction has ceased light the flame again and evaporate down to about 80 cc. Gradually add about 1500 cc. of water. Cool and make up to 2 litres. Mercuric sulphate reagent for tryptophane. See p. 89. Millon's reagent. See p. 39. It is usually purchased. Nessler's solution. Folin and Denis, Journ Biol. Ctiem., xxvi., p. 478. Phosphotungstic acid. Two per cent, in 5 per cent, sulphuric acid. Picric acid, saturated solution, about 1-2 per cent. Potassium ferricyanide. Saturated solution, prepared by grinding the solid with cold water in a mortar. Potassium ferrocyanide, 5 per cent. Roberts' reagent, see p. 304. Sodium hypobromite. Dissolve 100 grams, of caustic soda in 250 cc. of water. Cool. Cautiously add 25 cc. of bromine, cooUng thoroughly at intervals. It must be recently prepared. Soluble starch. 250 grams, of potato starch is placed in a litre flask. It is treated with a mixture of 375 cc. of water and 125 cc. of pure concentrated hydrochloric acid and the mixture thoroughly shaken until the whole of the starch has been wetted by the acid. It is allowed to digest at room tempera- ture for 8 days, being frequently shaken. The acid is then poured off, the residue repeatedly washed with distilled water and then filtered on a Buchner. To remove the last traces of acid, which inhibit the action of enzymes, it is advisable to suspend the starch in a buffer solution of Ph = 7. This can be prepared approximately by treating 50 cc. of 0-2 M. acid potassium phosphate (see p. 24) vrith 30 cc. of 0-2 N. soda and diluting to 500 cc. After standing for some time with frequent shakings the starch is again washed by decanta- tion with distilled water, filtered on a Buchner and dried in the air. Solutions are prepared in the manner described for starch paste (p. 120). Stokes' reagent, see p. 245. Sulphosalicylic acid, see p. 37. Tannic acid, see Almen's reagent. Tap grease. Heat together in a crucible on a sand bath i part of soft rubber (free from inorganic filUng matter), i part of paraffin wax and 2 to 3 parts of vaseline. Stir thoroughly until the rubber has completely dissolved. INDEX, Absorption spectra, 243 chart of, 376 Acetate bufiers, 28 Acetic acid, dissociation constant of, 12 Aceto-acetic acid, 314 estimation of, 349 preparation of, 316 removal of, 342 tests for, 316 Acetone, estimation of, 349, 351 tests for, 316 Achromic point method, 188, 190 Acid, excretion of, 274 Acidity, 14 estimation of, 275 of gastric juice, 196 of urine, 273 Acid haematin, 247 Acid haematoporphyrin, 248 Acidosis, 275, 315 Acid phosphates, 283 Acid, standard, 27, 384 Acrolein, 160 Active hydrochloric acid, 196 Adenine, 62, 298 Adjusting reaction, 277 Alanine, 68 Albumins, 45 boiling test for, 303 crystallisation of, 50 detection of, 367 heat coagulation, 42 in milk, 170 in urine, 302 preparation of, 47 properties of, 45 removal of, 48 serum, 47 tests for, 47 Albuminoids, 34, 58 Albuminuria, 302 Albumoses (see proteoses), 52 detection of, 369 formation of, 53 hetero, 53 in urine, 304 primary, 53 secondary, 54 separation of, 53 Alcohol, specific gravity of, 383 Aldoses, 40 Alkalies, standard, 26 Alkaline haematin, 247 Alkaline haematoporphyrin, 248 Alkaline tide, 273 Alkali reserve, 275 Alkaloidal reagents, 36 Allantoin, 292 Alloxan, 292, 295 Alloxantin, 295 Alpha-napthol test, iii, 112 Amines, 223 Amino-acids, 67 estimation of, 214, 333 in urine, 333 in various proteins, 71 methods of separation, 70 reactions of, 69 Ammonia, estimation of, 329 in urine, 274 specific gravity of, 382 Ampholytes, 31 Amphoteric electrolytes, 31 Amylase, 117, 186 Amylopectin, 117 Amylopsin, 220 Analysis of blood, 251 et seq. fluids, 365 gastric juice, 195 et seq. solids, 374 urine, 321 et seq. Anti-ferments, 186 Aqueous vapour, tension of, 380 Arabinose, 10 1 Arginine, 69 Asbestos filters, 393 Asparagine, 81 Aspartic acid, 8i Asymmetric, carbon atom, 147 INDEX. 