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 in Biological Chemistry 
 
HANDBOOK 
 
 FOR THE 
 
 BIO-CHEMICAL LABORATORY. 
 
 INCLUDING 
 
 METHODS OP PREPARATION 
 
 AND 
 
 NUMEROUS TESTS 
 
 ARRANGED ALPHABETICALLY, 
 
 BY 
 
 JOHN" A. MANDEL, 
 
 Professor of Chemistry at the Neio York College of Veterinary Surgeons, 
 
 and Assistant to the Chair of Chemistry, etc., at the Bellevue 
 
 Hospital Medical College and the College of 
 
 the City of New York. 
 
 FIRST EDITION'. 
 FIRST THOUSAND. 
 
 NEW YORK: 
 
 JOHN WILEY & SONS. 
 
 London : CHAPMAN & HALL, Limited. 
 
 1896, 
 
Copyright, 1896, 
 
 BY 
 
 JOHN A. MAN DEL. 
 
PREFACE. 
 
 In" this little handbook an attempt has been made to give 
 concise directions for preparing the most important sub- 
 stances that enter into the composition of the fluids and 
 tissues of the animal body. The methods herein presented. 
 are compiled from the most recent and important works on 
 physiological chemistry; and in certain instances two or three 
 procedures are given for obtaining the same result. 
 
 The two hundred or more tests are arranged in alphabetical 
 order ; and the name of the scientist who suggested the test, 
 or the name under which it is ordinarily known, is given in 
 each case. My most earnest desire in compiling this hand- 
 book has been both to facilitate general work in bio-chemical 
 laboratories and to afford the student an opportunity to have 
 conveniently at hand all the necessary facts in modern 
 scientific testing, so that loss of time in consulting works of 
 reference might be reduced to a minimum. 
 
 John A. Mandel. 
 
 College op the City of New York, 
 Januar}'-, 1896. 
 
 iii 
 
LIST OF PREPARATIONS, 
 
 PAGE 
 
 Adenin , . . . 55 
 
 Albuminates 14 
 
 Allantoin. . . 51 
 
 Arnylopsin , 36 
 
 Artificial gastric juice ........ 32 
 
 Aspartic acid 40 
 
 Bilirubin 28 
 
 Biliverdin 29 
 
 Carbon monoxide hse moglobin . 21 
 
 Casein 13 
 
 Cholalic acid 25 
 
 Cholesterin 30 
 
 Creatin 53 
 
 Creatinin „ . 54 
 
 Dextrose 4 
 
 Fatty acids 7 
 
 Fibrin 15 
 
 Fibrin ferment, 22 
 
 Fibrinogen 11 
 
 Glucose o... 4 
 
 Glutamic acid 41 
 
 Glycocholic acid 23 
 
 Glycocoll 26 
 
 Glycogen ... 1 
 
 Glycuronic acid 60 
 
 Guanin. 55 
 
 Haemoglobin , . . . . 21 
 
 Hippuric acid 50 
 
 Hypoxanthin 55 
 
 Indol 41 
 
 Inosit 6 
 
 Lactic acid (fermentation) ..... 44 
 
 PAGE 
 
 Lactic acid (sarco) 45 
 
 Lactose 2 
 
 Lecithin 19 
 
 Leucin 37 
 
 Maltose 3 
 
 Methaemoglobin , 22 
 
 Myosin 11 
 
 Nuclein 17 
 
 Nucleinic acids 18 
 
 Ov-albumin 9 
 
 Ov-vitellin 12 
 
 Oxyhemoglobin , 20 
 
 Pepsin 31 
 
 Peptones » . . 16 
 
 Protagon 46 
 
 Ptyalin 31 
 
 Rennin 34 
 
 Serum albumin 8 
 
 Serum globulin 10 
 
 Skatol 42 
 
 Soap 8 
 
 Steapsin. 37 
 
 Taurin 27 
 
 Taurocholic acid D . . 24 
 
 Trypsin 35 
 
 Tyrosin 37 
 
 Urea 47 
 
 Uric acid 48 
 
 Urobilin... 58 
 
 Vitellin... 12 
 
 Xanthin 55 
 
HANDBOOK FOR BIO-CHEMICAL 
 LABORATORY. 
 
 Glycogen, C 6 H 10 O 5 . 
 
 Preparation. — 1. Kill a large well-fed rabbit by cutting its 
 throat, open the abdomen immediately and remove the liver. 
 After weighing, cut it up in rather large pieces and quickly 
 throw them in boiling water (about 400 c.c. to 100 grms. liver), 
 and let boil for half an hour. Then remove the pieces and 
 grind them up finely in a mortar, return to the boiling water 
 and add caustic potash solution (3-4 grms. KOH to 100 grms. 
 liver). Now warm on the water-bath, and allow it to concen- 
 trate until you have 200 c.c. of liquid for every 100 grms. liver. 
 If a scum forms on the surface, place the liquid in a beaker, 
 covering it with a watch-glass, and heat it until all has dis- 
 solved, then put aside to cool. Neutralize with HOI and pre- 
 cipitate the albuminous bodies by the alternate addition of HC1 
 and a solution of potassio-mercuric iodide * (Brilcke reagent) 
 in small portions. The addition of potassio- mercuric iodide 
 must be continued until no further precipitate occurs. If 
 the liquid at last remains milky, nearly neutralize with 
 caustic soda, and then treat with HOI again. Filter off the 
 precipitate of albuminous bodies through thick filter-paper, 
 and wash by removing the precipitate from the filter by means 
 of a spatula and place it with water containing HC1 and 
 
 * This solution is prepared by saturating a boiling, not too concen- 
 trated (10^) solution of potassium iodide with pure mercuric iodide and 
 filtering after cooling. 
 
 1 
 
2 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 potassio-mercuric iodide and then return to filter. This 
 treatment must be repeated at least four times. The 
 several filtrates are united and, while stirring, treated with 2 
 vols. 96$ alcohol, which precipitates the glycogen, and allow 
 it to stand in a cool place overnight. Filter off: the precipitate 
 and wash first with 62$ and then with 98$ alcohol. This 
 glycogen generally contains but a trace of albumins, but if re- 
 quired more pure, dissolve it while still moist in a little warm 
 water, add some HC1 and potassio-mercuric iodide after al- 
 lowing to cool, and proceed as above. Lastly, wash the glyco- 
 gen, which has been previously treated with absolute alcohol, 
 a couple times with ether, and allow it to dry in the air or 
 over sulphuric acid. (E. Kulz.) 
 
 2. Briicke's method consists in precipitating the albumin- 
 ous bodies from the watery extracts by HC1 and potassio- 
 mercuric iodide without previously extracting with- caustic 
 potash, and then proceeding as above directed. 
 
 Properties. — Glycogen is a white amorphous powder, easily 
 soluble in hot water, yielding an opalescent solution, which 
 when allowed to evaporate on the water-bath forms a pellicle 
 over the surface which disappears again on cooling. The 
 solution is dextro-rotatory, (a) D — -\- 211° (Kulz). On boil- 
 ing with dilute mineral acids, or by the action of diastatic 
 enzymes (ptyalin, diastase), it is readily converted into malt- 
 ose, isomaltose, and dextrose. Its solution does not reduce 
 Fehling's solution on boiling, but holds copper oxyhydrate in 
 solution in alkaline liquids. With a solution of iodine glyco- 
 gen solutions are colored wine-red which disappears on heat- 
 ing. Glyoogendoes not ferment with yeast. 
 
 Lactose, C 13 H aa O n +H 3 0. 
 
 (MILK-SUGAR.) 
 
 Preparation. — The sweet whey obtained after the precipi- 
 tation of casein from milk (seepage 13) is heated to boiling, 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 3 
 
 filtered, evaporated to dryness with magnesium carbonate, and 
 the residue extracted with alcohol. Exhaust the part insol- 
 uble therein with hot water, filter, and evaporate the filtrate 
 to a syrupy consistency, and allow to stand in a cool place un- 
 til the lactose crystallizes out. If the syrup is at all colored, 
 the solution must be decolorized by passing the solution 
 through animal charcoal. 
 
 Properties. — Lactose crystallizes in rhombic prisms which 
 contain a molecule of water of crystallization. It is soluble 
 in 6 parts cold and 2.5 parts hot water. It has only a faint 
 sweet taste. It is insoluble in ether or absolute alcohol. 
 Aqueous solutions are dextro-rotatory, (a) D = -J- 52.5°. 
 Milk-sugar combines with bases ; the alkali combinations are 
 insoluble in alcohol. Solutions of lactose reduce Fehling's 
 solution, but less powerfully than dextrose. On warming 
 with phenyl-hydrazine acetate it gives on cooling a yellow 
 precipitate of phenyl-lactosazon, C 24 H 32 N 4 9 . By boiling 
 with water, or more readily on boiling with acids, or by 
 means of inverting ferments, as in the alimentary canal, it 
 takes up water and is converted into a glucose called galac- 
 tose. It undergoes alcoholic fermentation by the action of 
 certain schizomycetes, producing lactic acid at the same time. 
 
 Maltose, C 12 H 22 11 + H 2 0. 
 
 Preparation. — 500 grms. potato-starch are thoroughly 
 mixed with 2.5 litres cold water and converted to a paste by 
 heating on the water-bath. Allow this paste to cool to 
 60-65° C, and add a watery extract of 30-35 grms. of air-dried 
 malt made at 40° C. Keep at 60° C. for an hour, then boil, 
 filter, and evaporate to syrup in a flat porcelain dish. This 
 is extracted several times with boiling 90$ alcohol. If no 
 crystals of pure maltose are at your disposal, boil a portion of 
 the syrup with absolute alcohol, filter, evaporate to thin syrup, 
 and allow it to stand in thin layers until it crystallizes, which 
 
4 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 generally takes place in a few days. In the meantime distil 
 most of the alcohol off from the main portion, evaporate the 
 residue to a thick syrup, and on cooling stir into this a few 
 crystals of the pure crystallized maltose. After three to five 
 days the syrup will have crystallized to a stiff mass of crystals. 
 These are rubbed to a thin paste with methyl alcohol, 
 drained on paper, and washed once with methyl alcohol, 
 pressed, washed again with methyl alcohol, and purified by 
 further crystallization. For this purpose dissolve 500 grms. 
 of the dried, pressed maltose in 15 c.c. water on the water- 
 bath, add 130 c.c. 90$ alcohol, boil, filter, and allow to cool. 
 No syrup should separate out. Add a few crystals of pure 
 maltose, and shake often, until the entire liquid after a few 
 hours crystallizes into a thick mass of crystals. After draining 
 the crystals they may be recrystallized from methyl alcohol, 
 which is done by heating 50 grms. of the crystals with 12 c.c. 
 water until all has dissolved, and adding 300 c.c. methyl alco- 
 hol; boil, filter, and allow to cool. Shaking facilitates crystal- 
 lization considerably. (Soxhlet.) 
 
 Properties. — Maltose crystallizes generally in microscopic 
 needles containing 5$ (1 mol.) water of crystallization. The 
 dried crystals are hygroscopic, specific rotatory power being 
 (a) D— + 137°. Maltose reduces alkaline solutions of copper, 
 bismuth, and other metallic salts, but its reducing power as 
 measured by Fehling's solution is i less than that of dextrose. 
 With phenyl-hydrazine acetate it gives after heating for l£ 
 hours clusters of yellow crystals, C 24 H 32 N 4 9 ,meltingat 206° O. 
 Maltose is easily and completely fermented by yeast. When 
 heated with very dilute sulphuric acid, maltose yields dex- 
 trose. The diastatic enzymes act in the same way. 
 
 Dextrose, C 6 H 12 6 . 
 
 Preparation. — Warm a mixture of 1.5 litres 90$ alcohol 
 and 60 c.c. strong HC1 on the water-bath to 45° C, and 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 5 
 
 gradually add 500 grms. powdered cane-sugar, stirring all 
 the while and taking care that the temperature does not 
 rise above 50° C. After two hours the sugar will have dis- 
 solved and will be inverted into dextrose and lasvulose ; allow 
 it to cool, and place it in a cold place until crystallization com- 
 mences, which occurs in from six to eight days. The crystal- 
 lization may be facilitated by constant stirring. If pure anhy- 
 drous crystals of glucose are at hand, add a few grammes to 
 the cold solution and stir well. In this case the crystalliza- 
 tion will begin in a few hours, and is complete in 36 hours. 
 The crystals thus obtained are well drained, then washed free 
 from HC1 by 90$ alcohol, then with absolute alcohol, and 
 now dried at a moderate heat. To completely purify these 
 crystals boil them for five to ten minutes with pure methyl 
 alcohol* (sp. gr. 0.810 at 20° C), filter, quickly cool, and the 
 glucose crystals will separate out. (Soxhlet). 
 
 Properties. — Dextrose (glucose, grape sugar) is readily sol- 
 uble in water, sparingly soluble in alcohol, and insoluble in 
 ether. It crystallizes from an aqueous solution in white 
 spheroidal masses, and from alcohol in transparent anhydrous 
 prisms. Its solutions rotate the ray of polarized light to the 
 right ; (a) D — -\- 52.6°. In alkaline solutions dextrose 
 reduces salts of silver, bismuth, mercury, and copper. Under 
 the influence of yeast it is converted into alcohol and carbon 
 dioxide. It may also undergo lactic-acid fermentation under 
 the influence of certain bacterial growths. With a mixture 
 of 2 parts- phenyl-hydrazine hydrochloride and 3 parts sodium 
 acetate a watery solution of glucose gives, when heated on 
 the water-bath, a precipitate of fine yellow needles (phenyl 
 glucosazon, C 18 H 2i N 4 4 ), melting at 204°-205° O. 
 
 * The purest methyl alcohol is mixed withjabout 20f water, and about 
 four-fifths distilled off on the water-bath. This distillate has, as a 
 rule, the above specific gravity, and is immediately used. 
 
6 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Inosit, 6 H 12 6 . 
 
 Preparation. — 1. Make a watery extract of 2 lbs. chopped 
 meat, remove the albuminous bodies by coagulating at boil- 
 ing heat. This is filtered and the filtrate precipitated by 
 sugar of lead, and again filtered and washed. This filtrate is 
 boiled with basic lead acetate and allowed to stand 24-48 
 hours. The precipitate thus obtained, which contains all the 
 inosit, is decomposed in water by H 2 S. The filtrate is strongly 
 concentrated, treated with 2-4 vols, hot alcohol, and the 
 liquid removed as soon as possible from the tough and flaky 
 masses which ordinarily separate. If no crystals separate 
 from the liquid within 24 hours, then treat with ether until 
 the liquid has a milky appearance and allow it to stand. In 
 the presence of a sufficient quantity of ether, crystals of inosit 
 separate within 24 hours. The crystals thus obtained, as also 
 those which are obtained from the alcoholic solution directly, 
 are recrystallized by redissolving them in very little water 
 and the addition of 3-4 vols, alcohol. 
 
 2. Inosit may also be prepared from green beans by evap- 
 orating the watery extract to a syrupy consistency and pre- 
 cipitating with alcohol. The precipitate is dissolved in water 
 and the inosit allowed to crystallize out. (Volil.) 
 
 Properties. — Inosit crystallizes in large, colorless, rhombic 
 crystals of the monoclinic system, or, if not pure and if only 
 a small quantity crystallizes, it forms fine crystals similar to 
 cauliflower. The crystals melt at 217° 0. It dissolves in 6 
 parts water at the ordinary temperature, and the solution has 
 a sweetish taste. It is insoluble in strong alcohol and in 
 ether. Inosit does not ferment with beer yeast, but is cap- 
 able of lactic-acid fermentation. It dissolves copper oxhy- 
 drate in alkaline solutions, but does not reduce on boiling. 
 It gives negative results with Moore's or Boetger-Almen's 
 bismuth test. Its solutions have no action on polarized light. 
 Inosit gives no combination with phenyl-hydrazin acetate. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. ? 
 
 Fatty Acids, C n H 2n 2 . 
 
 Preparation. — Dissolve 20 grms. caustic potash in 100 c.c. 
 absolute alcohol, placing the vessel in cold water as consider- 
 able heat is generated. When all has dissolved that will, 
 decant the clear solution from the sediment. Now heat on 
 water-bath 50 grms. mutton tallow or leaf lard with 50 c.c. 
 alcohol in a flask connected with a return condenser. Continue 
 the application of heat until all the fat has melted; now add 
 the potash solution, and gently boil for one half to one hour. 
 When the liquid in the flask does not give a cloudiness when 
 added to water, then all the fat has been converted into soap. 
 Filter through a cotton plug and dilute this liquid with 500 to 
 600 c.c. water,, Boil over the naked flame until all the odor of 
 alcohol has disappeared, and add dilute sulphuric acid (1 to 4) 
 until the solution has a marked acid reaction. Allow this to 
 stand on the boiling water-bath until the separated fatty- 
 acids have collected on the surface as an oily layer. Now 
 allow to cool, filter through a wet filter, wash the fatty acids 
 with cold water, and crystallize the same from hot 80^ alco- 
 hol. The oleic acid (0 18 H 34 2 ) remains nearly entirely in the 
 mother liquid, while the palmitic (C 16 H 32 2 ) and stearic acid 
 (C 18 H 36 2 ) forms the crystals. Determine the melting-point 
 of the mixture, then dissolve the same in cold alcohol, and 
 fractionally precipitate this solution with an alcoholic solu- 
 tion of sugar of lead (3 to 4 fractions are sufficient). Each 
 precipitate is shaken witn ether and the fatty acid obtained 
 on the evaporation of the ether. Determine the melting- 
 point of each fraction, and a different melting-point will be 
 found for each, showing that the fatty acids obtained from 
 the fat consists of a mixture. The first lead precipitate con- 
 tains the stearic acid. 
 
 Stearic acid melts at 69.2° C. 
 
 Palmitic acid melts at 62° C. 
 
8 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Soap. 
 
 Preparation. — Dissolve 50 grms. fatty acids (page 7) in 
 100 c.c. alcohol by warming on the water-bath. Gradually 
 add an alcoholic solution of caustic soda (10 grms. NaHO in 
 100 c.c. alcohol) to this solution until a very faint alkaline 
 reaction is obtained. Heat on water-bath for 15 minutes, 
 transfer to flat porcelain dish, and evaporate off the alcohol 
 on the water-bath. When nearly all alcohol is off, add 30 c.c. 
 water and continue the evaporation, stirring all the while. 
 The product thus obtained when dry will be a neutral soap. 
 
 Serum Albumin. 
 
 Preparation. — 1. Defibrinated ox blood (or human transu- 
 dations) is filtered through washed linen (free from starch) 
 and allowed to stand in the cold in a tall vessel until the red 
 blood-corpuscles have settled to the bottom. The clear serum 
 is carefully drawn off by means of a siphon and saturated at 
 30° O. with magnesium sulphate, filtered at the same tem- 
 perature, and washed with a saturated solution of magnesium 
 sulphate. Saturate the filtrate with sodium sulphate (or 
 ammonium sulphate) at 40° C, whereby the serum albumin 
 is precipitated. This precipitate is collected on a filter, 
 pressed between paper, dissolved in water, reprecipitated by 
 sodium sulphate (or ammonium sulphate), and the process 
 repeated several times. The solution in water is now freed 
 from salts by means of dialysis, using large amounts of dis- 
 tilled water. The serum albumin may be obtained from this 
 dialyzed solution by evaporating the solution to dryness at a 
 gentle heat, or, better, by precipitating with an excess of 
 strong alcohol, filtering, washing with alcohol, and finally 
 with ether, and then drying by exposure to the air. 
 
 When precipitating the serum albumin by means of alcohol 
 filter immediately, press between paper, and remove the alco- 
 hol from the precipitate by means of ether. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 9 
 
 2. Serum albumin may also be precipitated from the fil- 
 trate, after the precipitation of the serum globulin, by means 
 of acetic acid — about \%. Filter after a few hours, press the 
 precipitate between filter-paper, dissolve in water, neutralize 
 by the addition of alkali, and remove salts by means of dial- 
 ysis. The serum albumin is obtained from this salt-free solu- 
 tion as above directed. 
 
 Properties. — In the dry state serum albumin forms a trans- 
 parent, gummy, brittle, hygroscopic mass or a white powder, 
 readily soluble in water, forming a clear solution with a spe- 
 cific rotatory power, for a solution saturated with NaCl, of 
 (a) D = — 62.6° to 64.6°. The coagulation temperature is 
 -f 70° to 75° C, but varies with the varying concentration 
 and the amount of salts. Its solutions are precipitated by 
 alcohol and ether. 
 
 Ov-Allbumin. 
 
 Preparation. — 1. The white of several hen's eggs (free 
 from yolk) is subdivided by cutting with a scissors or by 
 beating violently, filtered through linen (free from starch), 
 and then treated with an equal amount of water. A precipi- 
 tate will form, and this must be removed by filtration. Sat- 
 urate the filtrate at 20° 0. with very finely powdered mag- 
 nesium sulphate (MgS0 4 -f- 7H 2 0), which is clone by adding 
 small portions of the salt at a time and constantly stirring. 
 After completely saturating, remove the precipitated globu- 
 lins by filtration, and thoroughly dialyze the filtrate until a 
 portion removed and treated with Ba01 2 does not give any 
 reaction for sulphates. Evaporate the solution (which greatly 
 increases in volume during dialysis) at 40°-50° 0. in a flat 
 dish, and allow this concentrated solution to undergo dialysis 
 again; and, lastly, evaporate to dryness at the above-mentioned 
 temperature. 
 
 2. Ov-albumin may also be obtained by saturating the fil- 
 
10 HANDBOOK FOR BIO CHEMICAL LABORATORY. 
 
 trate from the magnesium sulphate with sodium sulphate 
 (or ammonium sulphate) at 20° C. The precipitate of ov- 
 albumin is filtered off and pressed between filter-paper, dis- 
 solved in water, again precipitated with sodium sulphate (or 
 ammonium sulphate), and after repeating this process several 
 times the salts are removed by dialysis, and the salt-free 
 solution evaporated to dryness at 40° C, or in a vacuum. 
 
 Properties. — The ov-albumin remains as a yellowish, trans- 
 pareDt mass soluble in water, precipitated from its solutions 
 by alcohol, and quickly converted into coagulable albumin. It 
 is not precipitated from its watery solution by MgS0 4 , but 
 completely precipitated by NH 4 S0 4 . Its 1-3$ solution con- 
 taining some salt coagulates at about 56° C. It is not pre- 
 cipitated by ether, and has a specific rotatory power of (a) 
 I) = -35.5°. 
 
 Serum Globulin or Paraglobulin. 
 
 1. Faintly acidify blood serum (prepared as directed on 
 page 8) with a few drops acetic acid, and dilute with 10-20 
 vols, of water. The serum globulin will separate as a fine 
 flocculent precipitate, which is filtered and further purified 
 by dissolving it in a dilute common-salt solution or in water 
 by the aid of the smallest possible amount of alkali, and then 
 reprecipitating by diluting with water or by the addition of a 
 little acetic acid. On repeating this twice the serum globulin 
 is carefully dried in the air. (Al. Schmidt.) 
 
 2. Serum may also be precipitated from blood serum by 
 means of magnesium or ammonium sulphate added to satu- 
 ration. Filter and wash with one-half saturated solution of 
 the salt used, and purify the precipitate by means of dialysis. 
 As ammonium sulphate is removed by dialysis with difficulty, 
 it is best to use magnesium sulphate. "When great purity is 
 required the precipitate may be redissolved by adding distilled 
 water, reprecipitating by saturating with the salt again, and 
 purifying this by thorough dialysis. (Ilammarsten.) 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 11 
 
 Properties. — Serum globulin is insoluble in water, but 
 soluble in dilute salt solutions (XaCl, (XHJ 2 S0 4 , MgSOJ. 
 Its solution in dilute solutions of (lifH 4 ) a S0 4 , or MgSO^ are 
 completely precipitated by saturating these solutions with the 
 respective salt, but it is incompletely precipitated by NaCl. 
 The coagulation temperature with 5-10$ XaCl in solution is 
 75° C. Specific rotatory power for a solution containing salt 
 is (a) D = - 47.8. 
 
