ADVANCED CHEMICAL PHYSIOLOGY 311 



Digestion is allowed to proceed until the bromine water reaction is 

 maximal. The digest is then boiled, cooled and filtered, and H 2 SO 4 

 added to the nitrate, so as to bring the amount of H 2 SO 4 in the latter 

 to 56 per cent. If any precipitate is hereby formed it should be 

 filtered off. The clear filtrate is then mixed with an excess of an acid 

 solution of mercuric sulphate (10 per cent, mercuric sulphate dissolved 

 in 10 per cent. H 2 SO 4 ) and filtered. This reagent may precipitate, 

 besides tryptophan, some tyrosine and cystine. 



From tyrosine the precipitate is freed by washing it with 56 per cent. 

 H 2 SO 4 , the mercury compound of tyrosine being very soluble in this. 

 From cystine (which is scanty in a digest of casein) the tryptophan is 

 separated by reprecipitation. For this purpose the washed mercury 

 precipitate is suspended in water and decomposed with H 2 S gas. To 

 complete this reaction the suspension must be saturated with the gas, 

 then warmed and saturated again. The HgS precipitate is filtered off, 

 the filtrate warmed to rid it of H 2 S, then acidified to 56 per cent. 

 H 2 SO 4 , and the mercuric sulphate reagent added to it until a small 

 permanent precipitate is produced. This is mainly cystine, and is 

 filtered off. The tryptophan in the filtrate is then completely pre- 

 cipitated by mercuric sulphate, and the resulting precipitate treated 

 exactly like the first one. 



In this way a solution of tryptophan in 5-6 per cent. H 2 SO 4 is 

 obtained. The H 2 SO 4 is now precipitated by adding Ba(OH) 2 water in 

 the heat and filtering. Great care should be taken that the filtrate 

 contains no excess either of H 2 SO 4 or of Ba(OH) 2 . The watery solution 

 of tryptophan is then mixed with half its bulk of alcohol and evaporated 

 on a waterbath. During evaporation small quantities of alcohol are 

 added from time to time to prevent the browning which occurs if watery 

 solutions of tryptophan are heated alone. Evaporation proceeds till 

 crystallisation commences, when the basin is removed and allowed to 

 stand. The crystals (glistening plates) are collected on a filter, and, 

 to purify them, may be recrystallised. 



A solution of the crystals gives the bromine and the glyoxylic reactions 

 very distinctly, and if the crystals be heated in a test tube indol and 

 Rcatol (see p. 238) are evolved. 



Tryptophan is the mother substance of indol, which, along with its 

 methyl derivative scatol, is largely responsible for the faecal colour. 

 These bodies are produced from tryptophan by bacterial growth 

 (see p. 238). 



Preparation of Cystine (Folin). Place 50 gms. wool in a 500 c.c. 

 flask, add 100 c.c. cone. HC1 and place on a waterbath until all dissolved. 

 In order to prevent undue loss of fluid close mouth of flask with a cork 

 carrying a 3-foot length of glass tubing to act as a simple condenser. 

 When completely dissolved boil very gently over a small flame for three 

 to four hours. Add solid sodium acetate (100-130 gms.) until congo 

 red paper no longer turns blue. Allow mixture to stand three to five 

 days (the longer the better up to three weeks), then filter on a Buchner 

 funnel and wash with cold water. Dissolve precipitate in 150 c.c. 

 water + 5-10 c.c. cone. HC1, add about 20 gms. purified bone charcoal 

 and boil 510 minutes. Filter again with suction, heat filtrate to 

 boiling, neutralise hot by adding very slowly hot concentrated sodium 

 acetate solution, taking care not to add an excess (test constantly with 

 congo red paper). Precipitate formed is cystine. If crystals not white 

 repeat solution and treatment with charcoal. Note crystal form and 