397 Asymmetric compounds, resolution of, 151 Atomic weights, 380 Autolysis, 228 Bacteria, nutrient media for, 226 Bacterial decomposition, 222 Bang's method for chlorides, 257 glucose in blood, 253 Barfoed's test, 108, 114, 115 Beckmann's method, 8 Bence-Jones' protein, 304 Benedict's method for creatine, 341 for sugar, 127 for sugar in blood, 251 for sugar in urine, 347 for sulphur, 359 Benedict's sulphur reagent, 359 Benzidine method for sulphates, 360 test for blood, 306 Bertrand's method tor sugar, 126 ;3-iininazol-ethylamine, 223 Bial's test, 313 Bile, 265 Bile pigments, 267 in urine, 307 Bile salts, 265 action on lipase, 158 in urine, 308 Bilirubin, 267 Biliverdin, 267 Biuret, 41, 287, 291 Biuret reaction for proteins, 40 Blood, chlorides, 257 coagulation of, 234 detection of, 305 glucose in, 249 haemolysis of, 239 in urine, 305 laking of, 239 non-protein nitrogen of, 261 plasma, 234 serum, 234 Boiling points, 383 Bread, 174 Bromine reaction for trytophane, 94, 217 Briicke's reagent, 37 "Buffers," 17 Buffer solutions, standard, 27 Burettes. 385 Cadaverine, 225 Calcified milk, 206, 213 Calcium phosphates in milk, 172 in urinary sediments, 319 in urine, 283 Calcium salts in clotting of blood, 234 in clotting of milk, 208 in heat coagulation of proteins, 43. 45 in milk, 172 in urine, 280, 284 Cane sugar, 116 estimation of, 142 test for, 117 Caramel, 105 Carbamide, see urea Carbohydrates, 100 detection of, 369 estimation of, 125 in proteins, 34, 57 Carboxy-haemoglobin, 295 Carmine fibrin, 204 Casein, 166, 167 action of rennin on, 167, 207 digestion of, 87, 213 iso-electric point, 11 solution for enzyme work, 213 Caseinogen, 34 Cerebrosides, 165 Charcoal, adsorbent, 356 Cheese, 172 Chlorides, detection of, 284 estimation of, 199, 257, 355 in blood, 257 in urine, 281, 355 Cholesterol, 161, 239, 269 preparation oif, 162 reactions of, 162 Choline, 164, 165 Chromic period, 191 Chromoproteins, 34 Claisen flasks, 99 Cleaning fluid for glass, 394 Clotting, see coagulation Coagulation of blood, 234 of milk, 207 of proteins, by alcohol, 37 of proteins, by heat, 42 Co-ferments, 185 Cole and Onslow comparator, 21, 276 on nutrient media, 226 398 INDEX. Cole's apparatus for automatic delivery, 19 1 reading burettes, 386 standard heating power, 136 Cole's method for acidity of urine, 275 amino-acids, 215 diastase, 193 formol titration, 333 lactose, 139 micro-Kjeldahl, 261 sugar in blood, 253 total nitrogen, 327, 329 uric acid, 343 Cole's test for bile pigments, 267, 307 glucose, 109 lactose in urine, 313 sugar in urine, 310 Collagen, 58 Collodion solution, 3 Colloids, I electrical properties of, 9 precipitation of, 10, 11, 13 Colorimeters, Duboscq's, 388 Kober's, 389 Colorimetric determination of Ph, 29 Colour reactions of proteins, 38 Comparators, large, 276 small, 20 Congo red, 22 papers, 394 Copper ammoniacal, preparation of, 291 Copper salt, of aspartic acid, 82 of glycine, 77 Creatine, 178 conversion into creatinine, 179 estimation of, 340, 341 from meat extract, 178 in urine, 298 Creatinine, 178, 298 estimation of, 338 Jaffe's test, 179, 300 origin of, 298 preparation of, 299 Weyl's test, 179, 301 Zinc chloride compound, 300 Cresol, 223, 282 Cryoscopy, 5, 272 Cyanuric acid, 287, 291 Cysteine, 41 Cystine, 41, 61 . m urine, 319 preparation of, 82 properties of, 83, 84 