 Fibrinogen. 
 
 Preparation. — Precipitate salt plasma with an equal volume 
 
 of a saturated solution of XaCl (33$). The precipitate thus 
 obtained is pressed between filter-paper, redissolved in an 8$ 
 salt solution, the filtrate precipitated by a saturated salt solu- 
 tion as above, and after precipitating in this way three times, 
 the precipitate at last obtained is filtered, pressed between 
 filter-paper, and finely divided in water. These operations 
 should be performed rapidly, as prolonged contact with a half- 
 saturated salt solution renders the precipitate of fibrinogen 
 very insoluble. The fibrinogen dissolves by the aid of the 
 small amount of XaCl contained in itself, and the solution 
 may be made salt free by dialysis with very faintly alkaline 
 water. [Hammarsten.) 
 
 Properties. — Fibrinogen has the general properties of the 
 globulins, namely, insoluble in water, but soluble in dilute 
 neutral salt solutions. It is precipitated unchanged from 
 these solutions on sufficiently diluting with water. On heat- 
 ing it coagulates. When dissolved in a 5-10$ XaCl solution it 
 coagulates at 52° to 55° C , and the faintly alkaline 'or nearly 
 neutral weak solution coagulates at 56 C. Its specific rota- 
 tory power for sodium light is — 52.5°. 
 
 Myosin. 
 
 Preparation. — 1. Finely chopped meat is extracted by 5$ 
 magnesium-sulphate solution. The filtered extract is then 
 
12 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 treated with MgS0 4 in substance until 100 c.c. of the liquid 
 contains about 50 grms. of the salt. The so-called paramyosin 
 or musculin separates. The filtered liquid is now treated 
 with magnesium sulphate until each 100 c.c. of the liquid 
 holds 94 grms. MgS0 4 in solution. The myosin which now 
 separates is filtered, dissolved in water by the aid of the re- 
 tained salt, precipitated by diluting with water, and, when 
 necessary, purified by redissolving in dilute salt solution and 
 precipitating with water. (Halliburton.) 
 
 2. Myosin may also be prepared by treating finely chopped 
 meat, which has first been soaked in cold water until the 
 muscles are white, with a 10-20$ ammonium-chloride solu- 
 tion, allowing to stand for a few hours, stirring now and then. 
 Filter this solution off and dilute with about 20 vols, water. 
 The myosin separates in flakes which gradually settle to the 
 bottom. This is washed 3 to 4 times by decantation, but not 
 oftener, as then the myosin becomes insoluble. Dissolve now 
 in ammonium-chloride solution and the myosin obtained 
 therefrom, either by reprecipitating by diluting with water, or 
 by removing the salt by dialysis. (Danilewshy.) 
 
 Properties. — Myosin has the general properties of the 
 globulins. It is completely precipitated by saturating with 
 NaCl, also by MgS0 4 , in a solution containing 94$ of the salt 
 with its water of crystallization. Like fibrinogen it coagulates 
 at 56° O. in a solution containing common salt, though the 
 coagulation temperature may vary for myosins of different 
 origin, and also for the same myosin in different salt solutions. 
 It is soluble in dilute alkalies. Myosin decomposes hydrogen 
 peroxide. 
 
 Ovo-Yitellin. 
 
 Preparation. — Shake the yolks of two eggs with 200 c.c. acid- 
 free ether in a stoppered cylinder, then add 5 c.c. alcohol. A 
 sticky, stringy precipitate will be formed. Remove the ether 
 as well as possible and add 100 c.c. of a 10$ common salt 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 13 
 
 solution. On shaking the precipitate it dissolves in the com- 
 mon salt solution, yielding a cloudy liquid ; place the solution 
 in a separatory funnel and shake with an equal volume of 
 ether. It will remain clear or nearly so. Draw off the 
 watery solution and allow it to stand until the next day, 
 when a cloudiness will have appeared; this is settled byre- 
 shaking again with ether. Draw off the watery liquid again, 
 measure it, and dilute with 10 vols, of water. The very fine 
 precipitate produced is filtered off the following day, washed 
 with water and then with alcohol. To further purify the 
 substance thus obtained place the precipitate in a flask, boil 
 with absolute alcohol on the water-bath, filter, wash with alco- 
 hol, then with ether, and lastly subdivide the mass in a flat 
 dish and allow to dry over sulphuric acid or in a vacuum. 
 (SalJcowsJci.) 
 
 Properties. — Vitellin is insoluble in water, but soluble in 
 dilute neutral salt solutions. It is soluble in hydrochloric acid 
 of 1 p.m. and in very dilute solutions of alkalies or alkali 
 carbonates. The coagulation temperature for the solution 
 containing NaCl lies between 70° and 75° 0. It yields nuclein 
 when digested with pepsin and hydrochloric acid. Vitellin 
 obtained as above should contain only 0.95$ phosphorus. 
 
 Casein. 
 
 Preparation. — 200 c.c. fresh milk are diluted with 800 c.c. 
 water and treated with acetic acid so that the dilute milk con- 
 tains 0.75-1 p.m. acetic acid. (Hydrocloric acid may also be 
 used.) The casein hereby precipitated is quickly washed with 
 water by decantation and rubbed with water in a mortar so that 
 it is as fine as possible. Dissolve it with the least possible quan- 
 tity of a 0.1$ caustic-soda or ammonia solution, continually 
 stirring, and taking care that the liquid, does not became alka- 
 line but neutral. The milk-white liquid is filtered through 
 several folds of filter-paper, when it will become water-clear 
 
14 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 with only a slight bluish opalescence. After diluting with 
 water it is again precipitated by acetic acid (or hydrochloric 
 acid) as above directed, and the precipitate again ground 
 finely, washed on a filter with water, and then dissolved in caus- 
 tic soda as above. This is repeated once or twice. Then the 
 washed precipitate is gently pressed, quickly rubbed to a 
 paste with 97$ alcohol, transferred to a filter, washed first 
 with alcohol, then with ether, pressed, then dried in a mortar 
 after having finely divided it. The last traces of ether are 
 removed in a vacuum or over sulphuric acid. (Hammarsten.) 
 Properties. — Casein forms a white, dusty, insoluble powder 
 which reddens moist blue litmus-paper. It is readily soluble 
 in dilute alkalies and acids. It is completely soluble in 0.2$ 
 hydrochloric acid, and if this is digested at 38-40 u 0. with 
 pepsin a gradual cloudiness is formed and a precipitate of 
 nuclein is produced. Casein solutions do not coagulate on 
 boiling, but are covered, as milk, with a skin. Casein isr pre- 
 cipitated from neutral solutions or from milk by NaCl or 
 MgS0 4 in substance without changing its properties. Metallic 
 salts, such as copper sulphate, completely precipitate casein 
 from neutral solutions. Casein coagulates with rennet or 
 chymosin in the presence of lime-salts. 
 
 Albuminate (Alkali). 
 
 Preparation. — Beat up the white of an egg finely and filter 
 through a piece of clean linen, and treat the filtrate with 
 a solution of 1 grm. caustic potash in a little water, contin- 
 ually stirring. The solution will be immediately, or after 
 some time, converted into a gelatinous mass. This is cut into 
 pieces, washed a few times with water, then dissolved in warm 
 water, allowed to cool, and precipitated by acetic acid, wash- 
 ing the precipitate with alcohol and ether. This precipitate 
 of alkali albuminate appears as a flaky, amorphous, white 
 substance, nearly insoluble in water as well as NaCI solutions, 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 15 
 
 but readily soluble in alkalies Na 3 C0 2 , Na 2 IIP0 4 , as well as 
 dilute hydrochloric acid. It does not coalgulate on applying 
 heat to its solutions. 
 
 Albuminate (Acid). 
 
 Preparation. — Digest the white of two eggs with hydro- 
 chloric acid (0.5$), aud let it stand or apply gentle heat ; then 
 dilute with twice its volume of water. Collect the precipi- 
 tate, dissolve it in hot water, and carefully neutralize the solu- 
 tion with Na a C0 3 ; finally wash well with water, and the prod- 
 uct will be pure acid albuminate. 
 
 Fibrin, 
 
 Preparation. — Whip freshly-drawn ox-blood with a bunch 
 of twigs; the fibrin adheres to the twigs and entangles but a 
 few blood-corpuscles. The mass is washed for a long time in 
 a stream of running water until nearly white, and then with a 
 bfo common salt solution, and again with water. When free 
 from NaCl extract with alcohol and then with ether, and pre- 
 serve in a solution of equal parts glycerine and water. 
 
 Properties. — Fibrin is soluble with difficulty in a 5-10$ 
 common salt or saltpetre solution or similar solutions of 
 MgS0 4 or other neutral salts. In the presence of enzymes or 
 by putrefaction it may dissolve. It is insoluble in water, alco- 
 hol, or ether. Fibrin decomposes hydrogen peroxide. Solu- 
 tions of fibrin are precipitated by lead acetate, copper sulphate, 
 and mercuric chloride. Weak HOI (0.2$) causes fibrin to 
 swell up into a transparent jelly, while stronger acids dissolve 
 it in a time with the formation of acid albumin or syntonin 
 and albumoses. 
 
16 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Peptone, 
 
 Preparation. — 1. Digest 2000 grms. washed fibrin, but not 
 boiled, with 5 litres of a solution of purified pepsin (see Pep- 
 sin) obtained from 600 grms. of the removed mucous mem- 
 brane of the fundus of the stomach of two pigs and contain- 
 ing 0A<f o HC1. This mixture is allowed to digest for a fort- 
 night at 37°-40°C., so as to insure as complete a conversion of 
 the albumoses into peptones as possible. A little thymol 
 (0.25$) may be added to prevent putrefaction. The forma- 
 tion of peptones takes place much more quickly with trypsin 
 digestion ; therefore it may be used instead of pepsin. After 
 complete digestion the solution is filtered through linen and 
 should not contain any albuminates or coagulable albumin. 
 Exactly neutralize the filtrate with soda and heat to boiling. 
 While boiling hot precipitate by saturating with ammonium 
 sulphate. Allow to cool, and separate the precipitated albu- 
 moses and the ammonium sulphate, which has crystallized 
 out, by filtration. The filtrate is again heated to boiling, 
 and made strongly alkaline with ammonia and ammonium 
 carbonate and saturated with ammonium sulphate while boil- 
 ing. Filter when cold, and boil the filtrate until the odor of 
 ammonia has entirely passed off ; now saturate again with 
 ammonium sulphate while hot, and acidify with acetic acid. 
 When cold filter and strongly concentrate the filtrate, stirring 
 constantly, and when cold decant the liquid from the salts which 
 have crystallized out. A great part of the remaining ammon- 
 ium sulphate may be removed by careful fractional precipitation 
 with alcohol (\ vol.), so that at last a solution rich in peptones, 
 containing alcohol and some little ammonium sulphate, is ob- 
 tained. The alcohol is driven off by boiling the solution, 
 and the ammonium sulphate decomposed by boiling with 
 barium carbonate. The filtrate is freed from the excess of 
 barium by the careful addition of dilute sulphuric acid. This 
 last filtrate, which must not contain any sulphuric acid, is 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 17 
 
 strongly concentrated, and the peptones precipitated by the 
 addition of alcohol. The peptones thus obtained are quickly 
 dried in a desiccator connected with an air-pump. (Eilhne.) 
 
 2. The solution obtained after the digestion of fibrin with 
 pepsin as described in method 1 is first neutralized with 
 sodium hydrate, filtered through linen, and the filtrate acidi- 
 fied with acetic acid and concentrated considerably. Now 
 precipitate the albumoses by saturating with amonium sul- 
 phate, filter and press, and boil the solution with barium hy- 
 drate, and finally with barium carbonate and a large quantity 
 of water, until ammonia can no longer be detected. Eemove 
 the barium sulphate by filtration through cloth bags, and the 
 filtrate evaporated after washing the precipitate. The barium 
 peptone thus obtained is decomposed by a slight excess of 
 sulphuric acicl, the barium sulphate filtered off, the solution 
 concentrated, the free acid neutralized with ammonia, and. 
 after cooling add 6^ sulphuric acid (previously diluted). 
 Now precipitate with a large excess of phospho-tungstic acid, 
 filter, wash first with 6$ sulphuric acid, then with a large 
 quantity of water, after which the compound is decomposed 
 by means of barium hydrate and the excess of barium com- 
 pletely removed from the filtrate by the careful addition of 
 sulphuric acid. The solution of peptones thus obtained has a 
 distinct acid reaction, and the solid peptone may be obtained 
 therefrom by repeated precipitation and boiling with alcohol. 
 (Kilhne and Chittenden.) 
 
 Nuclein. 
 
 Preparation. — 1. Pure casein is dissolved in water contain- 
 ing 2 p.m. HOI and filtered, and the filtrate digested with pep- 
 sin at 40° C. After a time a precipitate of nuclein appears; this 
 is filtered off, washed with warm water, and purified by re- 
 peated solution in a lfo solution of sodium carbonate and re- 
 precipitated after filtration by the addition of dilute hydro- 
 
18 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 chloric acid. The nuclein thus obtained is washed with 
 water and extracted with alcohol and then ether. Dry over 
 sulphuric acid. 
 
 2. Suspend some beer-yeast in water and wash by decanta- 
 tion, and then add it to water containing 5 p.m. HC1. After 
 some time add a slight excess of soda. Filter immediately 
 through a rapid filter and allow to flow into dilute HOI, when 
 a precipitate will fall to the bottom of the flask. Wash by 
 decantation first with dilute hydrochloric acid, then with 
 water, and then with boiling alcohol. Dry in a vacuum. 
 
 Properties. — The nucleins are colorless, amorphous, insol- 
 uble or very slightly soluble in water. They are insoluble in 
 alcohol and ether. They are more or less readily soluble in 
 alkalies ; in dilute mineral acids they are insoluble or dissolve 
 with difficulty. On boiling with caustic alkali they decom- 
 pose and alkali phosphates are formed. On fusing with soda 
 and saltpetre they give alkali phosphates also. 
 
 Nucleinic Acids. 
 
 Preparation. — Treat 1000 c.c. well-washed yeast with 3250 
 c.c. of a 3f caustic-soda solution, and allow to digest at the 
 temperature of the room for 5 minutes. Neutralize with hy- 
 drochloric acid, and add an excess of acetic acid. Filter off 
 the precipitated albuminous bodies and measure the filtrate. 
 Now add HC1 to filtrate so that it contains 3-5 p.m., and 
 then add an equal volume alcohol which has previously been 
 acidified to the same extent (3-5 p.m.). The impure nu- 
 cleinic acid thus precipitated is filtered off, dissolved in am- 
 moniacal water, and purified by treating as above, namely, 
 acetic acid, hydrochloric acid, and alcohol. The purified 
 product is dried in desiccator. (Altmann.) 
 
 Properties.— -Nucleinic acids are white, amorphous, and 
 acid in reaction, readily soluble in ammoniacal or alkaline 
 water, and are not precipitated therefrom by an excess of 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 19 
 
 acetic acid, but readily by a slight excess of hydrochloric acid, 
 especially in the presence of alcohol, They are insoluble in 
 alcohol and ether. 
 
 Lecithin. 
 
 Preparation. — The yolk of several eggs free from white are 
 violently shaken with ether until the ether does not dissolve 
 any more. The several etheral extracts are freed from ether 
 by distillation, and the residue dissolved in petroleum ether 
 and filtered. The filtrate is transferred to a separatory funnel 
 and shaken several times with 75$ alcohol; the alcoholic ex- 
 tracts are united, and allowed to stand until entirely clear. 
 Separate any petroleum ether present, and filter. The last 
 portions of petroleum ether are distilled off, and the residue 
 is allowed to stand in a cold place for several days, when a 
 precipitate will be produced. The liquid is separated from the 
 precipitate by decantation and then filtration. Decolorize the 
 solution by boiling with animal charcoal, filter, and evaporate 
 to syrupy consistency at 50-60° C. Dissolve the syrup in 
 ether, filter, and evaporate the filtrate, when the lecithin will 
 be obtained in a nearly pure form. To further purity, dis- 
 solve in as little absolute alcohol as possible, and on cooling 
 this solution in a freezing mixture to —5° to — 15° C. the 
 lecithin will be deposited. This is filtered while cold, and 
 dried in a vacuum over sulphuric acid. (E. Gilson.) 
 
 Properties. — Lecithin is a soft, waxy, mouldable, not mark- 
 edly crystalline mass, which when placed on a microscope slide 
 with water forms oily drops, appearing under the microscope 
 like worms, called "myeline forms." It is easily decom- 
 posed by acids and alkalies, especially by the latter. On 
 fusion with soda and saltpetre on platinum foil it yields 
 a mass in which phosphoric acid can be easily detected. It 
 combines with acids and bases. The combination with HOI 
 gives a double salt with PtCl 4 , which is insoluble in alcohol, 
 
20 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 but soluble in ether, and which contains 10.2$ platinum. It 
 is soluble in chloroform, carbon disulphide, benzol, and fatty 
 oils. 
 
 Oxyhemoglobin. 
 
 Preparation. — Defibrinated blood (best from the dog or the 
 horse) is mixed with at least 10 times its volume of a salt solu- 
 tion (1 vol. saturated solution with 9 vols, water), and allowed 
 to stand in a cool place for a couple of days until all the blood- 
 corpuscles have settled to the bottom. Eemove the superna- 
 tant liquid by means of a siphon, and transfer the blood-cor- 
 puscles to a flask. Add 2 vols, water, and then shake with an 
 equal volume of ether. After a time remove the ether by de- 
 cantation, and allow the ether retained by the blood solution 
 to evaporate in an open dish in the air. Filter quickly 
 through a folded filter, cool the solution to 0° C, and add while 
 stirring J of its volume of alcohol which has been cooled to 
 0° O. and allow to stand for several days at —5° to —10° C. 
 The crystals which separate are filtered off, pressed, and re- 
 crystallized by first quickly dissolving them in not too much 
 water at 20° to 30° C, cooling to 0° C, precipitating with cold 
 alcohol as above, and allowed to stand at —5° to —10° 0. This 
 recrystallization is repeated several times. Lastly, the crystals 
 are washed with cooled water containing alcohol (^ vol. alco- 
 hol), and dried in vacuum at 0° 0. or a lower temperature. 
 {Eoppe Seyler.) 
 
 Properties. — Oxyhemoglobin from various kinds of blood 
 has different crystalline forms. It is insoluble in ether, chlo- 
 roform, benzol, and carbon disulphide. Crystals of oxyhemo- 
 globin are insoluble in absolute alcohol. Its solution in water 
 is not precipitated by many metallic salts, but is precipitated 
 by sugar of lead. On heating the watery solution it decom- 
 poses at 60°-70° C, and sj)lits into haematin and albumin. It 
 is also decomposed by acids, alkalies, and many metallic salts. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 21 
 
 Dilute solutions show a spectrum with two absorption-bands 
 between the Fraunhofer lines D and E. The one band, a, 
 which is narrower but darker and sharper, lies on the line D; 
 the other, broader, less defined and less dark band, /3, lies at 
 E. Oxyhemoglobin solutions are reduced by reducing solu- 
 tions such as ammonium sulphide, ammoniacal ferro-tartrate 
 solution {Stokes reduction liquid) yielding a characteristic 
 spectrum of hemoglobin. 
 
 Haemoglobin. 
 
 Preparation. — Fill a cylinder nearly full with a dilute 
 watery solution of oxyhemoglobin, and add a few drops of a 
 putrefied extract of meat. Close up hermetically, and allow to 
 stand some time at the ordinary temperature. The oxyhemo- 
 globin will be reduced to hemoglobin, and if the solution is of 
 the proper concentration a crystallization of hemoglobin may 
 occur in the cylinder at lower temperatures. The solution 
 of hemoglobin is more purple than a solution of oxyhemo- 
 globin, and shows a spectrum with only one absorption-band 
 occupying the space between the Fraunhofer lines D and E. 
 
 Carbon-monoxide Haemoglobin. 
 
 Preparation. — Pass a current of carbon monoxide (pre- 
 pared by heating a mixture of sulphuric and oxalic acids and 
 passing the gases through a wash-bottle containing KOH), 
 through a watery solution of oxyhemoglobin. The color of 
 the solution becomes lighter, due to the formation of carbon- 
 monoxide hemoglobin. The spectrum of the solution is not 
 changed, but it differs from the oxyhemoglobin in that it 
 cannot be reduced by reducing solutions into hemoglobin. 
 The crystals are isomorphous to the oxyhemoglobin crystals, 
 but are less soluble. 
 
22 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 MethaeniogloMn. 
 
 Preparation. — On treating a concentrated solution of oxy- 
 hemoglobin with a sufficient quantity of a concentrated 
 solution of potassium ferricyanide to give the mixture a 
 porter-brown color, methaemoglobin will be formed. After 
 cooling to 0° C. add J vol. alcohol cooled to 0° C, and allow 
 the mixture to stand a few days in the cold. The crystals ob- 
 tained may be easily purified by recrystallization from water 
 by the addition of alcohol. (Pig's blood is better adapted for 
 the preparation of methsemoglobin than dog's blood.) 
 
 Properties. — Methasmoglobin crystallizes in brownish-red 
 needles, prisms, or six-sided plates. It dissolves readily in 
 water, aud this solution becomes red on the addition of alkali. 
 The spectrum of methasmoglobin shows three absorption- 
 bands, one in the red about half way between C and D, the 
 other two between D and E, which resemble the position of the 
 oxyhemoglobin lines, but on careful measurement are found 
 to be different. 
 
 Fibrin Ferment. 
 
 Preparation. — Blood-serum is mixed with 10 to 15 times 
 its volume of absolute alcohol ; by this means the proteids are 
 precipitated, as also the ferment. The precipitate is allowed 
 to stand a few months with the alcohol, which renders the 
 proteids insoluble. Filter the precipitate off and dry in de- 
 siccator over sulphuric acid. The ferment is separated from 
 the other bodies by extracting with water, which dissolves the 
 ferment. {A. Schmidt.) 
 
 Properties. — A solution of the ferment will coagulate peri- 
 cardial and similar fluids or solutions of fibrinogen. It is 
 most active at about 40° 0. It is diminished in action at 0° C, 
 and entirely destroyed on heating its solutions to 73°-75° 0. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 23 
 
 Glycocliolic Acid, C 2e II 49 N0 6 , 
 
 Preparation. — 1. The precipitate obtained by treating an 
 alcoholic solution of dried decolorized bile with ether is dis- 
 solved in water, and dilute sulphuric acid added until a per- 
 manent and dense turbidity is produced. Allow this liquid 
 to stand a few hours, when glycocliolic acid will separate out 
 as fine silky needles. Collect on filter, wash with water, 
 press between folds of filter-paper, and then dissolve in alcohol, 
 using as little alcohol for solution as possible. Add many 
 times its volume of ether, and glycocholic acid will separate 
 out as long silky needles. 
 