Cytosine, 63 Deposits in urine, 319 Dextrins, 118 detection of, 370 distinction from glycogen, 121 formation of, 118, 121 malto, 119 reactions of, 121 stable, 118 Dextrose, loi see glucose Diabetes, 250, 308 Dialysed iron, 10 Dialysing membranes, 2 Dialysis, 2, 3 > of serum, 48 Diastase, urinary, 362 Diffusion, 2 Digestion, of carbohydrates, 187, 220 of fats, 157 of nudeo-proteins, 5o of proteins by erepsin, 218 of proteins by pepsin, 53, 201 of proteins by trypsin, 212 Dilution, efiect of, on reaction, 18 Disaccharides, 113, 221 Dissociation constant, 18 Distillation in vacuo, 73, 99 Distributor, 191 Dreyer's dropping pipette, 24 Duboscq's colorimeter, 3SS Dulcite, 104 Dunstan's test for glycerol, 160 Earthy phosphates, 2S3, 367 Edestin method for pepsin, 204 Egg-white, 49 Egg albumin, 49 crystallisation of, 50 Ehrlich's test for indol, 227 Electrolytes, action of, on colloids, 13 on heat coagulation, 43 on ptyalin, 187 Emulsification, 156, 157 Emulsins, 184 Emulsoids, i Enantiomorphs, 150 INDIiX. 399 Enterokinase, 210 Enzymes, 183 amylopsin, 220 autolytic, 228 detection of, 371 erepsin, 218 lactase, 221 lipase, 158 maltase, 221 optimum Ph for, 32, 184 oxidase, 230 pepsin, 201 ptyalin, 186 rennin, 207 reversible action of, 185 sucrase, 221 trypsin, 210 tyrosinase, 232 urease, 287 Erepsin, 218 Erythrodextrin, 118 Esbach's albuminometer, 362 reagent, 36 Ethereal sulphates, 281 detection of, 285 estimation of, 359, 361 preparation of, 285 Euglobulin, 46 Fats, 153 digestion of, 157 emulsification of, 156 estimation of, 169 in cheese, 172 in milk, 169 saponification of, 155 Fatty acid, 160 Fehling's method for estimation of glucose, 171 Fehling's solution, preparation, 106 Fehling's test, 106 Fermentation test, 311 Ferments, see enzymes Fibrin, ferment, 236 Fibrinogen, 234 Flour, 173 Fluoride plasma, 237 Folin and Denis on non-protein nitrogen, 263 •on sugar in milk, 172 Folin and McEllroy method for sugar, 129 Folin's fume-absorber, 387 Folin's method for ammonia, 330 for creatine, 340 for creatinine, 339 for cystine, 82 for sulphates, 358 Folin-Schaffer method for uric acid, 343 Folin's test for uric acid, 296 Formol titrations, 214 Fraunhofer's lines, 243 Free hydroi;hloric acid, 196, 200 Freezing points, 5, 7 Fructose, loi, iii Fruit sugar, see fructose Fuld's method for pepsin, 204 Fume-absorber, 387 Furfurol, 112 Fusion mixture, 64 Galactolipin, 165 Galactose, loi, 165 Gastric juice, 194 acidity of, 198 active hydrochloric acid, 196 analysis of, 197 free hydrochloric acid, 196 in disease, 198 mineral chlorides of, 200 total chlorides of, 199 Gelatin, 58, 369 Gerhardt's test, 316 GUadins, 34, 173 Globulins, detection of, 367 eu-, 46 in muscle, 176 in serum, 46, 47 preparation of, 46, 47 properties of, 45 pseudo-, 46 Gluconic acid, 104 Glucoproteins, 57 Glucosazone, no Glucose, loi, 103 estimation of, 125, 251, 346 fermentation of, 311 in blood, 249 in urine, 308, 346 phenyl-osazone of, no preparation of, 105 properties of, 104, in Glucosides, 103, 184 Glutaminic acid, 78 Glutelins, 34, 173 ' 400 INDEX. Gluten, 173 Glycerol, 154, 159 Glycerophosphoric acid, 164 Glycine, 72 Glycine ester hydrochloride, 76 Glycocholic acid, 265 Glycogen, 123, 182 estimation of, 123 identification of, 370 preparation, 124 reactions of, 124 Glycoproteins, 34 Glycosuria, 250, 309 Glycuresis, 309 Glycuronic acid, 104 Glyoxylic reaction, 39, 95 reagent, 39 Gmelin's reaction, 269 Graham and Poulton on estimation of creatinine, 339 Graham on gastric contents, 198 Grape-sugar, see glucose Guanine, 62, 65 Guiaconic acid, 232 Guiacum tincture, preparation of, 232 Gunsberg's test, 197 Haematin, 247 Haematoporphyrin, 248 in urine, 279 Haematuria, 305 Haemin, 249 Haemochromogen, 248 Haemoglobin, 240 in urine, 305 Haemolysis, 238 Haser's coefi&cient, 272 Hay's test for bile salts, 267, 308 Heat coagulation, 42 Heating power, standard, 136 Heller's test for albumin, 49, 303 for blood, 305 Hepatic disease, ammonia in, 301 creatinine in, 298 urea in, 286 urobilin in, 278, 280 Hetero-albumose, 53 Hetero-xanthine, 298 Hexosans, 117 Hexoses, 10 1 Hippuric acid, 77 Histamine, 223 Histidine, action of bacteria on, 223 preparation of, 84 properties of, 86 Histones, 34 Hopkins-Cole method for uric acid, 343 test for proteins, 39 Hopkins' method for uric acid, 343 test for lactic acid, 180 Huppert-Cole test for bile pigments, 267 Hurtley's test for aceto-acetic acid, 317 Hydrochloric acid, active, 196 in gastric juice, 195 normal, 384 specific gravity of, 381 Hydrogen-ions, concentration of, 14 estimation of, 29 Hydrolysis of fats, 155, 158, i6i of proteins, 70, 72, 84, 98 of starch, 118, 121 Hypobromite, action on urea, 288, 366 method for urea, 336 preparation of, 290 Hypoxanthine, 62, 65, 180, 29 Indican, 318 Indicators, 19 preparation of, 24 ranges of, 22 Indol, 225 aldehyde, 94 from tryptophane, 227 mother substance of, 227 tests for, 227 Indoxyl, 318 Internal compensation, 152 Intestinal extracts, 218 Inuhn, III Invertase, see sucrase Invert sugar, in, 116 Iodine value, 156 Iron in haemoglobin, 241 in urine, 280 Isoelectric point, definition of, 11, 31 determination of, 1 1 of various substances, 33 Isoleucine, 98 INDEX. 401 Jaffe's test ior creatinine, 179, 300 for indican, 318 Jones, W., on nucleic acid, 64 Kataphoresis, g Kephalin, 165 Kerasin, 165 Keratin, 59 Ketoses, 100 Kjeldahl's method, 321 Kober's colorimeter, 389 Lactalbumin, 170 Lactase, 221 Lactic acid, 180 Hopkins' test for, 180 in gastric contents, 195 in muscle, 181 Uffelmann's test for, 181 Lactosazone, 115, 171 Lactose, 115, 171 Cole's test for, 313 distinction from glucose, 115, 171 estimation of, 139, 172 in milk, 171 in urine, 313 osazone, 115, 171 Laevulose, loi, 11 1 see also fructose Laking of blood, 238 Larrson's method for chlorides, 356 Lecithin, 163 Leucine, 97 preparation of, 98 properties of, gg Liebermann-Burchard test for cholesterol, 162 Ling's method for sugar, 141 Ling's indicator, 141 Lipase, 158 Ijpines, 153 Logarithms, see back cover Long's coefficient, 272 Lysine, 69 Maltase, 113, 184, 221 in pancreatic extracts, 221 intestinal, 221 Malto-dextrin, iig Maltosazone, 115 Maltose, 113 action of enzymes on, 221 distinction from glucose, 114 estimation of, 129, 131, 134 preparation of, 113 Mannite, 104 Mannose, loi Meat, see muscle, 175 Meig's method for fat, 169 Mellanby, E., on origin of creatinine, 298 Mellanby, J., on amylopsin, 220 on estimation of trypsin, 214 on pancreatic extracts, 212 on trypsin, 211 Mercuric nitrate, action on urea, 290 action on proteins, 35 preparation of, 395 Mercuric sulphate reagent, for acetone bodies, 349 for tryptophane, 84 Mercury green, rotations with, 147 Mesotartaric acid, 152 Metaproteins, 51 detection of, 367 Methaemoglobin, 246 Mett's tubes, 205 method for pepsin, 202, 205 Micro-balance, 392 Micro-methods for chlorides in blood, 257 for sugar in blood, 253 for total nitrogen, 329 Milk, 166 calcified, 206 clotting, 167, 207 composition of, 167 estimation of fat in, 169 estimation of lactose in, 172 inorganic constituents of, 172 Milk sugar, see lactose Millon's reaction, 38, 97 reagent, 38 Mineral chlorides of gastric juice, 196, 200 Molecular weight of casein, 166 of egg-albumin, 7 of