 2. Decolorized extract of ox-bile is dissolved in water and 
 the solution treated with a solution of lead acetate. A pre- 
 cipitate of lead glycocholate is produced. Collect on a filter, 
 wash, drain, and mix with alcohol. To this add a solution of 
 sodium carbonate in excess and evaporate to dryness. This 
 converts the lead glycocholate into sodium glycocholate. 
 Kemove the sodium glycocholate from the lead glycocholate 
 by extracting the residue w r ith absolute alcohol. Distil off 
 the alcohol nearly to dryness, and evaporate to dryness in a 
 porcelain dish. Dissolve this residue in water, decolorize 
 this solution by animal charcoal, and the glycocholic acid is 
 precipitated from the solution by the addition of dilute sul- 
 phuric acid. The acid thus obtained is purified by crystalli- 
 zation from boiling water on cooling, or by the addition of 
 ether to its solution in absolute alcohol. 
 
 3. Fresh ox-bile is treated with a few drops HC1, which 
 causes the precipitation of the mucoid substances. Filter, 
 and to each 190 c.c. filtrate add 5 c.c. strong HOI. Place in 
 a stoppered cylinder and add 30 c.c. ether for every 100 c.c. 
 bile, shake well, and allow to stand in a cool place. After a 
 time the mixture is found to be converted into a mass of 
 crystals of glycocholic acid. Filter and wash with ice-water 
 until the washings are colorless ; they are then dissolved in 
 
24 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 the smallest possible quantity of boiling water, which deposits, 
 on cooling, crystals in a colorless and pure state. (Hufner.) 
 Properties. — Glycocholic acid crystallizes in fine, colorless 
 needles or prisms. It is soluble with difficulty in water 
 (about 300 parts cold and 120 parts boiling water). Readily 
 soluble in strong alcohol, but with greater difficulty in ether. 
 The solutions have a bitter taste, but at the same time a 
 sweetish taste. The salts of the alkalies and alkaline earths 
 are soluble in alcohol and water, while the glycocholates of the 
 heayy metals are either insoluble or only sparingly soluble in 
 water. It is a monobasic acid. The solution of the alkali- 
 salts in water is precipitated by lead acetate, copper, ferric 
 salts, and silver nitrate. On boiling with acids or alkalies 
 it splits into cholalic acid and glycocoll. Glycocholic acid 
 or its salts are dextro-rotatory. Specific rotation of the acid 
 dissolved in alcohol is (a) D = + 29°. 
 
 Taurocholic Acid, C 26 H 45 lSrS0 7 . 
 
 Preparation. — Agitate dog's bile with animal charcoal and 
 alcohol, allow to settle, decant through filter. The clear 
 filtrate is evaporated to dryness and dissolved in a little hot 
 absolute alcohol and precipitated by the addition of ether. 
 This crystalline precipitate of sodium taurocholate is filtered, 
 dissolved in water, and precipitated by lead acetate and am- 
 monia, filtered, washed well, and then boiled with absolute 
 alcohol, filtered while hot, and the filtrate decomposed with 
 H 2 S until no further precipitation of PbS. occurs. This is 
 filtered, the filtrate concentrated by evaporation at a gentle 
 heat and then precipitated by an excess of water-free ether. 
 The precipitate consists of taurocholic acid which after a 
 time becomes converted into needle-shaped crystals possess- 
 ing a silky lustre. If the precipitate refuses to crystallize, 
 after a time add a drop or two of alcohol. (Parke.) 
 
 Properties. — Taurocholic acid crystallizes in needles which 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 25 
 
 rapidly deliquesce in the air. It is readily soluble in water 
 and alcohol, yielding a solution with a bitter-sweetish taste. 
 Its salts are, as a rule, readily soluble in water, and the solu- 
 tions of the alkali salts are not precipitated by copper sulphate, 
 silver nitrate, or sugar of lead, but are precipitated, on the 
 contrary, by basic lead acetate. The aqueous solution of the 
 alkali salts foam like soap. On boiling with acids and alkalies 
 it splits into cholalic acid and taurin. Both aqueous and alco- 
 holic solutions of taurocholic acid are dextro-rotatory. Spe- 
 cific rotation of its alcoholic solution is (a) D = + 25°. 
 
 Cholalic Acid, C 24 H 40 O 5 . 
 
 Preparation. — 1. 500 c.c. bile are treated with 75 grms. 
 barium hydrate and the mixture boiled for 24 hours on the 
 sand-bath. The flask should be connected with a return con- 
 denser. Allow the liquid to cool, and filter. Add concen- 
 trated hydrochloric acid to the filtrate, which decomposes the 
 barium cholalate, depositing impure cholalic acid. These 
 crystals are separated from the mother-liquor and pressed, and 
 dissolved in a solution of caustic soda; the solution is then 
 mixed with 30 grms. animal charcoal and allowed to stand for 
 a feAV days. It is next filtered and the filtrate decomposed with 
 hydrochloric acid, filtered again and washed thoroughly and 
 the substance from the filter dissolved by the smallest possible 
 quantity of hot alcohol. The alcoholic solution is treated with 
 water until a slight turbidity appears. On the liquid being 
 cooled cholalic acid separates in the form of hard, transparent 
 tetrahedra or in clumps of radiating needles. For further 
 purification recrystallize from methyl alcohol. {Mylius.) 
 
 2. Mix ox-bile with one fifth of its weight of a 30$ caustic- 
 soda solution and boil for 24 hours on water-bath, having the 
 flask connected with a return condenser. Now saturate the 
 liquid with C0 2 and evaporate to dryness. Extract the 
 residue with strong alcohol, which dissolves the sodium salts 
 
26 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 of cholalic acid as well as those of choleic and stearic acids. 
 Now dilute with water until it does not contain more than 
 20f o alcohol, and add a dilute solution of barium chloride as 
 loug as a precipitate occurs. Filter, aud add to the filtrate, 
 which should yield no further precipitate with Ba01 2 , hydro- 
 chloric acid. Allow the precipitate of cholalic acid to stand 
 with the liquid and it will be found to become crystalline; 
 purify by recrystallization from ethyl alcohol and then from 
 methyl alcohol. (Mylius.) 
 
 Properties. — Cholalic acid crystallizes with 1 molecule of 
 water in rhombic plates or prisms, or in larger rhombic tetra- 
 hedra or octahedra with 1 mol. alcohol of crystallization. 
 They are quite insoluble in water (4000 parts cold and 750 
 parts boiling), rather soluble in alcohol, but soluble with 
 difficulty in ether. It is somewhat soluble in glycerin and 
 almond-oil. Its solutions have a bitter-sweetish taste. The. 
 w T ater-free acid melts at 195° 0. In the free state, as 'well as 
 in combination the acid is dextro-rotatory. The specific 
 rotatory power of the sodium salt is (a) D = -j-31.4°. The 
 watery solution of the alkali-salts, when not too dilute, is 
 precipitated immediately or after some time by sugar of lead 
 or by barium chloride. The barium salt, (C 24 H 39 5 ) 2 Ba, 
 crystallizes in fine, silky needles, which are soluble in 30 parts 
 cold and 23 parts boiling water, but more soluble in alcohol. 
 
 Grlycocoll or Glycocine, C 2 H 5 NO,. 
 
 Preparation. — 1 part hippuric acid is boiled from 10 to 12 
 hours, in a flask connected with a return condenser, with 4 
 parts dilute sulphuric acid (1 to 6). After allowing to cool 
 pour the contents of the flask into a porcelain dish and allow 
 to stand for 24 hours. Filter off the benzoic acid and wash 
 with cold water; concentrate the filtrate by evaporation, and 
 shake with ether to remove any traces of benzoic acid. 
 Dilute this acid solution and just neutralize with barium 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 27 
 
 hydrate, which precipitates the sulphuric acid ; allow the 
 precipitate to settle, wash by decantation with boiling water, 
 and concentrate the solution by evaporation. If an excess of 
 Ba(OII) 2 has been added, remove this by passing C0 2 through 
 the solution, boiling and then filtering, and allowing to stand 
 until crystals commence to form. The crystals are removed 
 from the mother-liquor and this concentrated further, and 
 the process repeated as long as crystals continue to separate 
 out. The glycocoll is purified by recrystallization from water. 
 Properties. — Glycocoll occurs as fine, hard, colorless, 
 rhombic crystals or four-sided prisms. The crystals have a 
 sweetish taste, are soluble in 4.3 parts cold water, very slightly 
 soluble in spirits of wine, and insoluble in cold absolute alco- 
 hol and ether. It combines with acids, bases, and neutral 
 salts. Glycocoll dissolves copper hydroxide in alkaline solu- 
 tion, but does not reduce at the boiling temperature, but de- 
 posits on cooling dark blue needles if the liquid is sufficiently 
 concentrated. Its solutions possess an acid reaction. When 
 heated to 232°-236° 0., glycocoll becomes brown, evolves gas- 
 bubbles, and melts. A solution of glycocoll is colored red by 
 ferric chloride. Its combination with HC1 is soluble in water 
 and alcohol. 
 
 Taurin, C 2 H 7 NS0 3 . 
 
 Preparation. — Mix ox-bile with strong hydrochloric acid so 
 as to precipitate the mucoid substances, filter, and boil the 
 filtrate for several hours. After allowing to cool and to de- 
 posit the dyslysin and choloidic acid concentrate the liquid 
 on the water-bath, filter so as to remove any common salt or 
 other substances which may have separated. The filtrate is 
 treated with strong alcohol, which precipitates the taurin with 
 some KaCl. This precipitate is washed with alcohol, dried, 
 and dissolved in the smallest quantity of boiling water possi- 
 ble. On cooling the taurin deposits in fine four-sided prisms 
 which may be further purified by recrystallization from water. 
 
28 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Properties. — Taurin crystallizes in colorless, often large, 
 shining, 4-6-sided prisms, which dissolve in 15-16 parts water 
 at the ordinary temperature, but rather more readily in warm 
 water. It is insoluble in alcohol and ether. Taurin is taste- 
 less and its solutions are neutral to test-paper. On boiling 
 with strong caustic alkalies it yields acetic acid and sul- 
 phurous acid but no alkali sulphides; but this decomposition 
 does not occur on boiling with HOI or HN0 3 . Taurin com- 
 bines with metallic oxides such as mercuric oxide. Metallic 
 salts do not precipitate solutions of taurin. 
 
 Bilirubin, 16 H 18 ]Sr 2 3 or 32 H 36 N 4 6 . 
 
 Preparation. — 1. Finely-powdered gallstones (of oxen) are 
 first extracted with ether and then with boiling water to remove 
 cholesterin and bile-acids. The residue is treated with dilute 
 hydrochloric acid, which sets the pigment free. The insoluble 
 powder is thoroughly washed with water and alcohol and then 
 dried and boiled with chloroform until entirely extracted. 
 Evaporate the chloroform solution on the water-bath and treat 
 the powdered residue with absolute alcohol and ether, which 
 removes the lilifuscin. Dissolve the remaining residue, insolu- 
 ble in alcohol, in chloroform; precipitate the bilirubin from 
 this solution by the addition of alcohol, and repeat this several 
 times. The bilirubin is finally dissolved in chloroform and 
 allowed to evaporate spontaneously, when crystals of bilirubin 
 separate. 
 
 2. Dilute bile (preferably from the dog) with water, and 
 precipitate with milk of lime. Pass carbon dioxide through 
 the mixture, filter and wash. Suspend the precipitate in water, 
 decompose it with hydrochloric acid and shake with chloro- 
 form, care being taken to avoid an excess of air. The chloroform 
 solution is evaporated to a very small volume, precipitated by 
 alcohol, and purified by repeated solution in chloroform and 
 reprecipitating with alcohol. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 29 
 
 Properties. — Bilirubin occurs partly amorphous and partly 
 crystalline, as rhombic plates whose obtuse angles are often 
 rounded. It is insoluble in water, almost insoluble in ether, 
 and very sparingly soluble in alcohol; easily soluble in chloro- 
 form, especially in the warmth, and less soluble in benzol, carbon 
 disulphide, amyl alcohol, fatty oils, and glycerin. Its solutions 
 show no absorption-bands and have, even on diluting greatly 
 (1 to 50,000), a decided yellow color. The combinations with 
 alkalies are insoluble in chloroform. Bilirubin forms com- 
 pounds with bases such as sodium, calcium, barium, lead, and 
 silver in ammoniacal solution. On allowing an alkaline solu- 
 tion of bilirubin to stand in contact with the air it gradually 
 absorbs oxygen, and green biliverdin is formed. 
 
 BiliTerdin, C, 6 H 18 N 2 4 or C^H^O.. 
 
 Preparation. — 1. Biliverdin is readily prepared by expos- 
 ing a thin layer of an alkaline solution of bilirubin to the air or 
 by passing a current of oxygen through the alkaline solution 
 until it has acquired a bright green color. Add hydrochloric 
 acid, which precipitates the biliverdin, filter, and wash the pre- 
 cipitate with water until no reaction for HC1 is obtained in the 
 filtrate. Dry the precipitate, dissolve in alcohol, and separate 
 the pigment by the addition of water to the alcoholic solution. 
 Any bilirubin present may be removed by extracting with 
 chloroform. 
 
 2. Add Pb0 2 to an alkaline solution of bilirubin and stir. 
 The liquid assumes a dark green color in a few minutes; now 
 acidify with acetic acid, which precipitates a compound of bili- 
 verdin with lead. This combination is decomposed by means 
 of alcohol containing sulphuric acid in solution. The alcoholic 
 solution containing the biliverdin is separated from the lead 
 sulphate by filtration and the filtrate poured into water, which 
 precipitates the biliverdin. Collect the precipitate on a filter 
 
30 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 and purify by repeated solution in alcohol and reprecipitating 
 with water. 
 
 Properties. — Biliverdin is amorphous, but it has occasionally 
 been obtained as green rhombic plates with tunicated ends. It 
 is insoluble in water, ether, pure chloroform, benzol, but 
 readily soluble in ethyl alcohol, methyl alcohol, glacial acetic 
 acid, and in chloroform containing alcohol. It dissolves in 
 alkalies, yielding a brownish-green solution which is precipi- 
 tated by acids, as well as by calcium, barium, and lead salts. 
 Solutions of biliverdin exhibit no definite absorption-bands 
 with the spectroscope. An absorption occurs from the red 
 toward the violet end of the spectrum. 
 
 Cholesterin, C 26 H 44 + H 2 0. 
 
 Preparation. — Powdered gallstones are first boiled with 
 water and then repeatedly boiled with alcohol. The solution is 
 filtered quickly through a heated filter, and the cholesterin 
 separates out in a fairly pure condition as the filtrates cool. 
 The crystals are washed with cold alcohol and then boiled 
 with an alcoholic solution of caustic soda, which saponifies 
 the fats present. After the evaporation of the alcohol, extract 
 the cholesterin from the residue by means of ether, which 
 dissolves the cholesterin, but not the soaps; filter and evap- 
 orate the ether, and purify the cholesterin by recrystallization 
 from a mixture of alcohol and ether. 
 
 Properties. — Cholesterin crystallizes from anhydrous ether 
 or chloroform as needles containing no water of crystallization, 
 but from alcohol as rhombic transparent plates whose sides 
 and angles often appear broken and whose acute angle is 
 7G° 30' or 87° 30', with 1 mol. water of crystallization. It melts 
 at 145° C. when free from water of crystallization. Cholesterin 
 is insoluble in water, dilute acids, and alkalies. It is readily 
 soluble in boiling alcohol; also in ether, glycerin, chloroform, 
 and benzol, as well as the volatile and fatty oils. Its solutions 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 31 
 
 are laevo-rotatory, the specific rotatory power being (a) D = 
 - 31.6°. 
 
 Ptyalin, 
 
 Preparation. — 1. Mixed human saliva is strongly acidulated 
 with phosphoric acid and the mixture carefully neutralized by 
 the careful addition of lime-water, which produces a copious 
 precipitate of calcium phosphate, and which carries down with 
 it a large proportion of the proteids present, together with all 
 the ptyalin. On extracting the precipitate, after filtration, 
 with a volume of water equal to that of the saliva originally 
 employed, the enzyme passes into solution and is obtained 
 therefrom by the addition of absolute alcohol, which precipi- 
 tates it in the form of white flocculi, which when dried in 
 vacuo appears as a white powder. (Cohnheim.) 
 
 2. Saliva is diluted with an equal volume of water and 
 saturated with ammonium sulphate. The precipitate thus 
 formed is treated on the filter for 5 minutes with strong al- 
 cohol, removed from the filter, and further treated with ab- 
 solute alcohol for 1-2 days. It is now dried at 30° 0. and 
 yields, on extraction with a volume of water equal to that of 
 the original saliva, a solution which is actively zymolytic and 
 free from proteids. (Kraivkow.) 
 
 Pepsin. 
 
 Preparation. — 1. The mucous coat of the pig's stomach is 
 scraped with a watch-glass, and after carefully washing and 
 drying, by pressing between filter-paper, it is finely minced. 
 The mass is now digested with a 5^ solution of H 3 P0 4 at 35° C. 
 until nearly all is dissolved. Nearly neutralize the filtered 
 solution with lime-water, which causes a precipitate of calcium 
 phosphate, and which carries down with it the greater part of 
 the pepsin previously in solution. The peptones and para- 
 peptones remain in solution. The gelatinous precipitate is 
 carefully washed with water, pressed between filter-paper, 
 
32 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 suspended in water, and treated with HC1 until it just dis- 
 solves. The solution is then poured into a saturated solution 
 of cholesterin, made by dissolving it in a mixture of 4 parts 
 alcohol and 1 part ether. A precipitate of cholesterin will be 
 formed on the addition of the above acid solution, and this is 
 repeatedly shaken with the liquid, and the cholesterin will 
 mechanically carry down with it a part at least of the pepsin 
 in solution. Collect this on a filter, wash with water, then 
 with acetic acid, and lastly with water again until the wash- 
 water gives no further reaction for HC1 with AgN0 3 . 
 
 The moist cholesterin precipitate is shaken in a separatory 
 funnel with ether. Two layers will form — the upper an 
 ethereal solution of cholesterin and the lower an aqueous 
 solution of pepsin. Separate the two and shake the aqueous 
 solution repeatedly with ether until all traces of cholesterin 
 are removed. The turbid aqueous solution is now filtered, 
 and when acidulated possesses proteolytic activity. .When 
 allowed to evaporate spontaneously it leaves a grayish, amor- 
 phous, non-hygroscopic nitrogenous body. (BrilcJce.) 
 
 2. The finely divided mucous membrane of the fundus of 
 the stomach is extracted with glycerin, or, better, with 
 glycerin containing 1 p.m. HOI. Add 10 to 20 parts glycerin 
 for every part by weight of the mucous coat and allow to 
 stand for 8 to 14 days. Filter and precipitate the pepsin from 
 the glycerin extract by the addition of a large excess of abso- 
 lute alcohol. This may be purified by dialysis through parch- 
 ment paper, (v. Wittich.) 
 
 Artificial Gastric Juice. 
 
 Preparation. — 1. The mucous membrane from the cardiac 
 region of five or six pigs' stomachs is finely divided and then 
 digested for two weeks, at 40° 0., with two to three litres of- 
 0.5$ hydrochloric acid. At the end of this time all but traces 
 of albumoses have been converted into peptones, which are in 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 33 
 
 solution together with pepsin, although a small portion of for- 
 eign matters, nucleins, antialbumid, etc., remain undissolved. 
 The liquid is filtered and saturated with powdered (NH 4 ) 2 S0 4 , 
 when a precipitation of the albumoses with the pepsin occurs. 
 The precipitate is collected on a filter, washed with a saturated 
 solution of ammonium sulphate, and then dissolved in 0.2^ 
 HOI. Add 0.25^ thymol to this acid solution and dialyze in 
 running water until the whole of the (NH 4 ) 3 S0 4 is removed. 
 On opening the dialyzing tubes a precipitate is found, which 
 is soluble in 0.2^ HC1 and furnishes a very active gastric 
 juice. {Kilhne and Chittenden?) 
 
 2. The mucous coat of a freshly removed pig's stomach is 
 carefully washed with water and finely divided, or, if a calf s 
 stomach is employed, only the cardiac region is scraped with 
 a watch-glass or the back of a knife. The pieces of the 
 mucous coat or the slimy masses obtained by scraping are 
 rubbed up with pure quartz sand or glass powder, treated 
 with water, and allowed to stand for 24 hours in a cool place- 
 On filtering, an opalescent liquid is obtained, which, when 
 acidulated so as to contain 0.1-0.2 per cent HC1, possesses 
 powerful digestive activity. {Kilhne.) 
 
 3. The stomach of a pig is opened, emptied, and then the 
 surface cleaned with a wet sponge (running water will dissolve 
 out a considerable part of the pepsin). The mucous mem- 
 brane is removed from all but the pyloric end of the organ. 
 It is then freed from a portion of the water adhering to it by 
 pressing between dry cloths and then mincing. The finely 
 divided mucous membrane is then placed in two or three litres 
 of dilute HC1 containing from 6 to 10 c.c. strong HC1 per litre, 
 and the mixture is digested in an incubator at a temperature 
 of 35°— 15° 0. for a period varying from a few hours to a day. 
 If sufficient fluid be present and the mixture now and then 
 shaken, all ought to be dissolved in a few hours, leaving but a 
 small quantity of brownish flakes and some mucus undis- 
 solved, The liquid is filtered through paper, and then may 
 
34 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 be kept for several months without decomposition, retaining 
 active proteolytic properties. Such juice does not, however, 
 contain merely acid and pepsin, but considerable quantities 
 of albumoses and peptones. (Gamgee's Phys. Chem., vol. ii., 
 page 82.) 
 
 Milk-curdling Ferment of the Stomach. 
 
 (Reitntn or Ohtmosin.) 
 
 Preparation. — Out up five calves' stomachs finely, after 
 washing, and digest the mass for about 24 hours with a 0.5$ 
 common-salt solution, keeping the temperature at about 30° 
 C, or less. After digestion filter, measure the filtrate, and 
 acidify with hydrochloric acid, so that it contains 0.1$ of the 
 acid. A thick precipitate of mucous matter is formed, and 
 to facilitate its easy separation the liquid is kept at 20°-30° 
 C. and allowed to stand until all of the mucous matter has 
 separated, or centrifugal force may be used. Filter, measure 
 the filtrate, and acidify with HOI again so that the filtrate 
 contains 0.5$ acid, and now add powdered common salt until 
 no more salt dissolves. This supersaturated, acidulated salt 
 solution is brought to a temperature of 25°-30° C, and kept 
 at this temperature for 2-3 days, under constant agitation, 
 and then allowed to remain quiet for a day or so, the tem- 
 perature being raised to 30° to 35° O. Aflocculent substance 
 is obtained, which rises to the surface of the liquid or is sus- 
 pended therein. This is carefully filtered off, and then dried 
 at a temperature of about 28° 0. The product thus obtained 
 is the pure zymotic product. 
 
 Properties. — Eennin or chymosin is an amorphous, white, 
 gelatinous substance, greatly resembling aluminium hydrate. 
 It is without taste or smell, soluble in water, forming a limpid 
 or clear solution. Ohymosin has the property of curdling 
 large amounts of milk at 37° 0., or pure casein solutions 
 containing calcium salts. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 35 
 
 Trypsin. 
 
 Preparation. — 1. The fresh pancreas 01 an ox is freed from 
 adhering fat and connective tissue and then minced and di- 
 gested first with cold alcohol, and afterwards extracted with 
 boiling ether in an extraction apparatus. The insoluble resi- 
 due is then exposed to the air, so as to allow the ether to 
 evaporate, when there is left a white, friable mass. This 
 may be kept indefinitely, and made use of to prepare solu- 
 tions of trypsin. (Called Killings pancreas powder.) 
 