haemaglobin, 241 Molisch's test, 41 Monosaccharides, 100 Morner's reagent for tyrosine, 97 Mucic acid, 104 test for lactose, 314 Mucin, 57 in bile, 268 in saliva, 186 preparation of, 57 402 INDEX. Mucoid, 50 Mulder's test, 109 Murexide test, 295 Muscle, 175 extract, 176 Mutarotation, 102 Myosin, 176 preparation of, 177 Myosinogen, 176 Nephelometer, 3S9 Neumann's method for organic phosphorus, 169 Neutral salts, action of, on colloids, 13 on heat coagulation, 43 on ptyalin, 187 Neutral sulphur, 282 estimation of, 359, 361 Nicol's prism, 144 Nitric acid, action on proteins, 38, 49, 55 Nitrogen, distribution of, 270 estimation of, 321 non-protein, in blood, 260 Non-protein nitrogen of blood, estimation of, 260 Normal saline, 238 Normal solutions, preparation of, 26, 384 Nucleases, 61 Nucleic acid, 60 hydrolysis of, 65 preparation of, 64 Nucleohistone, 60 Nucleoproteins, 60 detection of, 368 in bile, 269 preparation of, 63 Nucleosides, 61 Nucleotides, 61 Nylander's test, 109 Oleic acid, 154 Oliver's test for bile salts, 267 Optical activity, 147 Optically active compounds, resolution of, 151 Organic phosphorus, detection of, 169 Osazone, of glucose, no of lactose, 115 of maltose, 115 preparation of, no Osmotic pressure, 3-7 of colloids, 7 of urine, 272 Ost's method for sugar, see Wood-Ost Ovo-mucin, 49 Ovo-mucoid, 50 Oxalate of calcium, 172, 319 urea, 288 Oxalate plasma, 237 Oxidases, 230 Oxy-butyric acid, 314, 350 Oxy-haemoglobin, 241 crystallisation of, 242 in urine, 305 spectrum of, 244 Palmer on acid excretion, 275 Palmitic acid, 154 Palmitin, 155 Pancreas, extract of, 88, 220 Parabanic acid, 292 Paracasein, 167 Paramyosinogen, 176 Paraxanthine, 298 Pavy's method for sugar, 125 Pentosans, 117 Pentoses, 10 1 in urine, 312 Pepsin, 201 action on protein, 53, 202 conditions of action of, 201 detection of, 204 distinction from rennin, 208 estimation of, 202 law of action of, 202 optimum reaction, 32, 201 products of action, 53, 202 Peptide linkage, 41, 202 Peptones, 52, 54, 56 detection of, 369 formation of, 202 reactions of, 56 removal from fluids, 58 Peroxidase, 231 Peters, Amos, method for sugar, 134 Pettenkofer's test, 266 Ph, 16 Ph, determination of, 30 Phenol, 223, 282 Phenol red, 22 Phenyl-alanine, 68 Phenyl-osazone, see osazone INDEX. 403 Phosphates, add, 283 acid potassium, 25 calcium, 283 distinction from proteins, 44 earthy, 283 estimation of, 354 iti milk, 172 in urine, 282, 319 stellar, 319 triple, 320 Phosphatides, 162 PhosphoUpins, 162 Phosphoproteins, 34 Phosphorus in proteins, detection of, i6g Phthalate, acid potassium, 25 Picramic acid, no preparation of, 251 Picric acid, purification of, 251, 338 Pigments, identification of, 371 of bile, 267 of blood, 242-249 of muscle, 175 of urine, 278 Piotrowsld's reaction, 40 Pipettes, Dreyer's dropping, 23 method of discharging, 385 Ostwald, 385 Plasma, fluoride, 237 oxalate, 237 salted, 235 Polarimetric estimation of sugar, 127 Polarimeter, description of, 145 Polarized light, 143 Polypeptides, 35 Polysaccharides, 100, 117 Potassium permanganate, standardisation of, 131 Potatoes, 173 Primary albumoses, 53 Prolamines, 34 Protamines, 34 Proteins, 33 action of alcohol on, 37 bacterial decomposition of, 223 carbohydrate groups of, 57 classification of, 33 colour reactions of, 38 conjugated, 34 crystallisation of, 50, 242 definition, 33 detection of, 367 general reactions of, 34 heat coagulation, 42, 44 hydrolysed, 35 hydrolysis of, 70, 72, 84, < 211, 21^, 218 in bile, 268 0, 202, in unne, 302, 304 of muscle, 176 osmotic pressure of, 7 peptic digestion of, 53, 202 percentage composition of, 