 One part by weight of the above pancreas powder is digested 
 at 40° C. for 4 hours with 5 parts of 0.1$ salicylic acid solu- 
 tion. The residue after being squeezed is further digested for 
 12 hours with 5 parts of 0.25$ Na 2 C0 3 solution, and the resi- 
 due is again squeezed out. The acid and alkaline extracts 
 are mixed together, the whole made up to contain 0.25-0.5$ 
 ]STa 2 C0 3 , and digested for at least a week in the presence of 
 0.5$ thymol. By this means all the first-formed albumoses are 
 fully converted into peptones ; this is essential. At the end 
 of the w T eek the fluid is allowed to stand in the cold for 24 
 hours, filtered, faintly acidulated with acetic acid, and satu- 
 rated with neutral ammonium sulphate. By this means all the 
 trypsin is separated out and may be collected on a filter, where 
 it is washed with (NH 4 ) 2 S0 4 (saturated solution) till free 
 from peptones. It is now finally dissolved off: the filter in a 
 little 0.25$ NA 2 C0 3 solution, to which thymol is added, and 
 thus an extremely active and pure digestive solution is ob- 
 tained. The ammonium sulphate present may be gotten rid 
 of by dialysis. (KiiJme.) 
 
 2. The pancreas is carefully removed from a dog killed 18 
 to 20 hours after a full meal and weighed. It is then pounded 
 up in a mortar with pure quartz sand or glass powder and 
 allowed to stand at the temperature of the laboratory for 24 
 hours and then mixed with 1 c. c. of 1$ acetic acid for every 
 gramme of pancreas, then for each part of the gland mass add 
 
36 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 10 parts glycerin and allow to stand for three days. After 
 this time filter and precipitate the filtrate with alcohol and 
 wash with alcohol. On now dissolving the precipitate in 
 water we obtain a solution which has powerful digestive ac- 
 tion. (Heidenhain.) 
 
 3. A watery infusion of the gland may be made only after it 
 has been exposed to the air for 24 hours, adding 5 to 10 parts 
 water for each part by weight of the gland. Salkowski sug- 
 gests the digestion of the finely divided gland at 40° C. with 
 water containing 5 to 10 c.c. chloroform per litre. After a few 
 days we obtain by this means an active solution of trypsin 
 which keeps. 
 
 Properties. — Trypsin is very soluble in water, but insoluble 
 in alcohol and glycerin. Its watery solution is not decom- 
 posed by long digestion at 40° C, and when evaporated it 
 yields a translucent, non-crystalline, yellowish solid residue. 
 Trypsin may be digested for a long time at 40° C. wiih caus- 
 tic soda without decomposing. 
 
 Diastatic Ferment of the Pancreas (Amylopsin). 
 
 Preparation. — 1. Finely divided pancreas, best after ex- 
 posure for 24 hours to the air, is dehydrated first by being 
 placed in strong alcohol and afterwards in absolute alcohol, 
 the action of which should be continued for some time. The 
 dry solid, separated from the alcohol, is then macerated in 
 glycerin. The glycerin solution is precipitated by the addi- 
 tion of alcohol, filtered, washed with alcohol, dried over sul- 
 phuric acid, and then dissolved in water, (v. Wittich.) 
 
 2. Precipitate the aqueous infusion of a pancreas which 
 has been treated with Mg 2 C0 3 with collodion, which carries 
 the proteids and the proteolytic ferment down with it in a 
 gelatinous form. The filtrate is concentrated by evapora- 
 tion in vacuo, and treated with strong alcohol, which throws 
 down a flocculent precipitate, Filter, and digest in a mixture 
 
HANDBOOK FOR BIO-CUEMICAL LABORATORY. 37 
 
 of equal parts alcohol and water, which dissolves the diastatic 
 ferment, a little ty rosin, and some salts, leaving some albumin 
 undissolved. The liquid is dialyzed, concentrated in vacuo, 
 and precipitated by absolute alcohol. The precipitate thus 
 obtained possesses feeble proteolytic properties, due to remain- 
 ing traces of the proteolytic ferment, but has intense diastatic 
 properties. (Danieleivski.) 
 
 The Fat-decomposing Ferment of the Pancreas 
 
 (Steapsin). 
 
 Preparation.— A perfectly fresh pancreas is crushed in a 
 mortar with glass powder and mixed with a solution com- 
 posed of 90 c.c. glycerin and 1 c.c. of a l/ solution of Na 2 C0 3 , 
 using 30 c.c. of the glycerin solution for every 3 grms. of the 
 pancreas. This is allowed to digest for not longer than four 
 to five days and then removed from the mass of the pancreas. 
 This extract has the property of decomposing neutral fats. 
 ( Griitzner. ) 
 
 Leucnr, C 6 H 13 N0 2 . 
 
 a. Amido-isobutyl Acetic Acid, 
 
 (GH 3 ) 2 0H.0H a .GH(]SrH 2 ).C00H. 
 
 Tyrosin, 9 H n NO,. 
 
 p. OXYPHENYL-AMIDO PKOPIONIC ACID, 
 
 ATT \P OH 
 
 Ue±1 * ] CH 2 .CH(NH 2 )COOH. 
 
 Preparation. — 1. These two bodies may be prepared in 
 large quantities by boiling albuminous bodies or albuminoids 
 with dilute mineral acids. Two parts hoof-shavings (| to 1 
 kilo in weight) are boiled for 24 hours with 5 parts concen- 
 trated sulphuric acid and 13 parts water, adding water from 
 time to time to replace that which has evaporated. After 
 
38 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 boiling it is diluted with water and gradually treated with 
 milk of lime with constant stirring until the liquid is neutral 
 or a little alkaline. Filter through a folded filter, the residue 
 on filter boiled with water several times, and after filtration 
 added to the main filtrates. The several filtrates are concen- 
 trated by evaporation and oxalic acid added so as to precipi- 
 tate all the lime; filter, boil the calcium oxalate precipitate 
 with water several times, filter and unite all filtrates, evapo- 
 rate until crystallization begins, and allow to cool. What first 
 crystallizes out consists chiefly of tyrosin with only a little 
 leucin. Filter this off and concentrate the liquid more, 
 when a second crystallization will form on cooling, which 
 consists of leucin with some tyrosin. This operation of evap- 
 orating and crystallizing is continued until no more crystals 
 are obtained. The several batches of crystals are united and 
 boiled with a large quantity of water and enough ammonia 
 to dissolve them. To this hot solution add lead acetate, con- 
 stantly stirring, until the precijntate formed is no longer 
 brown, but white; filter, heat the yellow filtrate to boiling, 
 neutralize or make faintly acid with dilute sulphuric acid, 
 filter while boiling hot, and allow to cool. Pure tyrosin is 
 deposited while leucin remains in solution. 
 
 The mother liquor from the tyrosin is treated with H 2 S, 
 filtered and concentrated and boiled a couple of minutes with 
 freshly precipitated copper oxyhydrate. A deep blue solution 
 is the result, which, if filtered and concentrated, deposits sky- 
 blue crystalline warts, and an insoluble cuprous compound of 
 leucin. This precipitate, as well as the crystalline warts, is 
 decomposed in hot water with H 2 S, filtered, and the filtrate de- 
 colorized when necessary with animal charcoal, strongly concen- 
 trated, and allowed to crystallize. The leucin which deposits 
 may be purified by recrystallization from boiling alcohol or 
 by precipitating it as leucin lead oxide, decomposing this" 
 precipitate when suspended in water with H 2 S, and evaporat- 
 ing the filtrate to point of crystallization. The tyrosin may 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 39 
 
 be purified by recrystallization from boiling water or from 
 ammoniacal water. 
 
 2. A large quantity of well-boiled fibrin is digested at 
 40° C. with a solution of Kuhne's dried pancreas containing 1 
 per cent salicylic acid and some thymol. After a day or two 
 the liquid is filtered, faintly acidified with acetic acid, and 
 boiled. After filtration concentrate the filtrate nearly to 
 syrupy consistency and set aside to cool. Considerable quan- 
 tities of leucin and tyrosin will have crystallized out after 24 
 hours. Remove the crystals and concentrate the mother-liquor 
 further if necessary, and precipitate the peptones with an 
 excess of hot alcohol, and filter while hot. On cooling crystals 
 of leucin will form if much is present. Pour off the mother 
 liquor, and wash with a saturated solution of ammonium sul- 
 phate. The mixed leucin and tyrosin resulting from these 
 operations must be separated and purified by the method of 
 Hlasiwetz and Habermann, as described in method Xo. 1. 
 
 The leucin may be simplest separated from the tyrosin by 
 boiling the yellow crystalline masses or crusts with alcohol, 
 which dissolves the leucin and leaves the tyrosin in great part. 
 After filtering and concentrating the filtrate leucin crystal- 
 lizes out and is purified by repeated crystallization from 
 alcohol. Tyrosin is obtained in a crystalline form by dissolv- 
 ing the residue insoluble in alcohol with water containing 
 ammonia, and allowing this solution, after filtration, to evap- 
 orate at ordinary temperatures. 
 
 Properties. — Leucin crystallizes when pure in shining 
 white, very thin, doubly refractive plates, nodular masses or 
 balls, possessing a greasy feel and which float on water. Pure 
 leucin dissolves in 27 parts cold water, and in 1040 parts cold 
 and in 800 parts boiling alcohol, though the solubility seems 
 to vary with the source of the leucin. On slowly heating it 
 melts at 170° C, and sublimes in white, woolly flakes, which 
 are similar to sublimed zinc oxide. Its specific rotatory power 
 is (a) D = -f- 17.5°. Solutions of leucin in water are not, as a 
 
40 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 rule, precipitated by metallic salts. The boiling hot solution 
 may, however, be precipitated by a boiling hot solution of 
 copper acetate. It forms crystalline compounds with sul- 
 phuric, hydrochloric, and nitric acids. 
 
 Pure tyrosin crystallizes in colorless, silky, fine needles, 
 which are often grouped in tufts or rosettes. From very 
 impure solutions it separates in part or wholly in nodules and 
 balls very like leucin. It is soluble with difficulty in water, 
 being dissolved by 2454 parts water at 20°C, and 154 parts 
 boiling water. It is insoluble in alcohol and ether. Tyrosin 
 is readily soluble in solutions of ammonia, caustic alkalies, and 
 alkaline carbonates. It is also soluble in dilute mineral acids, 
 with which it forms unstable compounds. 
 
 Aspartic Acid, C 4 H 7 N0 4 . 
 AMiDo-succnrco Acid, 2 H 3 (NH 2 )(CO.OH) 2 . 
 
 Preparation. — The mother-liquor from which leucin and 
 tyrosin have crystallized out (see page 38) is further concen- 
 trated and treated with a little alcohol, when, after some 
 time, new crystalline crusts will separate. These are dissolved 
 in water, and the solution boiled with freshly precipitated 
 cupric hydrate. On filtering the blue solution will deposit 
 the copper compound of aspartic acid, which occurs as light 
 blue needles. These are dissolved in HOI anddecomposed by 
 H 2 S, when white crystalline plates of aspartic acid will sepa- 
 rate out. This is purified by crystallization from boiling water. 
 
 Properties. — Aspartic acid crystallizes in rhombic prisms 
 which are sparingly soluble in cold water or alcohol, but 
 readily soluble in boiling water. Its solution, strongly acid 
 with nitric acid, is dextro-rotatory, (a) D = -f- 25.16°, but in 
 alkaline solution it is lsevo-rotatory. With copper it forms a 
 compound soluble in boiling water, but nearly insoluble in 
 cold water. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 41 
 
 Glutamic Acid, C 6 H 9 N0 4 . 
 Amido-pyrotartaric Acid, 3 H b (NH 2 )(COOH) 2 . 
 
 Preparation. — Boil 200 grms. casein (free from fat) with 
 GOO c.c. concentrated II CI and 500 grms. stannous chloride 
 for 3 days in a flask connected with a return condenser. After 
 diluting with 10 vols, water remove the tin by means of H 2 S, 
 and evaporate to syrupy consistency on water-bath. On stand- 
 ing in the cold the syrup will solidify into a mass of crystals; 
 these are stirred up and transferred to a filter connected with 
 a filter-pump, and as much mother-liquor sucked off as possi- 
 ble; then place the moist mass on an unglazed tile and placed 
 in desiccator until dry. Now recrystallize one or two times 
 from as little water as possible with the addition of cone. 
 HC1, and remove the mother-liquor as well as possible. The 
 crystals are now dissolved in a great deal of water and boiled 
 with an excess of lead oxyhydrate until the solution is free 
 from chlorine. Filter, precipitate the lead by H 2 S, filter again, 
 and evaporate the solution to point of crystallization. If these 
 crystals retain still small quantities of chlorine, treat the hot 
 solution with some silver oxide or carbonate; filter, treat the 
 filtrate with H 2 S, filter, and evaporate to crystallization. 
 
 Properties. — Glutamic acid crystallizes in the form of 
 small plates or rhombic tetrahedra or octahedra, which are 
 sparingly soluble in cold water, but readily in hot water. It 
 is insoluble in alcohol or ether, and melts at 135°-140°C. Its 
 dilute HC1 solution has a specific rotatory power of (a) D = + 
 31.1° to 31.6°. Dilute solutions reduce Fehling's solution. 
 When boiled with Cu(OH) 2 , glutamic acid separates in the 
 form of blue-colored prisms, which are soluble in 3400 parts 
 cold and 400 parts boiling water. 
 
 CH 
 
 / x \ 
 Indol, C 8 H 7 N = C 6 H 4 CH 
 
 NH 
 
42 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 C.CH 3 
 
 / ^ 
 
 Skatol, 9 H 9 N = C 6 H 4 OH 
 
 \ / 
 NH 
 
 or Methyl Indol. 
 
 Preparation. — Two kilogrammes of well-pressed blood fibrin 
 are placed in a large flask (12 litres capacity) treated with 8 
 litres water (containing 2 grms. KH 2 P0 4 and 1 grm. MgSOJ, 
 and well mixed with 200 c.c. of a cold saturated solution of 
 soda, then a few cubic centimetres of a putrefying infusion of 
 meat with some fragments of the decomposed meat. The flask 
 is closed with a stopper provided with a glass tube attached 
 with a rubber tube to a wash-bottle half full of water. 
 The rubber tube has a clamp which is left open during the 
 first days of the experiment. The mass is digested at 40° to 
 42° C. for 10 days, the flask being shaken from time to. time 
 and the clamp closed as soon as the evolution of gas has 
 ceased and only opened now and then so as to liberate the 
 accumulated gases. 
 
 After this time the entire liquid contents of the flask is 
 distilled off until the residue in the flask measures 1 to 1.5 
 litres. The strongly ammoniacal distillate is acidified with 
 HC1 and then precipitated by a solution of copper sulphate 
 and filtered. The clear filtrate is thoroughly shaken with 
 ether, which is best done by shaking fractions of -J litre at a 
 time in a separatory funnel drawing off the heavy liquid and 
 adding new portions of the filtrate to the ether in the funnel, 
 adding more ether from time to time. When one half of the 
 filtrate has been extracted with ether distill the ether from the 
 ethereal solution and use the ether for further extraction. The 
 entire ethereal extracts are distilled until about 500 c.c. are left. 
 This resi due is thoroughly shaken 2 times with a solution of 
 caustic soda to separate phenols and acids, and the ether now 
 distilled off at the lowest possible temperature. The oily 
 
HANDBOOK FOR BlO-CHEMlCAl LABORATORY. 43 
 
 residue, previously treated with some caustic-soda solution, is 
 distilled in a current of steam* until no more indol passes over. 
 
 The distillate is now shaken with ether, the ethereal solu- 
 tion distilled at the lowest possible temperature, and the 
 residue allowed to evaporate in a deep vessel until, on being 
 allowed to cool, it solidifies as a crystalline mass. The latter 
 is then dried in a desiccator over sulphuric acid. (E. and II 
 SalJco2ushi.) The crystals thus obtained, consisting of indol 
 and skatol, are dissolved in a very small quantity of absolute 
 alcohol and then treated with 8-10 vols, water. The skatol is 
 precipitated, but not the indol. The skatol is filtered off and 
 recrystallized from ether. The indol is obtained from the 
 watery solution by extracting it with ether and allowing the 
 ether to evaporate spontaneously. 
 
 The mass of crystals, consisting of indol and skatol, may be 
 separated from each other by means of fractional distillation 
 in a current of steam when the skatol passes over first. 
 
 Skatol may also be obtained from this mixture of crystals 
 by dissolving about 0.5 grm. in a few drops of benzol and 
 adding 1.5 grm. crystallized picric acid and sufficient benzol 
 to dissolve all while being heated in a covered beaker-glass. 
 On cooling the mass solidifies to a red crystalline mass. Xow 
 add 2 vols, petroleum ether and stir, filter after 24 hours, 
 wash with petroleum ether, and allow the crystals to dry 
 in the air. Place the crystals in a distilling flask and 
 distill with dilute caustic soda, which decomposes the indol, 
 while the skatol passes over and is condensed. This skatol is 
 purified by recrystallization from ether. 
 
 Properties. — Indol crystallizes from hot aqueous solutions 
 
 * The current of steam is best produced by filling a large flask two- 
 thirds with water which has been acidified with a few cubic centimetres 
 of dilute H 2 S0 4 and a few pieces of zinc, which by the action of the heat 
 develops a gentle current of hydrogen, which prevents bumping and 
 gives rise to a quiet, even generation of steam, which may be kept up 
 for hours. 
 
44 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 as small scales, which melt at 52° 0. It is tolerably soluble in 
 hot and less soluble in cold water ; readily soluble in alcohol, 
 ether, chloroform, benzol, and petroleum ether. It forms a 
 combination with picric acid, consisting of red needles, which 
 are decomposed on heating with caustic soda, but pass over 
 without decomposition when distilled with ammonia. Indol 
 has a peculiar excrementitious odor. 
 
 Skatol crystallizes in small plates, which melt at 95° C. It 
 is less soluble in water than indol, but in the presence of 
 steam it distils readily. It is readily soluble in alcohol, 
 ether, chloroform, and benzol. With picric acid it forms a 
 crystalline compound, which does not decompose on being 
 heated with caustic soda, but passes unchanged into the distil- 
 late. It has an intense fetid odor. 
 
 Ordinary or Fermentation Lactic Acid, 
 
 C 3 H e O s or OH 3 .OH.OH.COOH. 
 
 Preparation. — 330 grms. cane-sugar and 0.5 grm. tartaric 
 acid are dissolved in 1750 c.c. boiling water and allowed to 
 stand for two days. Then add 4 grms. putrefying cheese 
 (German Handkase), 440 grms. sour milk, and 135 grms. 
 zinc white, and allow this mixture to stand for eight to ten 
 days at 40° C. with constant shaking. After this time heat to 
 boiling, filter, and allow to cool, when zinc lactate will crystal- 
 lize out. This is purified by recrystallization and then de- 
 composed by H 2 S in hot aqueous solution, the solution fil- 
 tered, and the filtrate evaporated on the water-bath. The 
 syrupy residue is dissolved in ether (to separate any mannite), 
 and then the ether removed by distillation or by evaporation. 
 
 Properties. — Fermentation lactic acid has a similar appear- 
 ance to sarcolactic acid, namely, a colorless, faintly yellowish, 
 acid-reacting syrup, which mixes in all proportions with water, 
 alcohol, or ether. It is optically inactive. It forms salts 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 45 
 
 readily, which are soluble in water and most of them in alco- 
 hol. The zinc salt dissolves in 58 to 63 parts of water at 
 14-15° 0. and contains 18.18$ water of crystallization, but is 
 insoluble in alcohol. The calcium salt dissolves in 9,5 parts 
 water and contains 29.22$ (= 5mol.) water of crystalliza- 
 tion, 
 
 Sarcolactic Acid, 
 
 Paralactic Acid, C 3 H 6 3 orCH,.CH(OH).COOH, 
 
 Preparation, — Dissolve Liebig's extract of meat in 4 parts 
 of water and then add 8 parts 90$ alcohol, stirring all the 
 while. The mixture is allowed to stand and settle, and the 
 clear supernatant liquid is separated by decantation. To re- 
 move any lactic acid from the insoluble residue, mix it with 
 twice its weight of warm water and precipitate by adding 4 or 5 
 times its volume of alcohol. The alcoholic solutions thus ob- 
 tained are evaporated on the water-bath to a thin syrup, and 
 this latter again precipitated by the addition of 3 or 4 times its 
 volume of alcohol. The alcoholic solution is now evaporated 
 to dryness, the residue mixed with water, some dilute H 2 S0 4 
 added, and then shaken several times with ether. On evapo- 
 rating the ethereal solution a residue is obtained which con- 
 sists of a mixture of sarcolactic and fermentation lactic 
 acids. 
 
 To separate the two above acids they are dissolved in water 
 and boiled with zinc carbonate or zinc white, the solution 
 filtered, and the filtrate evaporated until crystals commence to 
 form. The liquid is now treated with 4 or 5 times its volume 
 of 90$ alcohol ; after some time the liquid becomes turbid 
 and deposits needles, consisting of zinc sarcolactate, the zinc 
 salt of the other acid remaining in solution. The crystals are 
 collected on a filter, washed with absolute alcohol, dissolved 
 in water, decomposed by H 2 S, filtered, concentrated, shaken 
 with ether, and the pure acid obtained on the evaporation of 
 the ethereal solution, 
 
46 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Properties. — Sarcolactic acid occurs as a syrup, which dis- 
 solves in water, alcohol, and ether readily. It is dextro-rota- 
 tory, but its salts, on the other hand, are laevo-rotatory. Its 
 zinc salt crystallizes with 2 mol. (12.9$) water of crystalliza- 
 tion as small four-sided prisms, which dissolve in 17.5 parts 
 water, its calcium salt containing 24.83-26. 21$ (4 or 4.5 mol.) 
 water of crystallization, and dissolving in 12.4 parts water. 
 
 Protagon. 
 
 Preparation. — An ox-brain as fresh as possible, with the 
 blood and membranes carefully removed, is ground fine and 
 then extracted for several hours with 85$ alcohol at 45° 0., 
 filtered while hot, and the residue extracted with warm alco- 
 hol until the filtrate fails to yield a precipitate at 0° 0. The 
 several alcoholic extracts are cooled to 0° C, and the precipi- 
 tates united and completely extracted with cold ether, which 
 removes cholesterin and lecithin-like bodies. The residue is 
 strongly pressed between filter-paper and allowed to dry over 
 sulphuric acid or phosphoric anhydride. The resulting mass 
 is powdered, moistened with water digested for many hours 
 with alcohol at 45° 0., filtered, slowly cooled to 0° 0. The 
 crystals may be purified by recrystallization. (Gamgee and 
 Blankenhom.) 
 
 Properties. — Protagon appears when dry as a white, loose 
 crystalline powder. It is soluble with difficulty in cold, but 
 more easily soluble in warm, alcohol and ether. At tempera- 
 tures above 55° 0. its solutions decompose. It swells up in 
 little water, decomposes partly, and gives myaline forms. It 
 is soluble in glacial acetic acid, which deposits crystalline 
 forms on cooling. When boiled with a solution of Ba(OH) 2 , 
 protagon decomposes into glycero-phosphoric acid, fatty acids, 
 and neurine. It melts at 200° C, forming a brown syrup. - 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 47 
 
 Urea, CH 4 N 2 or H a N.CO.NH 1 . 
 