33 phosphorus in, 169 precipitants, 36, 37 properties of, 35-39 removal of, 48, 56, 250, 260 sulphur in, 41 tryptic digestion of, 211 Proteoses, ses albumoses Prothrombin, 234 Proto-albumose, 53 Prout- Winter method for chlorides, 199 Ptyalin, action of, 119, 187 estimation of, 188 Purine bases, 62 in meat, 179 in urine, 298 Purpuric acid, 295 Putrescine, 225 Pyrimidine bases, 63 PyroUidone carboxylic acid, 80 Racemic compounds, 94, 151 Raistrick on bacterial decomposi- tion, 223 Ranges of indicators, 22 Reaction of fluids, 23 of urine, 273 Reduced alkaline haematin, 248 Reduced oxaUc acid, 39 Reducing sugars, 106 Removal of proteins, 10, 36, 37, 48, 56 Renal disease, 263, 272, 275 Rennet ferment, 207 Rennin, 207 distinction from pepsin, 209 Ribose, loi Roberts' test, 304 Rotatory power, specific, 146 Rothera's test, 316 Saccharic acid, 104 Saccharose, see cane sugar Safranine test, 109 Saliva, 186 404 INDEX. Salivary amylase, 183 SalkowsM's test for cholesterol, 162 Salted plasma, 236 Salting out, 161 Saponification, 154 Saponification value, 154 Sarcolactic acid, 180 Sarcosine, 178 Scatol, 223, 225 Scherer's method, 361 Schiff's test, 296 Schumm's test, 306 Schiitz-Borissow's law, 202 Scleroproteins, 34, 58 Secondary albumoses, 54 Sediments in urine, 319 SeMwanofi's test, 112 Serum, 234 Soaps, 154, 160, 161 Sodium hydroxide, standard solutions of, 26, 322, 384 Solids, analysis of, 374 Soluble myosin, 176 Soluble starch, 118, 395 Sorbite, 104 Sorensen's method, 70, 214 Soya bean method, 335 Specific gravity of alcohol, 403 ammonia, 382 hydrochloric acid, 381 milk, 167 potassium hydroxide, 382 sodium hydroxide, 382 sulphuric acid, 381 urine, 271 Specific oxygen capacity, 241 Specific rotatory power, 146 Spectroscope, 243 Spiegler's test, 304 Standard solutions of buffers, 26 hydrochloric acid, 27, 384 sodium hydroxide, 26, 322, 384 Starch, 117 cellulose, 117 digestion of, 119, 187, 370 grains, 119 hydrolysis of, 121 reactions of, 119, 174 paste, preparation of, 120 soluble, preparation of, 395 Steapsin, 5e«' lipase Stearic acid, 154 Stearine, 160 Stellar phosphates, 319 Stercobilin, 267 Sterols, 154 Stokes' fluid, 153 Sucrase, 116 Sucrose, 221 Sugars, 100 ■ estimation of, 125 in blood, 250 in urine, 308, 312 polarimetric estimation of, 127 tolerance for, 250 Sulphates in urine, 281 estimation of, 358 Sulphosahcylic acid, 37 test for albuminuria, 304 Sulphur estimation of, 358 in proteins, 41 in urine, 281 reaction for proteins, 41 Sulphuric acid, specific gravity of, 381 Sulphur test for bile salts, 266, 308 Suspensoids, i, 2 Synalbumose, 53 Tartaric acid, 152 Taurine, 265 Teichmann's crystals, see haemin Tensions of aqueous vapour, 380 Test meal, 193 Thio-albumose, 53 Thrombin, 236 Thrombokinase, 234 Thymine, 63 Tolerance for sugar, 250, 308 ToUen's test for glycuronic acid, 317 for pentoses, 312 Torsion balance, 392 Total nitrogen, estimation of, 322 Totani's reaction, 87 Triolein, 155 Tripalmitin, 155 Triple phosphates, 320 Tristearin, 155 Trommer's test for glucose, 105 Trypsin, 210 estimation of, 214 optimum reaction of, 211 preparation of, 212 products of action, 67, 211 INDEX. 405 Tryptic, 226 Tryptophane, 40, 87 action of bacteria on, 226 preparation, 87 properties of, 93, 217 Tyrosinase, 232 Tyrosine, action of bacteria on, 223 preparation of, 95, 218 properties, 96 Uffelmann's test for lactic acid, 181 Uracil, 63 Urates, 293 Urea, 286 constitution of, 288 detection of, 290 estimation of, 343 isolation of, 291 nitrate, 289 oxalate, 288 Uric acid, 292 crystals of, 294, 297, 319 estimation of, 343 in urine, 297 origin of, 63, 294 Urine, abnormal, 302 acidity of, 30, 273 albumin in, 302 average composition, 270 deposits in, 319 diastase in, 362 inorganic constituents, 280 osmotic pressure, 272 Ph , 30, 273 pigments, 278 proteins in, 302 reaction, 30, 273 specific gravity of, 271 sugar in, 308 total nitrogen of, 321 total solids of, 272 Urinometer, 272 Urobilin, 267, 278 tests for, 279 Urobilinogen, 267, 278 Urochrome, 278 Uroerythrin, 278 Urorosein, 279 Valine, 68 Volhard's method for chlorides, 199, 355 Weights and measures, 379 Werner on urea, 288 Weyl's test for creatinine, 179, 301 Wheat flour. 174 Whey, 207 Witte's peptone, 52 ' Wohlgemuth's method for diastase in urine, 362 for pvtalin, 188, 192 Wood-Ost's method for sugar, 126 131 Xanthine, 180 Xanthoproteic reaction, 38 Xylose, loi Printed by W Heffer and Sons Ltd., Cambridge, England LOGARITHMS. 10 n 12 13 14 15 16 17 18 19 20 21 22 23 24 25 25 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 0000 0043 0085 012S 0170 0414 0453 0492 0531 0559 0792 0S28 0864 0899 0934 1139 1173 1206 1239 1271 1461 1492 1523 1553 1584 1761 1790 1818 1847 1875 2041 2068 2095 2122 2148 2304 2330 2355 2380 2405 2553 2577 2601 2625 2648 2788 2810 2833 2856 2878 3010 3032 3054 3075 3096 3222 3243 3263 3284 3304 3424 3444 3464 3483 3502 3617 3636 3655 3674 3592 3802 3820 3838 3855 3874 3979 3997 4014 4031 4048 4150 4166 4183 4200 4216 4314 4330 4346 4362 4378 4472 4487 4502 4518 4533 4624 4639 4654 4669 4583 4771 4786 4800 4814 4829 4914 4928 4942 4955 4969 5051 5055 5079 5092 5105 5185 5198 5211 5224 5237 5315 5328 5340 5353 5366 5441 5453 5465 5478 5490 5563 5575 5587 5599 5611 5582 5694 5705 5717 5729 5798 5809 5821 5832 5843 5911 5922 5933 5944 5955 6021 6031 6042 6053 6054 6128 6138 6149 5160 6170 6232 6213 5253 6263 6274 6335 6345 6355 6365 6375 6435 6444 6454 6464 6474 5532 6542 6551 5561 6571 6628 6637 5646 6656 6665 6721 6730 6739 6749 6758 6812 6821 6830 5839 6848 5902 6911 5920 692S 6937 6990 6998 7007 7016 7024 7076 7084 7093 7101 7110 7160 7168 7177 7185 7193 7243 7251 7259 7257 7275 7324 7332 7340 7348 7356 0212 0253 0294 0334 0374 0607 0645 0682 0719 0755 0969 1004 1038 1072 1106 1303 1335 1367 1399 1430 1614 1544 1673 1703 1732 1903 1931 1959 1987 2014 2175 2201 2227 2253 2279 2430 2455 2480 2504 2529 2672 2695 2718 2742 2765 2900 2923 2945 2967 2989 3118 3139 3160 3181 3201 3324 3345 3365 3385 3404 3522 3541 3560 3579 3598 3711 3729 3747 3766 3784 3892 3909 3927 3945 3962 4055 4082 4099 4116 4133 4232 4249 4265 4281 4298 4393 4409 4425 4440 4456 4548 4564 4579 4594 4609 4698 4713 4728 4742 4757 4843 4857 4871 4885 4900 4983 4997 5011 5024 5038 5119 5132 S145 5159 5172 5250 5263 5275 5289 5302 5378 5391 5403 5416 5428 5502 5514 5527 5539 5551 5623 5635 5647 5658 5670 5740 575-' 5763 5775 5786 5855 5866 5877 588S 5899 5966 5977 5988 5999 6010 5075 6085 6096 6107 6117 6180 5191 6201 6212 6222 6284 6294 6304 6314 6325 6385 6395 5405 6415 6425 6484 6493 6503 5513 6522 6580 6590 6599 6609 6618 6675 6584 5593 6702 6712 6767 6776 6785 6794 6803 6857 6855 5875 6884 6893 6946 6955 6964 6972 6981 7033 7042 7050 7059 7067 7118 7126 7135 7143 7152 7202 7210 7218 7226 7235 7284 7292 7300 7308 7316 7364 7372 7380 7388 7396 Differences. 