 Preparation. — Evaporate \ to 1 litre of urine to syrupy 
 consistency on the water-bath, allow to cool in ice-water, treat 
 while stirring with 3 vols, nitric acid of sp. gr. 1.3 which 
 has previously been boiled and then coolod to 0° C. Allow 
 this mixture to stand for a few hours at 0° C. The crystals 
 of urea nitrate which form are transferred to a filter of glass 
 wool and washed several times with small amounts of ice-cold 
 pure concentrated nitric acid, then dissolved in as small a 
 quantity of hot water as possible, and reprecipitated by cone. 
 HN0 3 . These crystals are first drained, then dissolved in hot 
 water, and the solution decolorized by a little chlorine water, 
 and then treated with a small quantity of pure barium carbon- 
 ate until no more dissolves and effervescence ceases and the 
 solution is neutral. Now evaporate the solution to complete 
 dryness on the water-bath, pulverize the residue, and extract 
 it with cold absolute alcohol, which dissolves the urea, but not 
 the barium nitrate. The filtered alcoholic solution is decolor- 
 ized when necessary by animal charcoal and evaporated at a 
 medium heat to point of crystallization. 
 
 Synthetical. — Heat 250 grms. coarsely powdered potassium 
 ferrocyanide in a capacious porcelain dish over a naked flame 
 with constant stirring until all is changed into a white pow- 
 der, leaving no lumps having a yellow nucleus. Care must 
 be taken not to apply too strong a heat, as otherwise the pow- 
 der will turn brown. The mass thus obtained is finely 
 powdered and thoroughly mixed with one half (-J) its weight 
 of dried finely powdered manganese dioxide, and the mixture 
 heated on an iron dish under the draught, constantly stirring 
 until deflagration begins and the mass becomes doughy. The 
 heat is continued until a portion of the mass placed in water 
 and acidified with HC1 fails to give a blue precipitate with 
 ferric chloride. Then allow to cool, lixiviate with cold water, 
 add to the solution f of the weight of the dried potassium 
 
48 
 
 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 ferrocyanide of ammonium sulphate, filter, and evaporate at 
 about 60°-70° 0., at which temperature the ammonium cya- 
 nate is converted into urea. The crystals of potassium sul- 
 phate are removed from time to time and then the solution 
 evaporated to dryness. The urea is obtained from this residue 
 by extraction by absolute alcohol as above described. 
 
 Properties. — Urea crystallizes in needles or in long, colorless 
 four-sided, often hollow, anhydrous rhombic prisms, similar 
 to saltpetre. It has a neutral reaction, has a bitter taste, and 
 produces a cooling sensation on the tongue. It melts at 130°- 
 132° C, but partly decomposes at about 100° C. At ordinary 
 temperatures it dissolves in equal weights of water and in five 
 parts alcohol. Urea is readily soluble in amyl alcohol. It is 
 insoluble in anhydrous ether, chloroform, and in petroleum 
 ether. Urea combines with salts, such as NaCl, NH 4 C1, and 
 the chlorides of the heavy metals, such as mercury, gold, zinc, 
 copper, etc., producing crystalline combinations. It also com- 
 bines with acids, both inorganic and organic, forming crystal- 
 line salts. 
 
 Uric Acid, 
 
 C 6 H 4 N 4 3 or HN— CO 
 
 CO 0— NH 
 
 CO 
 
 HN-C— KH 
 
 Preparation. — 1. Powdered Peruvian guano is just heated 
 to boiling with 15-20 parts water in a porcelain dish, and 
 then dissolved by the careful addition of a small amount of 
 concentrated solution of caustic soda or potash, and now 
 boiled until the odor of ammonia has disappeared. Filter and" 
 saturate the filtrate with carbon dioxide, when a gelatinous, 
 ajtervvards granular, precipitate of acid potassium urate is 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 49 
 
 formed. The liquid is decanted after standing 24 hours, and 
 the precipitate washed with small quantities of ice-cold water, 
 and while still moist added to boiling dilute hydrochloric acid 
 and kept boiling for some time, continually stirring. After 
 cooling the separated uric acid is filtered, thoroughly washed 
 with cold water, and dried. If not quite pure, it can be redis- 
 solved in alkali, precipitated by C0 2 , and treated again as 
 above, or it may be added to water and dissolved in the cold 
 by the gradual addition of sodium amalgam, and after the re- 
 moval of the impurities by filtration the uric acid may be re- 
 precipitated by HOI. 
 
 2. Filtered normal urine is treated with 20-30 c.c. of 25$ 
 HOI for each litre of urine. After 48 hours collect the crys- 
 tals and purify them by redissolving in dilute alkali, decolor- 
 izing with animal charcoal, and reprecipitating with hydro- 
 chloric acid. Filter, wash with ice-cold water, and dry. 
 
 3. Uric acid may be prepared synthetically by melting 
 0.1-0.15 grms. glycocoll with 1-2 grms. urea in a test-tube 
 over a small flame. The mass is heated until solid, care be- 
 ing taken not to apply too strong (above 220° O.) a heat, also 
 not too low a heat. The cooled mass, which ought to be yel- 
 low or yellowish-brown, is dissolved in boiling water with the 
 addition of ammonia and filtered; the filtrate is precipitated 
 when cold with a mixture of magnesium mixture and ammo- 
 niacal silver nitrate solution. The precipitate is washed with 
 ammoniacal water, subdivided in hot water, and treated with 
 a solution of sodium sulphide. The filtrate from the silver 
 sulphide is acidified with HOI and concentrated, when on 
 cooling impure uric acid will crystallize out. Test these crys- 
 tals with the murexid test, and if they do not give the reac- 
 tion reprecipitate with ammoniacal silver solution and proceed 
 as above, when comparatively pure uric acid will be obtained. 
 It is better to make several fusions of glycocoll with urea, and 
 adding these together and obtaining the uric acid therefrom 
 in larger quantities. (J. Horbaczewski.) 
 
50 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Properties. — Pure uric acid is a white, odorless, and taste- 
 less powder consisting of very small rhombical prisms or 
 plates. It is insoluble in alcohol and ether, rather easily sol- 
 uble in boiling glycerin, very difficultly soluble in cold water 
 (14000-15000 parts), and difficultly soluble in boiling water 
 (1800-1900 parts). It is soluble in sulphuric acid in the cold 
 without decomposition, and readily soluble in many salts of 
 the alkalies, as in the caustic alkalies; ammonia, however, 
 scarcely dissolves it. Uric acid reduces alkaline solutions of 
 copper or silver, but not alkaline solutions of bismuth. 
 
 Hippuric Acid, 
 
 C 9 H 9 N0 3 or C 6 H 5 .CO.NH.CH a .COOH. 
 
 Preparation. — 1. Treat fresh horse or cow urine with milk 
 of lime until it is of a strong alkaline reaction; warm, filter, 
 evaporate the filtrate to a syrupy consistency, and acidify 
 strongly with HOI when cold. The hippuric acid thus pre- 
 cipitated is drained, washed with cold water, pressed between 
 filter-paper, dissolved in as small a quantity of boiling water 
 as possible, filtered, and the boiling-hot filtrate treated with 
 chlorine gas until the color of the solution is pale yellow. 
 Cool quickly, filter, wash the hippuric acid a few times with 
 cold water, and crystallize from boiling water after treating 
 the solution with animal charcoal. (TJi. Curtius.) 
 
 2. One litre of the fresh horse or cow urine is made faintly 
 alkaline with sodium carbonate and filtered, the filtrate 
 evaporated nearly to dryness, and the residue repeatedly ex- 
 tracted with cold, strong alcohol. The alcohol is nearly en- 
 tirely distilled off from the extracts the remaining watery 
 solution acidified with HOI, and then repeatedly extracted 
 with fresh portions of acetic ether (at least five times). The 
 acetic ether extracts are united, and washed by shaking with 
 water in a separatory funnel, and then evaporated at a moderate 
 temperature. The residue is repeatedly treated with petro- 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 51 
 
 leum ether, which removes the benzoic acid, oxyacids, fat, and 
 phenol, while the hippuric acid is left undissolved. The 
 residue is dissolved in a little warm water, treated with 
 animal charcoal, and evaporated at 50°-60° 0. to point of 
 crystallization. (Bunge and Schmiedeb'erg.) 
 
 Synthetical. — Hippuric acid may be prepared by adding a 
 few drops caustic-soda solution to a watery solution of glyco- 
 coll, shaking with benzoyl chloride, adding gradually an ex- 
 cess thereof and then making strongly alkaline with caustic 
 soda. Now precipitate with hydrochloric acid on cooling. 
 The precipitate of hippuric and benzoic acids are carefully 
 drained, pressed between filter-paper and washed with cold 
 water, dried, and extracted with petroleum ether, which only 
 dissolves the benzoic acid. The remaining hippuric acid is 
 recrystallized from hot water. (J. Baum.) 
 
 Properties. — Hippuric acid crystallizes in semi-transparent, 
 milk-white, long, four-sided rhombic prisms or columns, or 
 in needles on rapid crystallization. They dissolve in 600 
 parts cold water, but more easily in hot water. It is soluble 
 in alcohol, but with difficulty in ether. It is insoluble in 
 petroleum ether, benzol, and carbon disulphide. Hippuric 
 acid is readily soluble (about 12 times) in acetic ether. It 
 combines with bases, forming salts which are soluble in water 
 or alcohol. The combinations with the alkalies and the alka- 
 line earths are readily soluble in alcohol, while the silver, 
 copper, and lead salts are soluble with difficulty. On boiling 
 with caustic alkalies, mineral acids, or by continued heating 
 with water at 170°-180° 0. it splits into benzoic acid and 
 glycocoll. 
 
 Allantoin, 
 
 n tt at a r , A /NH.CH.NH.CO.NH a . 
 4 H 6 N 4 3 or CO^ NH co 
 
 Preparation. — 1. Evaporate the amniotic fluid of a cow to 
 pne sixth (£) its volume, or calf urine to syrupy consistency, 
 
52 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 and allow it to stand in a cold place for several days, when 
 allantoin and magnesium phosphate crystallize out, with ge- 
 latinous magnesium urate. Now dilute with water, stir, and 
 decant the liquid from the allantoin and magnesium-phos- 
 phate crystals. Eepeat this several times, and boil the crystals 
 with water after having added some animal charcoal; filter 
 while hot; faintly acidify the filtrate with HOI, which keeps 
 the phosphates in solution, and allow to crystallize. Purify 
 by recrystallization from water. ( Wohler.) 
 
 2. Make a thin paste of uric acid and water, heat nearly to 
 boiling, and add in small quantities a thin paste of finely 
 ground lead peroxide with water, continually stirring until 
 the chocolate-brown color of the mixture just disappears. 
 (Oare must be taken not to have an excess of lead peroxide.) 
 If the mass has become too thick, some water may be added 
 during the operation; now filter while hot, and allow to cool. 
 After crystallization the mother-liquor is concentrated by 
 evaporation so as to obtain more allantoin. The preparation 
 is purified by repeated crystallization from small amounts of 
 boiling water. (K Hoffmann.) 
 
 3. Treat 3 mols. uric acid, subdivided in water, with 1 mol. 
 potassium permanganate, taking care that the mixture does 
 not get warm. Filter quickly, supersaturate with acetic acid 
 and allow to stand 24 hours, when crystals of allantoin will be 
 formed. (K. Hoffmann.) 
 
 Properties. — Allantoin is a colorless substance, often crys- 
 tallizing in prisms, or, when prepared from calf urine, as small, 
 thin columns. It is soluble in 160 parts cold (20° C.) and 30 
 parts boiling water. It is insoluble in cold absolute alcohol 
 and ether. Allantoin combines with acids and bases, forming 
 salts. With silver oxide and mercuric oxide it forms im- 
 portant combinations. Allantoin reduces Fehling's solution 
 on continued boiling. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 53 
 
 Creating 
 
 C 4 H,N,0 9 + H a O or NH. : C [NHJ.N[CH,].CH,.COOH. 
 Syn. Methyl Guanidin Acetic Acid. 
 
 Preparation. — 1. Beef extract (Liebig's) is treated with 
 water and the albumin removed as far as possible by heating 
 to boiling, and filtering. The filtrate is precipitated by the 
 careful addition of basic lead acetate, taking care not to add 
 too great an excess of the precipitant. Filter, remove the 
 excess of lead acetate in the filtrate by H 2 S, and evajDorate the 
 filtrate to a small volume at moderate temperature. Allow 
 the solution to stand one week in a cool place, filter off the 
 separated crystals, wash with 88^ alcohol, and purify when 
 necessary by recrystallization. The crystals may be decolor- 
 ized by dissolving them in hot water to which animal charcoal 
 has been added, filtering,and allowing the filtrate to crystallize. 
 
 2. Finely chopped meat is mixed with one half its weight 
 of water, pressed, and the residue again extracted with the 
 same amount of water. This extract is freed from albumin 
 by heating to boiling and filtering, and adding a solution of 
 baryta water to the filtrate as long as a precipitate forms. 
 Filter off the precipitated phosphates, remove the excess of 
 baryta by means of carbon dioxide, filter, and evaporate the 
 filtrate to one twentieth of its volume on the water-bath. The 
 syrup is allowed to stand several clays in a cool place when 
 crystals will form. If a skin should form on the surface this 
 should be removed. 
 
 3. Chopped meat is treated with ether and allowed to stand 
 for a few days, when a strongly acid fluid separates out. This 
 is colored red, due to myohaematin. This watery fluid is sep- 
 arated and evaporated, when crystals of creatin will separate 
 out, which may be purified by recrystallization as above. 
 (Mac Jfu 7i7i.) 
 
 Properties. — Creatin crystallizes in hard, colorless, shining, 
 
54 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 monoclinic prisms containing 1 mol. water of crystallization, 
 which it loses when heated to 100° C. It dissolves in 74 
 parts water at the ordinary temperature, and in 9400 parts 
 absolute alcohol. It is insoluble in ether. Its watery solu- 
 tion is neutral in reaction, and has a bitter taste. Creatin 
 forms crystalline compounds with mineral acids and with 
 mercury. When heated with dilute mineral acids it is con- 
 verted into creatinin. It forms compounds with certain 
 metallic solutions. 
 
 Creatinin, 
 
 C 4 H 7 N 3 or NH : (/ NH ^ 
 
 \N(CH 3 ).CH 2 . 
 
 Methyl Guanldi^ Hydantoln". 
 
 Preparation. — 1. Evaporate several litres of urine to one 
 third or one quarter its volume, allow to cool, and decant liquid 
 from the precipitated salts, now precipitate with lead acetate 
 and filter. The excess of lead in the filtrate is removed by H 2 S 
 or soda solution; filter again, nearly neutralize with soda solu- 
 tion or acetic acid, heat to drive off H 2 S, and add a concentrat- 
 ed solution of mercuric chloride, which causes a precipitate. 
 Filter, suspend the precipitate in water and decompose it 
 with a current of H 2 S, and again filter. Decolorize the filtrate 
 with animal charcoal and evaporate to dryness. The mass is 
 recrystallized several times from strong alcohol. The creatinin 
 may be easily obtained pure from the creatinin hydrochloride 
 by treating with lead oxyhydrate and then crystallizing. 
 
 2. One-half to one litre of urine is treated with baryta mix- 
 ture (1 vol. saturated Ba(N0 3 ) 2 solution and 2 vols, satu- 
 rated baryta-water) until no further precipitate is formed, 
 filtered, and the filtrate evaporated on the water-bath to a 
 thin syrup. This is mixed with an equal volume alcohol and 
 allowed to stand in a cool place for 24 hours ; the salts which 
 separate out are removed by filtration and the filtrate treated 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 55 
 
 with 1 to 2 c.c. concentrated alcoholic solution of acid-free 
 zinc chloride. After a few days yellow crystals of creatinin 
 zinc chloride separate out ; these are filtered off, washed with 
 alcohol, dissolved in hot water and decomposed by boiling for 
 about half an hour with an excess of freshly precipitated 
 lead oxide or lead carbonate. Filter while hot, decolorize the 
 filtrate by boiling with animal charcoal, filter again, evaporate 
 to dryness, and extract the residue by strong alcohol in the 
 cold, which removes the creatinin. Creatin remains behind 
 undissolved. 
 
 3. Precipitate -§- to 1 litre of urine with phospho-tungstic 
 acid and hydrochloric acid, filter, and treat the precipitate 
 with caustic baryta. Filter, and remove the excess of baryta 
 by means of carbon dioxide, and filter again. Evaporate the 
 filtrate to dryness, extract the residue with strong alcohol and 
 evaporate the alcohol, when the creatinin will be obtained 
 impure. It may be purified as above described. 
 
 Properties. — Creatinin crystallizes in long, colorless, highly 
 refractive, monoclinic prisms, which do not become white 
 with loss of water when heated to 100° C. It dissolves in 
 11.5 parts cold water and 100 parts cold alcohol, though it is 
 more soluble in hot liquids. It is nearly insoluble in ether. 
 Creatinin combines with HC1 and certain metallic salts, form- 
 ing crystalline compounds. Creatinin acts as a reducing 
 agent, reducing Fehling's and Trommer's solutions; also 
 reducing mercuric oxide to metallic mercury, and yielding 
 oxalic acid and methyl guanidin. 
 
 Xanthin Bases. 
 
 Xanthln, C & H 4 N" 4 2 . 
 
 Hypoxanthin, C.IO^O. 
 
 ' 5 4 4 
 
 Adenin, C 5 H 6 N 6 . 
 
 Guanin, C 5 H 5 N 5 0. 
 
 Preparation. — 250 grms. finely chopped beef liver is placed 
 
56 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 in a large flask, provided with a glass stopper, and treated 
 with 2.5 litres chloroform water (2.5 litres tap water with 
 12.5 c.c. chloroform, and shaken until the latter is dissolved); 
 add 2.5 c.c. chloroform, and place this mixture in an incu- 
 bator and allow to digest at 40° C. for two to three days, con- 
 tinually shaking. After digestion transfer to a large dish 
 and heat to boiling, and continue boiling after acidifying with 
 acetic acid until the albumin is precipitated. Now filter, and 
 evaporate the filtrate to 800-1000 c.c. Make alkaline with 
 ammonia, filter off the precipitate formed, and completely 
 precipitate with a 3^ silver-nitrate solution. The gelatinous 
 precipitate formed consists of the silver combination of the 
 xanthin bases. Care must be taken not to have a silver- 
 chloride precipitate form, otherwise more ammonia must be 
 added. Filter off the precipitate, wash well, place the pre- 
 cipitate in a flask while still moist, and dissolve by the aid of 
 heat in nitric acid of sp. gr. 1.1 (equal volumes nitric acid 
 and water), adding a little urea to prevent the formation of 
 nitrous acid. The solution should be nearly clear. Filter 
 while hot and allow to stand for 24 hours. A precipitate of 
 guanin, adenin, and hypoxanthin silver nitrate will have 
 formed, while xanthin silver nitrate remains in solution. 
 Filter and wash with dilute nitric acid. 
 
 Add ammonia to the filtrate until alkaline, when a precipi- 
 tate of xanthin silver nitrate will form as a brownish or red- 
 dish flocculent precipitate. Filter, wash, suspend in water 
 with a few drops ammonia; heat, add a few drops ammonium 
 sulphide, stir, filter off the silver sulphide, and evaporate the 
 filtrate, when xanthin will be obtained. (The silver com- 
 pound may also be decomposed by HC1, but then on evapo- 
 ration xanthin hydrochloride is produced.) The xanthin 
 obtained as above is never quite pure. 
 
 The residue of hypoxanthin, guanin, and adenin silver- 
 compounds is suspended in water and decomposed with a 
 current of H a S, filtered, and the filtrate evaporated to a 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 57 
 
 small volume. This is made alkaline with ammonia and 
 allowed to stand, when a deposit of guanin will occur, while 
 the hypoxanthin and adenin (besides ammonium nitrate) 
 remain in solution. Filter off the guanin and wash with 
 water. The filtrate is evaporated to dryness on the water- 
 bath and the residue extracted with small amounts of water 
 to remove the ammonium nitrate, leaving the insoluble hypo- 
 xanthin and adenin. 
 
 Properties.— Xanthin is amorphous, or forms masses of 
 crystalline leaves. It is soluble with difficulty in water (in 
 14,151 to 14,600 parts water at + 16° 0. and in 1300 to 
 1500 parts at 100° C). It is insoluble in alcohol or ether, 
 but is dissolved by alkalies or acids. Xanthin forms a crys- 
 talline, difficultly soluble combination with HC1. With very 
 little caustic soda, xanthin gives a crystalline combination 
 which is readily soluble in more alkali. When dissolved in 
 ammonia, xanthin gives a gelatinous precipitate of xanthin 
 silver, with silver nitrate. This body is soluble in nitric acid. 
 A watery solution of xanthin is precipitated on boiling with 
 copper acetate. Xanthin is precipitated at the ordinary tem- 
 peratures by mercuric chloride and also by ammoniacal basic 
 lead acetate. 
 
 Hypoxanthin occurs as colorless, crystalline needles, which 
 are soluble in 300 parts cold and 78 parts boiling water. It 
 is nearly insoluble in alcohol, but is soluble in acids or alkalies. 
 The combination with HC1 is crystalline, but is more soluble 
 than the corresponding combination with xanthin. The 
 silver combination with hypoxanthin dissolves with great 
 difficulty in boiling nitric acid. Hypoxanthin picrate is diffi- 
 cultly soluble, but if a boiling solution is treated with a neu- 
 tral or faintly acid solution of silver nitrate, the hypo- 
 xanthin is quantitatively precipitated as a combination, hav- 
 ing the formula C.H.AgN^O.C.H^NOJ.OH. 
 
 Adenin crystallizes with 3 mol. water of crystallization in 
 long needles, which become opalescent when heated or ex- 
 
58 HANDBOOK FOR BlO-CHEMlCAL LABORATORY. 
 
 posed to the air. When the crystals are slowly heated with 
 insufficient water from a solution they become suddenly opa- 
 lescent at + 53° C. It is soluble in 10SG parts cold water, 
 and much more readily soluble in hot water. Adenin is 
 insoluble in ether, but somewhat soluble in hot alcohol. It is 
 readily soluble in acids or alkalies. It is more soluble in 
 ammonia than guanin, but less soluble than hypoxanthin. 
 The silver combination of adenin is difficultly soluble in 
 warm nitric acid, and deposits crystals of adenin silver nitrate 
 on cooling. It also gives a combination with picric acid 
 which is soluble with difficulty. 
 
 Guanin forms an amorphous, colorless powder, but crystal- 
 lizes in very small crystals from a solution in concentrated 
 ammonia if allowed to spontaneously evaporate. It is insolu- 
 ble in water, alcohol, or ether, readily soluble in mineral 
 acids or alkalies, but with difficulty in ammonia. The HC1 
 salt of guanin crystallizes readily, and on account of its action 
 on polarized light may be made use of in the identification 
 of guanin. Picric or metaphosphoric acids precipitate even 
 very dilute solutions of guanin. The silver combination is 
 very difficultly soluble in boiling nitric acid, and deposits 
 crystals on cooling. 
 
 Urobilin. 
 
 Preparation, — 1. Several litres of urine are acidified with 
 H 2 S0 4 , using 2 grms. for every litre; then saturated with 
 ammonium sulphate, filtered, and washed with a saturated so- 
 lution of ammonium sulphate faintly acidified with sulphuric 
 acid. The residue of urobilin is dissolved in alcohol, which 
 requires some time. The filtered alcoholic solution is mixed 
 with chloroform and a volume of water equal to the alcoholic 
 solution, well shaken together, and allowed to stand until the 
 chloroform has settled. The chloroform is removed by means" 
 of a separatory funnel, and washed with twice its volume of 
 water. The clear chloroform solution is now filtered through 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 59 
 
 a dry filter into a distilling flask and the chloroform distilled 
 off. The residue in the flask is washed with ether, which 
 only dissolves very little of the urobilin. This residue is 
 again dissolved in chloroform, filtered, and allowed to evapo- 
 rate at a very moderate temperature. (Melm.) 
 
 2. Precipitate the urine with basic lead acetate, wash the 
 precipitate with water, dry at the ordinary temperature, and 
 then boil it with alcohol, acidified with sulphuric acid. The 
 filtered alcoholic solution is diluted with water, now satu- 
 rated with ammonia, and then treated with a zinc-chloride 
 solution. This new precipitate is washed free from chlorine 
 with water, boiled with alcohol, dried, dissolved in ammonia, 
 and this solution precipitated with sugar of lead. This pre- 
 cipitate, which is washed with water and boiled with alcohol, is 
 decomposed by alcohol containing sulphuric acid; the filtered 
 alcoholic solution is mixed with -J vol. chloroform, diluted 
 with water and shaken repeatedly, but not too energetically. 
 The urobilin is taken up by the chloroform. This last is washed 
 once or twice with a little water and then filtered, leaving the 
 urobilin on evaporation of the chloroform. It may be further 
 purified, by treating with ether. 
 
 3. The urine is treated with ammonia in not too great ex- 
 cess, filtered, and the filtrate precipitated with a concentrated 
 watery or alcoholic solution of zinc chloride. If the filtrate 
 from this precipitate is still deeply colored, precipitate 
 by adding more ammonia and filtering. The voluminous, 
 generally red or reddish-brown precipitate is washed free 
 from chlorine by first using cold and then hot water, now 
 boiling with alcohol, and drying at a low temperature. The 
 mass is powdered and dissolved in ammonia, leaving a small 
 residue, and this ammoniacal solution is precipitated with 
 sugar of lead, and the red precipitate filtered off and washed 
 with water until the coloring matter commences to go into 
 solution. The precipitate is now digested with alcohol acidi- 
 fied with sulphuric acid, filtered, and the filtrate repeatedly 
 
60 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 shaken with its J- vol. chloroform and considerable water. The 
 chloroform is separated and washed once or twice with water 
 and the chloroform now distilled off. The residue, consisting 
 of impure urobilin, is washed with ether, which removes 
 considerable quantities of a red coloring matter, and leaves 
 the urobilin as brown amorphous masses. 
 
 Properties. — Urobilin, according to Jaffe, is amorphous, red, 
 dingy red or reddish yellow, according to the method of prep- 
 aration. It is readily soluble in alcohol, amyl alcohol, acetic 
 ether, and chloroform, but less readily in ether or water. It 
 is soluble in alkalies, and is incompletely precipitated from 
 the alkaline solution by the addition of acid. Its alkaline 
 solutions give insoluble combinations with salts of the heavy 
 metals, such as zinc and lead. A neutral solution of urobilin 
 gives a green fluorescence, and the acid solution shows a faint 
 absorption-band between b and F, which borders on F, or in 
 greater concentration extends over F. The alkaline solutions 
 show a darker or more .sharply defined absorption-band, almost 
 midway between b and F. 
 
 Glycuronic Acid, 
 
 C 6 H 10 O 7 . 
 
 Preparation. — Puree or Indian yellow is rubbed fine with 
 water in a mortar, forming a thick mass. This is acidified 
 with hydrochloric acid, filtered, aud thoroughly washed with 
 water. The euxanthic acid thus obtained is dissolved in hot 
 alcohol, filtered, and allowed to stand to crystallize as beautiful 
 yellow needles. This crystallization is repeated again so as to 
 obtain pure euxanthic acid. One part of the euxanthic acid 
 is treated with 150 parts water and heated, in a Papin's di- 
 gester supplied with a thermometer, first to boiling, allowing 
 the cover of the digester to be open. After closing the cover 
 heat for an hour, keeping the temperature at 120°-125 a C. 
 On cooling the liquid is filtered, leaving the crystals of unde- 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 61 
 
 composed enxanthic acid and euxanthon on the filter. These 
 are again treated with water as above. The filtrate is evapo- 
 rated in a flat dish, care being taken not to have too high a 
 temperature. When at a syrupy consistency it is allowed to 
 stand, when crystals of glycuronic acid anhydride are obtained. 
 {Thierf elder.) 
 
 Properties. — Glycuronic acid is not crystalline, but is ob- 
 tained only as a syrup. It dissolves in alcohol, and is easily 
 soluble in water. It forms crystalline salts with potassium 
 and sodium. The neutral lead salt is soluble in water, but 
 the basic salt is, on the contrary, insoluble. The acid is dex- 
 tro-rotatory (a)D = -f- 19.4, and reduces alkaline solutions of 
 copper, silver, and bismuth salts. It gives a crystalline com- 
 bination with phenyl hydrazine, melting at 114°-115° 0. 
 
LIST OF 
 
 IMPORTANT TESTS 
 
 ARRANGED IN ALPHABETICAL ORDER. 
 
LIST OF IMPORTANT TESTS ARRANGED IN 
 ALPHABETICAL ORDER. 
 
 Acetone. See Chautard, Gunning, Lieben, Legal, Le 
 Nobel, Materia, Penzolclt, Reynolds. 
 
 Aceto-acetic Acid. See Gerhardt. 
 
 Adamkiewicz's Reaction (Proteids). — Add the proteid to 
 a mixture of 1 vol. concentrated sulphuric acid and 2 vols, 
 glacial acetic acid. A reddish-violet color is obtained slowly 
 at the ordinary temperature, but more quickly on heating. 
 The liquid has also a feeble fluorescence, and gives an ab- 
 sorption band between the lines b and F in the solar spec- 
 trum. 
 
 Albumin - . See Axenfeld, Boedeher, Cohen, Furbringer, 
 Heller, Heynsius, Hindenlang, Johnson, McWilliam, Mehu, 
 Oliver, Pavy, Eeoch, Roberts, Rees, Raabe, Spiegler, Tanret, 
 Zouchlos. 
 
 Allen's Test (Phenol). — Add to one to two drops of the 
 liquid to be tested a few drops of hydrochloric acid and then 
 1 drop of nitric acid. Cherry-red color is produced, which 
 is intensified by gentle warming. Alcohol does not interfere 
 with the reaction. On supersaturating with caustic soda the 
 red liquid becomes dark brown, 
 
 65 
 
66 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Almen's Reaction (Blood). — Mix in a test-tube equal vol- 
 umes of tincture of gnaiacum and old turpentine which lias 
 become strongly ozonized by the action of air under the in- 
 fluence of light. Allow the liquid to be tested to flow down 
 gently on the surface of this mixture. If blood or blood- 
 coloring matters are present, a bluish-green and then a beau- 
 tiful blue ring appears where the two liquids come together, 
 and if shaken the liquid becomes more or less blue. Pus 
 gives a blue color with this mixture, but in this case the tinct- 
 ure of guaiacum alone, without turpentine, is colored blue. 
 
 Almen's Test (Glucose). — Heat liquid with a solution of 
 bismuth subnitrate dissolved in caustic soda and Rochelle 
 salts. If glucose is present, the liquid becomes dark, 
 cloudy, dark brown, or nearly black, and non-transparent. 
 After a time a black deposit appears. 
 
 Andreasch's Reaction (Cy stein). — Treat the hydrochloric 
 acid solution with a few drops dilute ferric chloride solution 
 and then ammonia. The liquid will become beautifully red, 
 darkening on shaking with air. 
 
 Axenfeld's Test (Albumin in Urine). — Acidify with formic 
 acid and add, drop by drop, a 0.1 per cent gold chloride solu- 
 tion and warm. The solution becomes first red, then purple 
 red, and, on the further addition of gold chloride, blue, and 
 lastly, a blue precipitate is .produced. The red coloration is 
 characteristic of albumin, while the blue and violet may be 
 produced by other bodies, such as glucose, glycogen, starch, 
 leucin, tyrosin, uric acid, urea, creatinin, etc. 
 
 Baeyer's Reaction (Glucose).— On boiling a glucose solu- 
 tion with ortho-nitrophenyl propiolic acid and sodium carbo- 
 nate indigo is formed. With an excess of glucose this blue 
 is converted into indigo white, 
 
HANDBOOK FOR BIO-CHEMICAL LARORATORY. 6? 
 
 Baeyer's Reaction (Indol). — A watery solution of indol 
 gives with fuming nitric acid a red liquid and then a red 
 precipitate of nitroso-indol nitrate, 16 H 13 (NO)N 21 HIs03. It 
 is better to first add two or three drops of nitric acid and 
 then a 2f solution of potassium nitrite, drop by drop. (Sal- 
 ic oivski.) 
 
 Bareoed ? s Reagent {Dextrose). — Dissolve 1 part copper ace- 
 tate in 15 parts water; to 200 c.c. of this solution add 5 c.c. 
 of acetic acid containing 38 per cent of glacial acetic acid. 
 On heating this reagent with a dextrose solution a reduction 
 of copper suboxide is produced, but not when heated with 
 lactose or maltose. 
 
 Baumann's Reaction {Dextrose). — If a watery solution of 
 grape sugar is treated with benzoyl chloride and an excess of 
 caustic soda, and shaken until the odor of benzoyl chloride 
 has disappeared, a precipitate of benzoic acid ester of dextrose 
 will be produced, which is insoluble in water or alkalies. 
 
 Baumann and Goldman's Test {Cystin). — Shake the solu- 
 tion of cystin in caustic soda withbenzoyl-chloride; a volumi- 
 nous precipitate of benzoyl cystin is produced. The sodium 
 salt precipitates as silky plates, which are readily soluble in 
 water, but nearly insoluble in an excess of caustic soda. 
 
 Berthelot's Test (Phenol). — On adding sodium hypo- 
 chlorite to an ammoniacal solution of phenol a beautiful blue 
 coloration is produced. 
 
 Bile Acids. See Drechsel, Mylius, Pettenkofer, Strassburg, 
 Udransky. 
 
 Bile Pigments. See Capranica, Dragendorff, Dumontpal- 
 Jier, Fl-eischly Gmelin, Huppert, Jolles, Le Nobel, Marechcd, 
 Rosenbach, Stokvis, Smith, Trousseau, Ultzmann, Vitalli. 
 
68 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Bilirubin. See Ehrlich. 
 
 Biuret Keaction (Proteids). See Piotrowski's Reaction. 
 
 Biuret Reaction ( Urea). — Heat urea in a dry test tube until 
 completely molten ; continue the heat for some time. When 
 cold, dissolve in water, add abundant caustic soda and a dilute 
 solution of copper sulphate, drop by drop. The solution be- 
 comes first pink, then reddish violet, and lastly bluish violet, 
 the more copper sulphate solution is added. 
 
 Blood. See Almen, Hoppe-Seyler, Hay em, Kobert, Laden- 
 dot 'ff, Pacini, Rubner, SalkoivsM, Sir uve, Wetzel, v. Zaleski. 
 
 Boas's Test (HOI in Contents of Stomach). — Dissolve 5 
 grms. pure resorcin and 5 grms. white sugar in 100 grms. di- 
 lute alcohol. A few drops of this reagent are spread out in a 
 thin layer upon a porcelain dish, and then gently heated. On 
 allowing a drop of the filtrate from the stomach to flow across 
 it, or a glass rod dipped in the solution touched to it, a deep 
 scarlet streak is developed. If the solution is very dilute, no 
 change is observed until the solution evaporates entirely to 
 dryness. 
 
 Boedeker's Reaction (Albumin). — Acidify the liquid with 
 acetic acid and add a solution of potassium ferrocyanide, drop 
 by drop. White precipitate of albumin will be formed. 
 
 Bottger's Test (Dextrose). — Make the liquid alkaline with 
 carbonate of soda or potash, add some solid bismuth subni- 
 trate and boil. The presence of dextrose is shown by the 
 darkening of the bismuth salt or a black precipitate. 
 
 Braun's Reaction- (Glucose). — Warm the glucose solution 
 with caustic soda or potash until it is yellow ; now drop into 
 this a dilute solution of picric acid, and heat to boiling. A 
 
HANDBOOK FOR BI0-CIIE.UICAL LABORATORY. 09 
 
 deep red coloration will be the result. Creatinin gives this 
 same reaction even in the cold, also acetone, though faintly. 
 
 Brucke's Reagent (Proteids). — Saturate a boiling 10 per 
 cent solution of potassium iodide with freshly precipitated 
 mercuric iodide; on cooling this is filtered, and the filtrate 
 employed with hydrochloric acid as a precipitant for the pro- 
 teids. 
 
 Capranica's Reactions (Guanin). — 1. A warm solution of 
 guanin hydrochloride with a cold saturated solution of picric 
 acid gives a yellow precipitate, consisting of silky needles. 
 
 2. With a concentrated solution of potassium chromate 
 guanin solutions give an orange-red crystalline precipitate, 
 very insoluble in water. 
 
 3. On the addition of a concentrated solution of potassium 
 ferricyanide to a guanin solution a prismatic, yellowish-brown 
 precipitate is formed. 
 
 Capranica's Reaction (Bile Pigments). — Shake the solution 
 with chloroform containing some bromine ; it becomes first 
 green, indigo blue, violet, yellowish red, and lastly colorless. 
 If the green or blue solution is shaken with HOI, the color is 
 taken up by the acid. 
 
 Cellulose. See Schulze, Scliiveitzer. 
 
 Chautard's Test (Acetone). — Pass sulphurous acid through 
 a solution of 0.25 grms. fuchsin in 500 c.c. water until the 
 solution is yellow in color. Add to a portion of this the liquid 
 to be tested for acetone. If present, the liquid will be colored 
 violet. 
 
 Cholesterin. See Liebermann, and Burchard, Obermiil- 
 ler, Salkoivski, Schiff, Schulze, Zwcnger. 
 
70 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Cholesterin. — 1. The crystal is treated with a mixture of 5 
 parts sulphuric acid and 1 part water, when colored rings are 
 produced, first a bright carmine red and then violet. 
 
 2. On the addition of a little iodine solution to the above 
 the crystals will be colored variously — blue, red, green, violet. 
 
 3. If a trace of cholesterin is gradually heated to dryness 
 with a few drops of nitric acid, a yellow spot is produced, 
 which turns red on the addition of ammonia. This red 
 color is not changed by the addition of caustic soda, thus dif- 
 fering from the murexid test for uric acid. 
 
 Ciamician and Magnanini's Test (Skatol). — On warming 
 skatol with sulphuric acid a beautiful purple-red coloration is 
 produced. 
 
 Cohen's Test {Albumin). — Add a solution of potassium 
 iodide and potassium bismuthic iodide to the acid solution of 
 albumin. Precipitation of the albumin occurs. Alkaloids 
 are also precipitated. 
 
 Oreatinin. See Jaffe, Kerner, v. Maschhe, Salhoioshi, 
 Weyl. 
 
 Crismer's Test {Glucose). — A solution of 1 part safranine 
 in 1000 parts water is decolorized or yields a pale yellow color 
 when heated to boiling with an alkaline solution of glucose. 
 Safranine solution is not decolorized when heated in alkaline 
 solution with uric acid, creatin, or creatinin. 
 
 Ctstein. See Andreasch. 
 
 Cystin. See Baumann and Goldmann, Liebig, Milller. 
 
 Davy's Test {Phenol). — Add 3-4 drops molybdic-sulphuric 
 acid (a solution of 1 part molybdic acid in 10 or more parts 
 cone, sulphuric acid) to 1-2 drops of the phenol solution. A 
 pale yellow or yellowish-brown coloration is the result, which 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. (I 
 
 passes to a chestnut or reddish brown and then to a beautiful 
 purple. Gentle heat facilitates the reaction. 
 
 Deniges's Test (Uric Acid). — If uric acid is converted into 
 alloxan by the careful action of nitric acid, and the excess of 
 nitric acid expelled by gentle warming and then treated with 
 a few drops sulphuric acid and also a few drops commercial 
 benzol (containing thiophen), a blue coloration is produced. 
 
 Dextrose. See Glucose. 
 
 Dietrich's Reaction (Uric Acid). — Add a solution of 
 sodium hypochlorite or hypobromite to the uric acid solu- 
 tion, when a red coloration is produced. This coloration 
 disappears on adding caustic alkali. 
 
 Donne's Test (Pus). — Stir a small piece of caustic potash 
 with the mass to be tested. If pus is present, the mass is con- 
 verted into a slimy tough material. 
 
 Dragendorff's Test (Bile Pigments). — -Place a few drops 
 of the urine on an unglazed porcelain surface and when it has 
 been absorbed add a drop or two of nitric acid. Several 
 rings of color will be produced if bile is present, chief 
 amongst these rings being the green ring, which is char- 
 acteristic of bile pigments. 
 
 Drechsel's Test (Bile Acids). — Treat the substance with a 
 little cane sugar and a few drops of a mixture of 5 vols. 
 syrupy phosphoric acid and 1 vol. water. Warm on water- 
 bath, when a beautiful red coloration is produced if bile acids 
 are present. 
 
 Dumontpallier's Test (Bile Pigments). See Smith's Test. 
 
 Eijkman's Test (Phenol). — Mix the phenol solution with a 
 few drops of an alcoholic solution of nitrous acid ethyl ether 
 
72 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 and an equal volume concentrated sulphuric acid. A red 
 coloration is the result. 
 
 Eiselt's Eeaction {Melanin in Urine), — Urine containing 
 melanin becomes dark-colored with oxidizing reagents, such 
 as concentrated nitric acid, potassium dichromate, and sulphu- 
 ric acid, as well as with free sulphuric acid. 
 
 Ehrlich's Eeaction {Bilirubin). — To a solution of biliru- 
 bin in chloroform add an equal volume or twice its volume of 
 a solution of sulpho-diazobenzol (1 grm. sulphanilic acid, 15 
 c.c. of hydrochloric acid, and 0.1 grm. sodium nitrite, diluted 
 to 1 litre with water). Then add as much alcohol as is 
 needed to render the solution clear. The liquid, which is of 
 a yellow color at first, assumes a beautiful red tint. On add- 
 ing HC1, drop by drop, the color changes first to violet and 
 then to an intense blue. On now carefully pouring into the 
 test-tube a solution of potassium or sodium hydrate three 
 zones of color are visible: near the alkaline solution, where 
 the reaction begins, the color is green; at the surface, where 
 the reaction is still acid, the original blue tint persists; 
 whilst intermediate betweent hese two zones is a red, neutral 
 zone. 
 
 Ewald's Test {Hydrochloric Acid in contents of stomach). 
 — Dilute 2 c.c. of a 10fo solution of potassium sulpho-cyanide 
 and 0.5 c.c. of a neutral solution of iron acetate to 10 c.c. 
 with water. Place a few drops of this ruby-red solution in a 
 porcelain dish and allow 1-2 drops of the liquid to be tested 
 to flow gently thereon. In the presence of HOI a faint violet 
 cloud is observed where the two liquids come in contact with 
 each other. On mixing the color becomes mahogany brown. 
 Peptones or salts do not interfere with this reaction. 
 
 Eehlltstg's Reagent {Glucose). — 1. Dissolve 34.65 grammes 
 pure copper sulphate in 1000 c.c. water. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 73 
 
 2. Dissolve L73 grammes Rochelle salts in 350 c.c. Avater, 
 adding 600 c.c. of a canstic soda solution of a specific gravity 
 of 1.12, and dilute to 1000 c.c. with water. For use mix equal 
 parts of the above solutions and dilute with an equal volume 
 of water. 
 Fehling's Solution. 
 
 10 c.c. = 0.0500 gramme dextrose, ]aevulose, or invert sugar. 
 
 10 c.c. = 0.0475 gramme cane-sugar (after inversion). 
 
 10 c.c. = 0.07143 gramme milk-sugar (lactose). 
 
 10 c.c. = 0.0807 gramme malt-sugar (ma]tose). 
 
 Fleischl's Reaction (Bile Pigments). — Treat the urine 
 with a concentrated solution of sodium nitrate and add con- 
 centrated sulphuric acid by means of a pipette. This latter 
 sinks to the bottom of the test-tube and produces the colora- 
 tion, as in Gmelin's test. 
 
 Frohde's Reaction (Proteids). — On heating a solid proteid 
 with sulphuric acid containing molybdic acid a beautiful dark 
 blue color is produced. 
 
 Frohst's Reagent. — Treat 1.5 grammes freshly precipitated 
 bismuth subnitrate with 20 c.c water, heat to boiling, and 
 then add 7 grammes potassium iodide and 10 c.c. hydrochloric 
 acid. 
 
 • 
 Furbrixger's Reagent (Albumin). — Gelatin capsules con- 
 taining the double salt of mercuric chloride and sodium chlo- 
 ride with citric acid. 
 
 Furfttrol Reaction (Proteids). — On heating proteids with 
 sulphuric acid furfurol is produced, which may be detected 
 by various means (see Molisch, Schultze.) 
 
 G-allois's Test (Inosit). — Evaporate the inosit solution to 
 incipient dryness and moisten the residue with a little mer- 
 curic nitrate solution, when a yellowish residue is obtained on 
 
74 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 drying. This yellow color becomes beautifully red on strongly 
 heating, but disappears on cooling, but reappears on gently 
 heating again. 
 
 Gentele's Test [Glucose). — If a glucose solution is added 
 to a solution of potassium ferricyanide made alkaline with 
 caustic soda or potash, it is decolorized with the formation of 
 potassium ferrocyanide on gently warming. Uric acid also 
 gives this same reaction even in the cold. 
 
 Gerhardt/s Eeaction {Aceto-acetic Acid). — Aceto-acetic 
 acid gives a wine-red coloration with a dilute, not too acid, 
 ferric chloride solution. In testing urine treat 1-15 c.c. with 
 ferric chloride as long as it gives a precipitate, filter the pre- 
 cipitate of ferric phosphate, and add some more ferric chloride 
 to the filtrate. In the presence of the acid a claret-red color 
 is produced. The urine may also be acidified with sulphuric 
 acid and shaken with ether (which takes up the acid). Now 
 shake the removed ether with a very dilute watery solution of 
 ferric chloride and the watery layer becomes violet red or 
 claret red. The color disappears on warming. 
 
 Gerhardt's Test {Urobilin). — Extract the urobilin from the 
 solution by shaking with chloroform. Treat this chloroform 
 extract with iodine solution and then a solution of caustic 
 potash, when a beautiful green fluorescence is the result. 
 
 Gerrard's Test {Glucose). — Add a 5$ solution of potassium 
 cyanide to Fehling's solution until the blue color just begins 
 to disappear. On heating this solution to boiling with a glu- 
 cose solution no precipitation of cuprous oxide is produced, 
 but the solution will be decolorized more or less. 
 
 Globulin". See Hammarsten, Pold. 
 
 Glucose. See Almen, Barfoed, Bottger, Baumann, Baeyer, 
 Br aim, Crismer, Felding, Gentele, Gerrard, Haines, v. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 75 
 
 Jalcscli, Knap2h LoiventUal, Moore, Molisch, Mulder, Nylan- 
 der, Penzoldt, Pavy, Roberts, Rubner, Soldani, Schiff, Trom- 
 mer, Wenders. 
 
 Gmelin's Test {Bile Pigments). — If nitric acid containing 
 some nitrous acid is carefully poured beneath a solution con- 
 taining bile pigments, a series of colored layers are obtained at 
 the juncture of the two liquids in the following order from 
 above downwards : green, blue, violet, red, and reddish yel- 
 low. The green ring must never be absent, and also the 
 reddish violet must be present at the same time, otherwise 
 the reaction may be confused with that for lutein, which gives 
 a blue or greenish ring. The nitric acid must not contain too 
 much nitrous acid, for then the reaction takes place too 
 rapidly and does not become typical. Alcohol must not be 
 present, because it gives a play of colors with the acid. 
 
 Griess's Reagent [Nitrous Acid). — A solution of metadiam- 
 ido-benzol (melting at 63° 0.) gives an intense yellow colora- 
 tion with dilute solutions containing nitrous acid which have 
 been acidified with a few drops sulphuric acid. 
 