1234 56789 4 8 12 17 21 25 29 33 37 4 8 11 15 19 23 26 30 34 3 7 10 14 17 21 24 28 31 3 6 10 13 16 19 23 26 29 3 5 9 12 15 IS 21 24 27 3 6 8 11 14 17 20 22 25 3 5 8 11 13 16 18 21 24 2 5 7 10 12 15 17 20 22 2 5 7 9 12 14 16 19 21 2 4 7 9 11 13 16 18 20 2 4 6 8 11 13 15 17 19 2 4 6 8 10 12 14 16 18 2 4 6 8 10 12 14 15 17 2 4 6 7 9 11 13 15 17 2 4 5 7 9 11 12 14 16 2 3 5 7 2 3 5 7 2 3 5 6 2 3 5 6 13 4 6 13 4 6 13 4 6 13 4 5 13 4 5 13 4 5 12 4 5 12 4 5 12 3 5 12 3 5 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 4 12 3 3 12 3 3 12 2 3 12 2 3 12 2 3 1 2 3 9 10 12 14 15 8 10 11 'l3 15 8 9 11 13 14 8 9 11 12 14 7 9 10 12 13 7 9 10 11 13 7 8 10 11 12 7 8 9 11 12 6 8 9 10 12 6 8 9 10 11 5 7 9 10 11 6 7 8 10 11 6 7 8 9 10 6 7 8 9 10 5 7 8 9 10 5 6 5 6 5 5 4 5 4 5 5 9 10 7 8 9 7 8 9 7 8 9 7 8 9 7 8 9 7 7 8 6 7 8 6 7 8 6 7 8 4 5 6 7 4 5 6 7 8 4 5 6 7 7 4 5 6 6 7 4 5 6 6 7 S 6 7 8 9 LOGARITHMS. I Differences. 12 3 4 5 6 7 55 56 57 53 59 GO 61 62 63 54 65 65 67 68 69 70 71 72 73 74 75 76 77 78 79 80 SI 82 83 84 85 86 87 88 83 90 91 92 93 94 95 96 97 98 99 7404 7412 7419 7427 7435 7482 7490 7497 7505 7513 7559 7566 7574 7582 75S9 7634 7642 7649 7657 7664 7709 7716 7723 7731 7738 7782 7789 7795 7803 7810 7853 7860 7868 7875 7882 7924 7931 7938 7945 7952 7993 8000 8007 8014 8021 8062 S069 8075 8082 8089 8129 8135 8142 8149 8156 8195 8202 8209 8215 8222 8261 8267 8274 8280 8287 8325 8331 8338 8344 8351 8388 8395 8401 8407 8414 8451 8457 8463 8470 8+75 8513 8519 8525 8531 8537 8573 8579 8585 8591 8597 8633 8639 8645 8651 8657 8592 8598 8704 8710 8716 8751 8756 8752 8768 8774 8808 8814 8820 8825 8831 8865 8871 8876 8882 8887 S921 8927 8932 8938 8943 8975 8982 8987 8993 8998 9031 9036 9042 9047 9053 9085 9090 9096 9101 9106 9138 9143 9149 9154 9159 9191 9195 9201 9206 9212 9243 9248 9253 9258 9263 9294 9299 9304 9309 9315 9345 9350 9355 9360 9365 9395 9400 9405 9410 9415 9445 9450 9455 94t)0 9455 9494 9499 95O4 9509 9513 9542 9547 9552 9557 9562 9590 9595 9500 9505 9609 9538 9643 9647 9652 9657 9585 9689 9694 9699 9703 9731 9736 9741 9745 9750 9777 9782 9786 9791 9795 9823 9827 9832 9836 9841 9868 9872 9877 9881 9886 9912 9917 9921 9926 9930 9956 9961 9965 9969 9974 7443 7451 7459 7466 7474 7520 7528 7536 7543 7551 7597 7504 7512 7519 7627 7572 7579 7585 7594 7701 7745 7752 7760 7767 7774 7818 7825 7832 7839 7846 7889 7896 7903 7910 7917 7959 7955 7973 7980 7987 8028 8035 8041 8048 8055 8096 8102 8109 8115 8122 8162 8159 8176 8182 8189 8228 8235 8241 8248 8254 8293 8299 8306 8312 8319 8357 8363 8370 8375 8382 8420 8426 8432 8439 8445 8482 8488 8494 8500 8506 8543 8549 8555 8561 8557 8503 8609 8615 8621 8627 8563 8669 8675 8681 8686 8722 8727 8733 8739 8745 8779 8785 8791 8797 8802 8837 8842 8848 8854 8859 8893 8899 8904 8910 8915 8949 8954 8960 8965 8971 9004 9009 9015 9020 9025 9058 9063 9069 9074 9079 9112 9117 9122 9128 9133 9165 9170 9175 9180 9186 9217 9222 9227 9232 9238 9269 9274 9279 9284 9289 9320 9325 9330 9335 9340 9370 9375 9380 9385 9390 9420 9425 9430 9435 9440 9-159 9474 9479 9484 9489 9518 9523 9528 9533 9538 9566 9571 9576 9581 9586 9514 9519 9624 9628 9533 9661 9665 9671 9675 9680 9708 9713 9717 9722 9727 9754 9759 9753 9768 9773 9800 9805 9809 9814 9818 9845 9850 9854 9859 9863 989g 9894 9899 9903 9908 9934 9939 9943 9948 9952 9978 9983 9987 9991 9996 2 2 3 4 5 5 6 7 2 2 3 4 5 5 6 7 2 2 3 4 5 5 6 7 1 2 3 4 4 5 6 7 1 2 3 4 4 5 6 7 1 2 3 4 4 5 6 6 1 2 3 4 4 5 6 6 1 2 3 3 4 5 6 6 1 2 3 3 4 5 5 6 1 2 3 3 4 5 5 6 1123 34556 112 3 3 4 5 5 6 112 3 3 4 5 5 6 112 3 3 4 4 5 6 1122 34456 1 2 2 1 2 2 12 2 12 2 12 2 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 4 4 5 6 3 4 4 5 5 3 4 4 5 5 3 4 4 5 5 3 4 4 5 5 3 3 4 5 5 3 3 4 5 5 3 3 4 4 5 3 3 4 4 5 3 3 4 4 5 1 2 2 3 3 4 4 5 1 2 2 3 3 4 4 5 1 2 2 3 3 4 4 5 1 2 2 3 3 4 4 5 1 2 2 3 3 4 4 5 1 2 2 3 3 4 4 5 1 1 2 3 3 4 4 5 1 2 2 3 3 4 4 1 2 2 3 3 4 4 1 2 2 3 3 4 4 1 1 1 1 1 1 1 1 1 1 2 112 1 2 1 2 1 2 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 4 4 2 3 3 3 4 1234 56789