 Grigg's Test (Proteids). — A solution of meta-phosphoric 
 acid gives a precipitate with all proteids with the exception 
 of the peptones. 
 
 Guanin. See Capranica. 
 
 Gunning's Test (Acetone). — Add an alcoholic solution of 
 iodine to the liquid to be tested and then ammonia. On 
 standing a precipitate of iodoform and a black precipitate 
 of iodide of nitrogen is formed, but this latter gradually dis- 
 appears on standing, leaving the iodoform visible. 
 
 Gunzburg's Eeagent (Hydrochloric Acid). — Dissolve 2 
 grms. phloroglucin and 1 grm. vanillin in 100 c.c. alcohol. 
 In testing for the presence of free HC1 add an equal amount of 
 
76 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 the above reagent to the liquid to he tested in a porcelain dish 
 and evaporate the mixture on the water-bath. In the pres- 
 ence of HC1 a delicate rose-red coloration is observed in the 
 residue in the porcelain dish. 
 
 H^emin. See Teichmann. 
 
 Hemoglobin. See Robert. 
 
 Haines's Solution {Glucose). — Dissolve 30 grains pure cop- 
 per sulphate in \ ounce distilled water and add -J- ounce pure 
 glycerin, mix thoroughly and add 5 ounces liquor of potassae. 
 
 Hammarsten's Test {Globulin). — Add powdered magne- 
 sium sulphate to the neutral solution until no more of the 
 salt dissolves. The globulin will be thus precipitated and 
 separated by nitration and washed with a saturated solution 
 of magnesium sulphate. 
 
 Hatem's Solution {Blood). — This solution is prepared by 
 dissolving 1 grm. sodium chloride, 5 grms. sodium sulphate, 
 0.5 grin, mercuric chloride, in 200 c.c. distilled water. It is 
 used in the microscopical examination of the form elements 
 of the blood. 
 
 Heller's Test {Albumin). — Float the liquid to be tested 
 on the surface of nitric acid. The presence of albumin is 
 shown by a well-defined white ring between the two liquids. 
 With this test even 0.02 p.m. albumin may be detected with- 
 out difficulty, 
 
 IIerzberg's Eeagent {Free Inorganic Acids). — Paper 
 moistened with a solution of Congo red and dried turns bluish 
 black or blue when moistened with hydrochloric acid. The 
 delicacy of this reaction is diminished by the presence of pro- 
 teids or salts in large quantities. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 77 
 
 Heynsius's Test (Albumin).— Strongly acidify the solution 
 with acetic acid and add a few cubic centimetres of a satu- 
 rated solution of sodium chloride and boil. In the presence 
 of albumin a flocculent precipitate is produced. 
 
 Hindeklang's Test (Albumin). — Add solid meta-phos- 
 phoric acid to the liquid to be tested, when a cloudiness or 
 precipitate is formed if albumin is present. 
 
 Hippuric Acid. See Lilche. 
 
 Hopmann's Test (Ty rosin). — Add a few drops Millon's 
 reagent to the solution to be tested and boil for a time. In 
 the presence of tyrosin the liquid becomes a beautiful red and 
 then yields a red precipitate. The test may also be applied 
 by first adding mercuric nitrate and boiling, and after this 
 adding nitric acid containing some nitrous acid. 
 
 Hofmeister's Test (Peptones). — A solution entirely free 
 from albumin gives a precipitate with an acetic acid solution 
 of phospho-tungstic acid. The phospho-tungstic acid may 
 be prepared by dissolving commercial sodium tungstate in 
 hot water and adding phosphoric acid until acid in reaction. 
 This liquid is strongly acidified with hydrochloric acid after 
 cooling and filtered after 24 hours. 
 
 Hopmeister's Test (Leucin). — On warming a solution of 
 leucin with mercurous nitrate a deposit of metallic mercury is 
 formed. 
 
 Hoppe SEYLER'sTest (Carbon Monoxide in Blood.) — Treat 
 the blood with double its volume of caustic soda solution of 
 1.3 sp. gr. Ordinary blood is converted into a dingy brown- 
 ish mass, which when spread out on porcelain is brown, with 
 a shade of green. Carbon monoxide blood gives under the 
 same conditions a red mass, which if spread out on porcelain 
 shows a beautiful red color, 
 
7S HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Hoppe Seyler's Test (Xanthin). — Add some chloride of 
 lime to some caustic soda in a porcelain dish and add the 
 xanthin to this mixture; at first a dark green and then quick- 
 ly a brownish halo forms around the xanthin-grains and then 
 disappears. 
 
 Huppert's Reaction (Bile Pigments). — Treat the solution 
 with milk of lime or with a solution of calcium chloride and 
 then precipitate with ammonia. This precipitate, containing 
 bilirubin calcium, is filtered, washed with water, transferred 
 while moist to a test-tube and treated with alcohol which has 
 been acidified with sulphuric acid, and heated to boiling for 
 some time, when the liquid becomes emerald green or bluish 
 green in color. 
 
 Hydrochloric Acid in Contents of Stomach. See Boas, 
 
 Eivald, Gunzburg, Herzberg, v. Jaksch, Mohr, Maly, Rabu- 
 teau, Reach, Sznbo, Ujfelmann, v. Velden, Witz. 
 
 Hydrogen Peroxide. See Wurster. 
 
 Hypoxanthin. See Kossel. 
 
 Indican. See Jaffe, MacMunn, Obermeyer, Weber, 
 
 Indigo Red. See Rosenbach, Rosin. 
 
 Indol. See Baeyer, Nenchi, Salkoivski. 
 
 Inosit. See Gallois, Seller er, Seidel. 
 
 Jacquemin's Test (Phenol). — Treat the solution with an 
 equal volume of anilin and then a solution of sodium hypo- 
 chlorite, when a blue coloration is the result. Acids turn the 
 liquid red and alkalies turn it blue again. 
 
 Jaffe's Test (Indican). — Treat 20 c.c. of the solution to be 
 tested with an equal volume of hydrochloric acid and add, by 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 79 
 
 means of a pipette, small amounts of a concentrated solution 
 of chloride of lime or -\ per cent potassium permanganate 
 solution, drop by drop, and after each drop shake the mixture. 
 In the presence of indican the mixture turns blue, due to the 
 production of indigo blue. An excess of oxidizing reagent, 
 especially chloride of lime, interferes with the reaction, and 
 must therefore be avoided. If 2-3 c.c. of chloroform are 
 shaken with the blue solution, it will be colored blue by the 
 indigo blue formed. 
 
 Jaffe's Reaction (Creatinin). — Treat the solution with a 
 rather concentrated watery solution of picric acid and a few 
 drops of caustic potash solution. In the presence of creatinin 
 a red coloration, lasting several hours, is produced on warm- 
 ing. Tli is color changes to yellow on the addition of acid. 
 Aceton and glucose give a similar reaction. 
 
 V. Jaksch's Test (Glucose). — Add in a test-tube containing 
 8-10 c.c. of the solution to be tested two knife-points of 
 phenyl hydrazin hydrochloride and three knife-points of 
 sodium acetate, and when the added salts do not dissolve on 
 warming, add more water. The mixture is heated in boiling 
 water for one hour. It is then poured into a beaker-glass of 
 cold water. In the presence of glucose a precipitate consist- 
 ing of groups of yellow needles of phenyl glucosazone is 
 formed. In doubtful cases determine the melting-point of 
 these yellow crystals to be 204-205° C. 
 
 V. Jaksch's Test (Melanin). — Add a few drops of a concen- 
 trated solution of ferric chloride to the liquid to be tested. In 
 the presence of melanin it turns gray, and on the addition 
 of more ferric chloride the precipitate, consisting of the color- 
 ing matter and the phosphates, is redissolved. 
 
 v. Jaksch's Test (TICl in Contents of Stomach). — Paper moist- 
 ened with a saturated, watery solution of benzo-purpurin 6 B. 
 
80 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 and dried gives with dilute solutions of HOI a beautiful 
 violet coloration. If the paper becomes dark blue, the solu- 
 tion contains more than 0.4 grm. HC1 in 100 c.c. of the 
 solution. 
 
 y. Jaksch's Test ( Uric Acid).— This consists in substituting 
 chlorine or bromine water or nitrous acid for the nitric acid 
 in the murexid test (see Murexid Test.) This reaction differ- 
 entiates between uric acid and the xanthin bases. 
 
 Johnson's Test {Albumin). — Float the acidified solution 
 on a cold saturated solution of picric acid. If albumin is 
 present, a precipitation of the albumin occursbetweenthetwo 
 liquids. 
 
 Jolles's Test {Bile Pigments in Urine). — Place 50 c.c. of 
 the urine in a stopper cylinder, add a few drops of 10$ hydro- 
 chloric acid and an excess of a barium chloride solution with 
 5 c.c. chloroform, and shake for several minutes. After 10 
 minutes remove the chloroform and the precipitate by means 
 of a pipette and place in a test-tube and heat on the water- 
 bath to about 80° O. After the evaporation of the chloro- 
 form decant the liquid from the precipitate carefully and 
 allow 3 drops concentrated sulphuric acid containing \ fuming 
 nitric acid to flow down the sides of the test-tube. In the 
 presence of bile pigments the characteristic coloration is pro- 
 duced. 
 
 Kerner's Reaction {Creatinin). — A solution of creatinin 
 acidified with a mineral acid gives a crystalline precipitate 
 with phospho-tungstic or phospho-molybdic acids, even in 
 very dilute solutions. 
 
 Knapp's Solution {Glucose). — Dissolve 10 grms. chemically 
 pure dry mercuric cyanide in 100 c.c. caustic soda solution 
 pf a specific gravity of 1-145 and dilute to 1 litre. On heating 
 
HANDBOOK TOE BIO-CHEMICAL LABORATORY. 81 
 
 [i glucose solution with the above solution diluted with water 
 a reduction of metallic mercury takes place. Ten c.c. of this 
 solution are reduced by 0.025 grm. glucose. 
 
 Robert's Test {Hamioglobin). — Shake the solution with 
 zinc powder or treat with a solution of zinc sulphate or acetate, 
 when a precipitate of zinc haemoglobin is formed. This pre- 
 cipitate when collected is colored red by alkalies. 
 
 Kossel's Test (Hypoxanthin). — Treat the solution with 
 zinc and hydrochloric acid and then make alkaline with 
 caustic soda or potash. In the presence of hypoxanthin the 
 solution becomes first ruby red and then brownish red in 
 color. 
 
 Lactic Acid. See Uffelmann. 
 
 Ladendorff's Test {Blood).— Treat the liquid with tinct- 
 ure of guaiacum and then with oil of eucalyptus, when the 
 lower layer becomes blue and the upper layer violet if blood 
 is present. 
 
 Landolt's Test {Phenol). — On treating the solution with 
 bromiue water a white crystalline precipitate of tribrom- 
 phenol (C 6 H 2 ,Br 3 ,OH) is produced. 
 
 Lang's Eoaction {Taurin). — On boiling a solution of 
 taurin with freshly precipitated mercuric oxide a white com- 
 bination occurs which appears as a precipitate. 
 
 Leoal's Test {Acetone). — Treat the acetone solution with a 
 few drops of a freshly prepared solution of sodium nitro- 
 prusside and then with caustic potash or soda solution. The 
 solution becomes ruby red in color, but if saturated with 
 acetic acid the color becomes carmine or purplish red. 
 Oreatinin gives the ruby-red color, with sodium nitro-prusside 
 
82 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 and alkali,, but this turns yellow, and then gradually green 
 and blue, when saturated with acetic acid. Ammonia may 
 be substituted for the caustic soda or potash and gives the 
 same reaction with acetone, but no reaction with creatinin. 
 
 Le Nobel's Modification of Legal's Test (Acetone). — 
 Instead of using caustic potash or soda with the sodium nitro- 
 prusside he suggests the use of ammonia, which produces a 
 ruby-red reaction with acetone, but not with creatinin. (See 
 LegaVs Test.) 
 
 Le Nobel's Test (Bile Pigments). — Treat the liquid with 
 zinc chloride and a few drops of tincture of iodine. A di- 
 chroitic play of colors is the result. 
 
 Leucln'. See Hoffmeister, Solierer. 
 
 Lieben's Test (Acetone). — When a watery solution of 
 acetone is treated with alkali and then a solution of iodine in 
 potassium iodide solution and gently warmed, a yellow pre- 
 cipitate of iodoform id formed, which is known by its odor and 
 by the appearance of the crystals (six-sided plates or stars) 
 under the microscope. 
 
 Liebermann'-Burchard's Test (Gliolesterin). — Dissolve 
 the substance in acetic anhydride and then add concentrated 
 sulphuric acid, when a beautiful violet color is produced, and 
 this passes quickly to green if cholesterin is present. 
 
 Liebermann's Test (Proteids). — Treat the proteid, pre- 
 viously washed with alcohol and ether, with concentrated 
 fuming hydrochloric acid, when a beautiful violet-blue colora- 
 tion is the result. This liquid gives an absorption-band be- 
 tween E and b. 
 
 Liebig's Test (Cystin). — Boil the substance with caujtic 
 alkali containing lead oxide. If cystin is present, a pre- 
 cipitate of blacklead sulphide is produced. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 83 
 
 Lowenthal's Test {Glucose). — On boiling a glucose solution 
 with a solution of ferric chloride dissolved in tartaric acid and 
 sodium carbonate it darkens and soon deposits a voluminous 
 precipitate of iron oxide. This test cannot be applied to the 
 urine, as all urines give it, 
 
 Lucre's Reaction (Hippuric Acid). — Evaporate the sub- 
 stance to dryness with nitric acid, when an intense odor of 
 nitro-benzol (oil of bitter almonds) is generated when the 
 residue is heated. 
 
 MacMunn's Test (Indican in Urine). — Equal parts of urine 
 and hydrochloric acid and a few drops nitric acid are boiled, 
 cooled, and agitated with chloroform. The chloroform is 
 colored violet and shows an absorption-band before D, due to 
 indigo blue, and another after D, due to indigo red. 
 
 Mac William's Test (Albumin). — Acid a concentrated wa- 
 tery solution of salicyl sulphonic acid to the acid-reacting 
 solution, when a cloudiness or precipitate will be formed in 
 the presence of albumin. In the presence of peptones or 
 albumoses the precipitate disappears on boiling, but reap- 
 pears on cooling. 
 
 Malerba's Test (Acetone). — A solution of dimethylpara- 
 phenylendiamine gives a red coloration with acetone, which 
 gives an absorption spectrum very similar to oxyhsemoglobin. 
 
 Maly's Test (HCl in Contents of Stomacli). — Place liquid 
 to be tested in a glass dish and add as much ultramarine to 
 make it just blue. Then cover the dish with a watch-glass 
 after having suspended a piece of lead- paper in the upper 
 part of the dish. On warming the mixture on the water- 
 bath after 15 minutes in the presence of HCl the blue color 
 of the mixture has changed to brown and the lead-paper will 
 have turned dark, due to the development of H ? S, 
 
84 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 Zinc sulphide may be substituted for the ultramarine, 
 using a knife-point of the powder to 20 c.c. of the filtered 
 contents of the stomach. 
 
 Mandel's Test (Proteids). — A 5 per cent solution of chro- 
 mic acid produces a precipitate with solutions of proteids. 
 If the solution is first made acid with acetic or citric acid, the 
 precipitate produced is flocculent and settles rapidly. It 
 produces a marked cloudiness with 1 part albumin dissolved 
 in 50,000 parts water. Chromic acid solution may be substi- 
 tuted for nitric acid in Heller's test ; using a 10 per cent 
 solution. 
 
 Marechal (Bile Pigments). See Smith's Test. 
 
 v. Maschke's Eeaction (Greatinin). — Dissolve the creatinin 
 in a cold saturated solution of sodium carbonate and add a 
 a few drops of Fehling's solution. An amorphous flocculent 
 precipitate is obtained in the cold, but better on warming 
 to 50-60° O. 
 
 Mehu's Test (Albumin). — Shake 100 vols, of the solution 
 with 2-3 vols, nitric acid and 10 vols, of a solution of 1 part 
 phenol and 1 part acetic acid in 2 parts 90$ alcohol. In the 
 presence of albumin a precipitate is produced. Instead of 
 nitric acid one half a volume of a saturated solution of 
 sodium sulphate may be used. 
 
 Melanin. See Eiselt, v. Jaksch, TJwrmdhlen, Zeller. 
 
 Michailow's Test (Proteids). — Treat the solution with 
 ferrous sulphate, and allow concentrated sulphuric acid to 
 flow under the solution, and then add carefully very little 
 nitric acid. Besides a brown ring, a blood-red coloration will 
 also be produced, 
 
HANDBOOK FOR 13IO-CIIEMICAL LABORATORY. 85 
 
 Millon's Reagent (Proteids). — Dissolve 1 part mercury in 
 2 parts nitric acid (sp. gr. 1.42), allow to stand some time, 
 and then apply heat. After complete solution of the mer- 
 cury add 1 vol. of this solution to 2 vols, of water. Allow 
 to stand a few hours and decant the supernatant liquid. 
 
 This reagent gives with solutions of proteid bodies a precipi- 
 tate which slowly at the ordinary temperature, but quickly at the 
 boiling-point, turns red, depending upon the amount of albu- 
 min. Solid albuminous bodies give the same reaction. This 
 reaction depends on the presence of the aromatic group in 
 the proteid, and is also given by tyrosin and other benzol 
 derivatives with a hydroxyl group in the benzol nucleus. 
 
 Mohr's Test (HClin Contents of Stomach). — A solution of 
 iron acetate (free from alkali acetates) so diluted as to have 
 only a light yellow color is treated with a few drops of a solu- 
 tion of potassium sulpho-cyanide. No change of color should 
 be produced, but if the filtered contents of the stomach are 
 added, and they contain free HC1, an intense red coloration 
 is the result. This color disappears on the addition of sodium 
 acetate. 
 
 Molisch's Test {Glucose). — 1. Treat -J to 1 c.c. of the solu- 
 tion with 2 drops of a 15-20$ alcoholic solution of a naph- 
 thol. The liquid becomes cloudy, due to the precipitation of 
 some of the naphthol, but on the addition of 1-2 c.c. concen- 
 trated sulphuric acid a beautiful deep violet coloration is pro- 
 duced, which forms a violet precipitate on diluting with 
 water. 
 
 2. Instead of employing a solution of a naphthol he also 
 suggests the use of a 15-20$ alcoholic solution of thymol, 
 applied as above. In the presence of glucose it is colored 
 ruby red and becomes carmine red on dilution with water. 
 
 Moore's Test ( Glucose). — If a glucose solution is treated with 
 about \ of its volume of caustic soda or potash and warmed, the 
 
86 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 solution becomes first yellow, then orange, yellowish brown, 
 and lastly brown, depending upon the amount of glucose 
 present. A faint odor of caramel is also observed, and this 
 is more pronounced if the solution is acidified. 
 
 Mulder's Test {Glucose). — Treat the solution with a solu- 
 tion of sodium carbonate and add a solution of indigo car- 
 mine. On heating in the presence of glucose the solution 
 becomes decolorized, and turns blue again on shaking with 
 air. 
 
 Mulder's Test, also Xantho-proteic Eeaction (Proteids). — 
 On treating proteids with concentrated nitric acid they are 
 colored yellow. On adding ammonia or carstic soda or potash 
 they turn orange yellow. 
 
 Muller's Test (Cystin). — Dissolve the cystin by boiling 
 with caustic potash, dilute with water when cold, and add a 
 solution of sodium nitro-prusside, when a violet coloration is 
 produced. This color changes rapidly to yellow. 
 
 Murexid Test (Uric Acid). — Heat the powder gently on a 
 watch-glass with a drop or two of strong nitric acid. A red 
 residue is produced, which, when cold, turns a purple red 
 when ammonia is added (purpurate of ammonium). When 
 caustic soda or potash is added to this, it becomes more blue 
 or bluish violet. Better results are obtained if the heating is 
 done over the water-bath, and not over a naked flame. 
 
 Mylius's Modification of Pett&nJcqfer's Test (Bile Acids). — 
 To each cubic centimetre of the alcoholic solution of bile 
 acids add 1 drop of furfurol solution and 1 c.c. concentrated 
 sulphuric acid, and cool when necessary, so that the test does 
 not become too warm. A red coloration is the result, and 
 this color does not disappear at the ordinary temperature, but 
 becomes more bluish violet in the course of a day. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. St 
 
 Nencki's Test (Indol). — Indol gives a pronounced red col- 
 oration with nitric acid containing nitrous acid. In concen- 
 trated solution a red precipitate may form. This reaction is 
 not given by skatol. 
 
 Nitrous Acid or Nitrites. See Griess. 
 
 Ntlander's Test (Glucose). — Dissolve 4 grms. Rochelle 
 salts in a solution of 10.33 grms. NaliO in 100 c.c. water. 
 Add to this 2 grms. bismuth subnitrate and digest on the 
 water-bath until as much of the bismuth salt is dissolved 
 as possible. On heating 10 vols, of a glucose solution with 1 
 vol. of the above solution for 2-5 minutes a black precipitate 
 or a dark coloration is the result. 
 
 Obermeyer's Test (Indican in Urine). — Precipitate the 
 urine with a lead acetate solution (1 to 5), being careful not to 
 add an excess of lead solution. Filter through a dry folded 
 filter and shake the filtrate with an equal volume of fuming 
 hydrochloric acid which contains 1-2 parts ferric chloride 
 solution to 500 parts of the acid. Continue shaking for 1 or 
 2 minutes, and then add some chloroform, which takes up 
 the indigo blue produced and is colored blue. 
 
 Obermuller's Test (Gliolesterin) . — Fuse the cholesterin with 
 2 or 3 drops propionic acid anhydride in a test-tube over a 
 small naked flame. On cooling the fused mass is first violet, 
 then blue, green, orange, carmine, and lastly copper red. 
 
 Oliver's Test (Albumin). — Mix equal parts of sodium 
 tungstate solution (1 to 4) and a saturated solution of citric 
 acid, (10 to 6). The urine is floated on this solution, and in 
 the presence of albumin a white ring is obtained at the con- 
 tact of the two liquids. 
 
 Pacini's Liquid (Blood). — One part corrosive sublimate, 2 
 parts sodium chloride, 13 parts glycerin, 11.3 parts distilled 
 
88 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 water. This mixture should stand 2 months. For use mix 
 1 part of this solution with 3 parts water and filter. 
 
 Pavy's Reagent {Albumin). — This is a practical dry reagent 
 for albumin. It consists of small disks or plates of citric acid 
 and sodium ferrocyanide. 
 
 Payy's Solution {Glucose). — Mix 120 c.c. of the ordinary 
 Fehling's solution with 300 c.c. of strong ammonia (sp. gr. 
 0.88) and with 400 c.c. more of caustic soda solution of sp. gr. 
 1.14. Now dilute to 1000 c.c. with water. One hundred c.c. 
 of this solution is reduced by glucose to the same extent as 
 10 c.c. of the ordinary Fehling's solution. This solution 
 becomes decolorized by boiling with glucose solution. 
 
 Pekzoldt/s Test {Glucose). — Dissolve 1 part diazobenzo-sul- 
 phonic acid in 60 parts of water, and to facilitate solution add 
 1 or 2 drops caustic potash. 
 
 In applying this test make some of the solution to be 
 tested strongly alkaline, and then add an equal volume of the 
 above solution of diazobenzol sulphonic acid. It is advisable 
 to do the same with a solution free from sugar. On allowing 
 to stand the mixture becomes yellowish red or light claret 
 red, then darker, and in the presence of considerable glucose 
 it becomes dark red and opaque. The red color has a bluish 
 shade. 
 
 Penzoldt's Test {Acetone). — A warm saturated solution of 
 orthonitrobenzaldehyde is treated with the liquid to be tested 
 for acetone and then made alkaline with caustic soda. In the 
 presence of acetone the liquid first becomes yellow, then green,, 
 and lastly indigo separates, and this may be dissolved with a 
 blue color by shaking with chloroform. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 89 
 
 Penzoldt and Fischer's Test (Phenol). — On treating a 
 strongly alkaline solution of phenol with a solution of diazo- 
 benzol sulphonic acid a deep red coloration is the result. 
 
 Peptones. See Hofmeister. 
 
 Petri's Test (Proteids). — On treating a proteid or peptone 
 solution with a solution of diazobenzol sulphonic acid only a 
 faint yellow coloration is produced, but on making the solu- 
 tion alkaline with caustic alkali the solution becomes orange 
 yellow to brown, according to concentration, and yields a red 
 froth on shaking. 
 
 Pettenkofer's Test (Bile Acids). — A small quantity of bile 
 in substance is dissolved in a small porcelain dish in con- 
 centrated sulphuric acid and warmed, or some of the liquid 
 containing the bile acids is mixed with concentrated sul- 
 phuric acid, taking special care in both cases that the tem- 
 perature does not rise higher than 60-70° C. Then a 10^ 
 solution of cane-sugar is added, drop by drop, continually 
 stirring with a glass rod. The presence of bile is indicated by 
 the production of a beautiful red liquid, whose color does not 
 disappear at the ordinary temperature, but becomes more blu- 
 ish violet in the course of a day. This red liquid shows a 
 spectrum with two absorption-bands, the one at F and the 
 other between D and E, near E. 
 
 Phenol. See Allen, Berthelot, Davy, Eijhnann, Jacque- 
 min, Landolt, Penzoldt and Fischer. 
 
 Piotrowski's Eeaction (Proteids), also called Biuret Ee- 
 action. — On heating a proteid with an excess of a concen- 
 trated solution of caustic soda and one or two drops of a 
 dilute solution of copper sulphate a violet color is produced 
 which deepens in tint on boiling. 
 
90 HANDBOOK FOR BlO-CHEMlCAL LABORATORY. 
 
 Piria's Test (Ty rosin). — Dissolve the substance in con- 
 centrated sulphuric acid and allow to stand for J an hour. 
 Dilute with water and neutralize the solution with BaC0 3 and 
 filter. On the addition of acid-free ferric chloride to the clear 
 filtrate a violet color is produced in the presence of tyrosin. 
 The reaction is impeded by the presence of free acid. 
 
 Pohi/s Test (Globulins). — He suggests to saturate the solu- 
 tion to one-half with ammonium sulphate, which precipitates 
 the globulins. Filter and wash with a one half saturated 
 solution of ammonium sulphate. 
 
 Proteids. See Adamkiewicz, Biuret, Brilcke, Frohde, 
 Furfurol, Grigg, Liebermann, Mandel, Millon, Mulder, Mi- 
 chailouy Petri, Piotroivski, Reichl, Schultze, Xanthoproteic. 
 
 Pus. See Donne. 
 
 Kaabe's Test (Albumin). — Place 1 c.c. of the liquid to be 
 tested in a test-tube and add a small piece of trichloracetic 
 acid. In the presence of albumin a white zone or ring will 
 be formed. The ring produced by uric acid is diffused and 
 not sharply defined. 
 
 Rabuteau's Test (HOI in Contents of Stomach). — Add 
 the filtered contents of the stomach to a solution containing 
 50 c.c. starch mucilage, 1 grm. potassium iodate, and 0.5 grm. 
 potassium iodide. In the presence of free HC1 it will become 
 blue. 
 
 Rees's Test (Albumin). — An alcoholic solution of tannic 
 acid precipitates small amounts of albumin. 
 
 Reichi/s Test (Proteids).— Add 2-3 drops of an alcoholic 
 solution of benzaldehyde to the proteid solution, and then 
 considerable sulphuric acid which has previously been diluted 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 91 
 
 with an equal volume of water. Lastly, add a few drops of a 
 ferric sulphate solution, when a deep blue coloration will be 
 produced in the cold after some time or immediately on 
 warming. Solid proteids are also colored blue by this reac- 
 tion. 
 
 Keoch's Test (Albumin). See Macivilliam. 
 
 Reoch's Test (HCl in Contents of Stomach). — A mixture 
 of citrate of iron and quinine and potassium sulpho-cyanide 
 is colored red by the gastric juice or contents of the stomach 
 containing free hydrochloric acid. 
 
 Reynold's Test (Acetone). — Precipitate HgO from a mer- 
 curic chloride solution by adding an alcoholic caustic potash 
 solution. To this freshly precipitated HgO add the liquid 
 to be tested for acetone, shake, and filter. In the presence 
 of acetone the filtrate contains mercury, due to the acetone 
 dissolving freshly precipitated HgO. The mercury is de- 
 tected in the filtrate by means of ammonium sulphide, which 
 turns black. 
 
 Robert's Test (Glucose in Urine). — Take the specific 
 gravity of the urine at a known temperature by means of a 
 urinometer or pyknometer supplied with a thermometer. 
 Now acidify slightly with tartaric acid and add a piece of 
 yeast the size of a pea and shake. Allow to stand at the 
 temperature of the room, or, better, at 20-25° C, for 24-48 
 hours. The fermentation by this time will be finished. Now 
 filter through a dry filter and cool to the same temperature 
 as you took the specific gravity before fermentation. Now 
 take the specific gravity again. 
 
 Each degree of specific gravity lost represents 1 grain of 
 glucose to the ounce of urine, or if the number of degrees 
 lost in specific gravity is multiplied by the factor 0.23 we 
 
92 HANDBOOK FOR BIO-OHEMICAL LABORATORY. 
 
 obtain the percentage of glncose or grammes per 100 c.c. of 
 urine. 
 
 Robert's Test {Albumin). — Allow the urine to flow on the 
 surface of a saturated common salt solution containing 5$ 
 HC1 of specific gravity 1.052. In the presence of albumin a 
 white ring or zone will form between the two liquids. 
 
 He also suggests a mixture of 1 part strong nitric acid and 
 5 parts saturated magnesium sulphate solution. It is to be 
 applied as above. 
 
 Rosenbach's Modification of Gmelin's Test (Bile Pig- 
 ments). — Filter the liquid through a very small filter. When 
 all liquid has passed through, apply to the inside of the filter 
 a drop of nitric acid which contains only very little nitrous 
 acid. A pale yellow spot will be formed, which is surrounded 
 by colored rings which are yellowish red, violet, blue, and 
 green. 
 
 Rosenbach's Test (Indigo Red or Indirubin). — On boiling 
 the liquid with nitric acid indigo blue is formed from the 
 indigo red. 
 
 Rosin's Test (Indigo Red or Indirubin), — Make the liquid 
 alkaline with sodium carbonate and extract with ether, which 
 is colored red by the indigo red. 
 
 Rubber's Test (Carbon Monoxide in Blood). — Shake the 
 blood for one minute with 4-5 volumes lead acetate solution. 
 If the blood contains CO, it will retain its bright red color, 
 while if it does not it will turn chocolate brown. 
 
 Rubber's Test (Glucose). — Treat the liquid with an excess 
 of lead acetate, filter, and add ammonia to the filtrate until 
 no further precipitate is produced. Warm gently, when the 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 93 
 
 precipitate produced by the ammonia will be gradually col- 
 ored pink. This coloration diminishes on standing. 
 
 Salkowski's Reaction {Cliolesterin). — Dissolve the sub- 
 stance in chloroform and then treat with an equal volume of 
 concentrated sulphuric acid. The cliolesterin solution be- 
 comes first bluish red, then gradually more violet red, while 
 the sulphuric acid appears dark red with a greenish fluores- 
 cence. If the chloroform solution is poured into a porcelain 
 dish, it becomes violet, then green, and finally yellow. 
 
 Salkowski's Modification of Hoppe Seyler's Test {CO 
 in Blood). — Dilute the blood to be tested with 20 vols, water 
 and add thereto an equal volume of a caustic soda solution 
 of sp. gr. 1.34. If the blood contains carbon monoxide, the 
 mixture will become milky in a few moments and then 
 bright red. On standing red flakes form, which collect on 
 the surface of the liquid. Normal blood treated in this way 
 gives a dirty brown coloration. 
 
 Salkowskl's Reaction {Creatinin). — If a few drops of a 
 freshly prepared very dilute solution of sodium nitro-prusside 
 are added to a dilute creatinin solution and then a few drops 
 of caustic soda, a ruby-red liquid is obtained which quickly 
 turns yellow {Weyl's reaction). If this yellow solution is 
 treated with an excess of acetic acid and heated, the solution 
 becomes first green and then blue, and finally a precipitate of 
 Prussian blue is obtained. 
 
 Salkowskl's Test (Indol). — Add a few drops nitric acid to 
 the indol solution and then, drop by drop, a 2f solution of po- 
 tassium nitrite. In the presence of indol a red color is pro- 
 duced, and lastly a red precipitate of nitroso-indol nitrate. 
 
 Scherer's Test (Inosit). — Evaporate the substance to dry- 
 ness on a platinum foil with nitric acid and treat the residue 
 
94 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 with ammonia and a drop of calcium chloride solution and 
 carefully re-evaporate to dryness. With inosit a rose-red 
 residue is obtained. 
 
 Scherer's Test (Leucin). — Carefully evaporate the leucin 
 with nitric acid on a platinum foil. No markedly colored 
 residue is left, but on gently warming this with a few drops 
 of caustic soda solution a color varying from a pale yellow to 
 a brown (depending on the purity of the leucin) is produced, 
 and on further concentrating over the flame it agglomerates 
 into an oily drop which rolls about on the foil. 
 
 Scherer's Test (Tyrosin). — Evaporate the substance care- 
 fully to dryness with nitric acid on a platinum foil. A beau- 
 tiful yellow residue (nitro-tyrosin nitrate) is obtained, which 
 gives a deep reddish-yellow color with caustic soda. 
 
 Schiff's Test {Uric Acid). — Dissolve the substance in 
 sodium carbonate and add silver nitrate solution, when a re- 
 duction of black silver oxide is obtained. If a drop of the 
 solution of the substance in sodium carbonate is placed on a 
 piece of filter-paper which has been previously treated with 
 silver nitrate solution, a reduction of black silver oxide will 
 also be formed on the paper. 
 
 Schiff's Test (Carbohydrate). — Strips of paper are dipped 
 in a mixture of equal volumes of glacial acetic acid and 
 xylidin, treated with very little alcohol, aud dried. On ex- 
 posing such paper to the furfurol vapors produced by treating 
 glucose with sulphuric acid the paper will be colored red. 
 
 Schiff's Reaction (Cholesterin).— Evaporate the substance 
 in a porcelain dish over a small flame with a few drops of a 
 mixture of 2-3 vols, concentrated hydrochloric or sulphuric 
 acid and 1 vol. of a medium solution of ferric chloride. In 
 
HANDBOOK FOR BlO-CHExMICAL LABORATORY, 05 
 
 the presence of cholesterin a reddish-violet residue is first ob- 
 tained and then a bluish violet. 
 
 Schiff's Test {Urea). — Place a drop of a concentrated 
 watery solution of furfurol on the crystal of urea and then a 
 drop of hydrochloric acid of sp. gr. 1.10. A change of color 
 from yellow, green, blue, to purple is obtained. Allantoin 
 gives this same reaction, but less intense and not so quickly. 
 
 Schroeder's Test ( Urea). — Place a crystal on a microscope- 
 slide and add a solution of bromine in chloroform. Urea will 
 not dissolve therein, but is decomposed with the develop- 
 ment of gas. 
 
 Schulze's Reagent {Cellulose). — Dissolve iodine to satura- 
 tion in a zinc chloride solution of sp. gr. 1.8 to which 6 parts 
 potassium iodide has been added. Cellulose turns blue with 
 this reagent. 
 
 Schulze's Test (Cholesteriu). — Evaporate the substance to 
 dryness on the water-bath in a porcelain dish with nitric acid. 
 A yellow residue is obtained with cholesterin, which turns 
 yellowish red on the addition of ammonia. 
 
 Schultze's Test (Proteids). — Add a few drops of a dilute 
 cane-sugar solution and then concentrated sulphuric acid to 
 a solution of the proteid and warm the mixture to 60° C, 
 when a beautiful bluish-red coloration is obtained. It is 
 important to keep the temperature at 60° 0. 
 
 Schweitzer's Reagent {Cellulose). — Sulphate of copper in 
 solution, to which some ammonium chloride has been added, is 
 precipitated with caustic soda ; the hydrated cupric oxide 
 thus obtained is washed and dissolved to saturation in 20$ 
 ammonia. It may also be prepared by pouring ammonia on 
 copper turnings, the requisite oxidation of the copper being 
 
96 HANDBOOK FOR BIO-CHEMICAL LABORATORY. 
 
 effected by drawing a current of air through the fluid in 
 which the turnings are immersed. Cellulose is soluble in the 
 above reagent. 
 
 Seidel's Reaction (Inosit). — Evaporate a small amount of 
 the substance to dryness in a platinum crucible with a little 
 nitric acid (sp. gr. 1.1-1.2), and treat the residue with ammo- 
 nia and a few drops of a solution of strontium acetate. In 
 the presence of inosit a greenish coloration is observed, to- 
 gether with a violet precipitate. 
 
 Skatol. See Ciamician and Magnanini. 
 
 Smith's Reaction {Bile Pigments). — Pour carefully over 
 the liquid to be tested tincture of iodine, whereby a green 
 ring appears between the two liquids. 
 
 Soldani's Solution (Glucose). — Dissolve 15 grms. copper 
 carbonate in 1400 c.c. water and add 416 grms. potassium 
 bicarbonate. On heating a glucose solution with the above 
 solution a reduction of copper suboxide is obtained. 
 
 Spiegler's Test (Albumin). — Remove mucin from the 
 solution by the addition of acetic acid, filter, and treat the 
 filtrate with a solution prepared by dissolving 8 grms. mer- 
 curic chloride, 4 grms. tartaric acid, in 200 c.c. water and 
 adding 20 grms. glycerin thereto. In the presence of albumin 
 a white ring is obtained between the two liquids. 
 
 Stokvis's Test (Bile Pigments). — Treat 20-30 c.c. of urine 
 with 5-10 c.c. of a solution of zinc acetate (1 to 5). The pre- 
 cipitate is washed on a small filter with water and then dis- 
 solved in a little ammonia. Filter, and the filtrate gives, 
 after standing in the air, a peculiar brownish-green color, and 
 shows the three absorption-bands of bilicyanin, the first between 
 (J and D, the second at D, and the third between D and E, 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 9? 
 
 Stokes's Reagent [Reducing Oxyhemoglobin). — To a solu- 
 tion of ferrous sulphate add some citric or tartaric acid and 
 enough ammonia to make it alkaline. 
 
 Strassburg's Test (Bile Acids). — Treat the liquid with 
 cane-sugar and dip a strip of filter-paper in this liquid. Dry 
 this carefully over a gas or alcohol flame and place a drop of 
 sulphuric acid thereon. In the presence of bile acids a red 
 coloration is produced on the paper. The liquid must be free 
 from albumin for this test. 
 
 Struve's Test (Blood in Urine). — Treat the urine with 
 ammonia or caustic potash, and then add tannin and acetic 
 acid until the mixture has an acid reaction. In the presence 
 of blood a dark precipitate is formed. Filter, dry, and obtain 
 the characteristic hgemin crystals from the dry residue by the 
 addition of ammonium chloride and glacial acetic acid. (See 
 Teichmann. ) 
 
 Szabo's Test (HOI in Contents of Stomach). — Equal 
 volumes of \io solutions of ammonium sulpho-cyanide and of 
 sodic-ferric tartrate are mixed. On adding liquid containing 
 HOI to this solution, which is pale yellow, a brownish-red 
 color is produced. 
 
 Tanret's Test (A Tbumm).— Dissolve 3.32 grms. potassium 
 iodide and 1.35 grms. mercuric chloride (4 mols. KI to 1 mol. 
 HgCl 2 ) in 20 c.c. acetic acid and dilute to 60 c.c. When 
 this reagent is added to an albumin solution, a white precipi- 
 tate is produced. 
 
 Tatjrin. See Lang. 
 
 Teichmann's Test (Haimin Crystals).— Place a few parti- 
 cles of the dry residue on a microscope-slide, add a grain of 
 common salt, and cover with a cover-glass. Now add some 
 
98 HANDBOOK FOR BtO-CIlEMICAL LABORATORY. 
 
 glacial acetic acid under the cover-glass and warm gently not 
 to boil the liquid. In the presence of blood-coloring matters 
 the characteristic dark brown, long, rhombic crystals of hasmin 
 are obtained. If no crystals appear after the first warming, 
 warm again, and if necessary add some more acetic acid. 
 
 Thormahlen's Test {Melanin in Urine). — Add sodium, 
 nitro-prusside, caustic potash, and acetic acid to the urine to 
 be tested, and in the presence of melanin a deep blue colora- 
 tion is the result. 
 
 Trommer's Test {Glucose). — Make the liquid strongly alka- 
 line with caustic soda and add a not too concentrated solution 
 of copper sulphate, drop by drop, until, a little of the copper 
 hydrate formed remains undissolved on shaking. Now warm, 
 and in the presence of glucose a yellow reduction of hydrated 
 suboxide of copper is first formed and then red suboxide sepa- 
 rates even below the boiling-point. If too little copper salt 
 has been added, the test will be yellowish brown in color, as in 
 Moore's test ; but if an excess of the copper salt has been 
 added, the excess of hydrate is converted on boiling into a 
 dark brown hydrate, which interferes with the test. 
 
 Trousseau's Test {Bile Pigments). See Dumontpallier, 
 Smith. 
 
 Tyrosin". See Hoffmann, Piria, Scherer, Udransky, 
 Wurster. 
 
 Udransky's Test {Tyrosin).— Dissolve a particle of the 
 substance in 1 c.c. of water and add 1 drop of a 0.5$ watery 
 furfurol solution and then 1 c.c. concentrated sulphuric acid. 
 The mixture becomes faintly red. Care should be taken not 
 to have the mixture get above 50° 0. 
 
 Udransky's Test {Bile Acids).— Treat 1 c.c. of a watery or 
 alcoholic solution of the substance with 2 drop of a 0.1$ 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 99 
 
 watery solution of furfurol and allow 1 c.c. concentrated sul- 
 phuric acid to flow underneath this mixture. Care should be 
 taken to keep the mixture cool, in the presence of bile acids 
 a red coloration is obtained. The red color should have a 
 shade of blue (violet) to be characteristic of bile acids. 
 
 UffelmanVs Test (Lactic Acid in Contents of Stomach), — 
 Mix 10 c.c. of a 4^ carbolic acid solution and 20 c.c. water and 
 add a few drops ferric chloride solution, when an amethyst- 
 blue solution is obtained. With lactic acid this solution is 
 colored yellow. 
 
 Uffelmann's Test (HOI in Contents of Stomach). — Dip 
 strips of filter-paper in an amyl alcohol extract of huckle- 
 berries and dry. When the contents of the stomach contains 
 HC1, it will turn this paper pink. 
 
 Ultzm ann's Eeaction (Bile Pigments). — Treat the solution 
 with caustic potash and mix, and then acidify with hydro- 
 chloric acid. The solution becomes emerald gieen, due to 
 the formation of biliverdin. 
 
 Urea. See Biuret, Schiff, Schroeder. 
 
 Uric Acid. See Deniges, Dietrich, v. JaJcsch, Murexid, 
 Schiff. 
 
 Urobilin. See Gerhardt. 
 
 v. d. Velden's Test (HCl in Contents of Stomach). — A 
 watery or alcoholic solution of Tropaeolin 00, which is }^ellow 
 in color, turns ruby red or deep brownish red with free hydro- 
 chloric acid. Paper moistened with the above solution may 
 be used for the test. 
 
100 HANDBOOK FOE BIOCHEMICAL LABORATORY. 
 
 Vitalli's Test (Bile Pigments.) — Treat the solution with 
 a few drops of a solution of potassium nitrite and then some 
 dilute sulphuric acid, when a beautiful green color will be 
 obtained. This green color changes to yellow after a time, 
 but first turns red or blue. 
 
 Weber's Test (Indican in Urine). — Treat 30 c.c. of the 
 urine with an equal volume hydrochloric acid and 1-3 drops 
 dilute nitric acid and heat to boiling. The solution becomes 
 dark, and if shaken with ether, when cold, the ether will be 
 colored red to violet, while a blue foam is observed on the top 
 of the ether. 
 
 Weidel's Eeaction (Xantliiri). — A little of the substance 
 is dissolved in fresh chlorine water containing some nitric 
 acid and evaporated on the water-bath to dryness. On expos- 
 ing the white or yellowish residue to the vapors of ammonia, 
 under a bell- jar, a red or purple violet color is produced. 
 
 Wender's Test (Glucose). — Dissolve 1 part commercial 
 methylene blue in 3000 parts distilled water. On making this 
 solution alkaline with caustic potash and heating with a 
 glucose solution the blue color disappears and the solution 
 becomes decolorized. 
 
 Wetzel's Test (CO in Blood). — Dilute the blood with 4 
 vols, water aud treat with 3 vols, of a \<f tannic acid solution. 
 Tn the presence of carbon monoxide the blood becomes car- 
 mine red, while normal blood gradually becomes gray. 
 
 Weyt/s Eeaction (Creatinin).— Add a few drops of a 
 freshly prepared solution of sodium nitro-pfusside to the 
 solution of creatinin and then a few drops of caustic soda. A 
 ruby-red liquid is obtained, which quickly turns yellow again. 
 The solution of creatinin zinc chloride may also be used. 
 
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 101 
 
 Witz's Test (HCl in the Content* of the Stomach). — A watery 
 solution of methyl-anil in violet is first rendered blue, then 
 green, and ultimately decolorized by dilute inorganic acids. 
 
 Wurster's Test (Tyrosin). — A boiling watery solution of 
 tyrosin is colored red when treated with lfo acetic acid and a 
 sodium nitrite solution, drop by drop. 
 
 Wurster's Test (Tyrosin). — Dissolve the tyrosin in hot 
 water and to the hot solution add some dry chinon. The 
 solution becomes deep ruby red, which remains for 24 hours 
 and then passes to brown. 
 
 Wurster's Test (Hydrogen Peroxide). — Paper soaked with 
 a solution of tetramethylparaphenylendiamine turns blue 
 violet with hydrogen peroxide. 
 
 Xanthin. See Hoppe Seyler, Weidel. 
 
 Xantho-proteic Eeaction (Proteids). See Mulder. 
 
 v. Zaleski's Test (Carbon Monoxide in Blood). — Mix 2 c.c. 
 of the blood with 2 c.c. water and 3 drops of a \ saturated 
 copper sulphate solution. With normal blood a greenish- 
 brown precipitate is produced, while if the blood contains CO 
 a brick -red precipitate is obtained. 
 
 Zeller's Test (Melanin in Urine). — A urine containing 
 melanin gives, when treated with bromine water, a yellow pre- 
 cipitate, which gradually turns black. 
 
 Zouchlos's Test (Albumin). — The reagent consists of 100 
 parts 10$ potassium sulpho-cyanide solution and 20 parts 
 acetic acid. When this reagent is added, drop by drop, to a 
 solution of albumin, a marked cloudiness is observed. 
 
 Zwenger's Test (Cholesterin). See Cholesterin Reactions 
 No. 1. 
 
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