ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 61 



cooling ; these are filtered off, washed with alcohol, dried, and 

 weighed. 



The last portions of the lysine in the mother liquor from the picrate 

 can be obtained by acidifying with sulphuric acid, extracting the picric 

 acid with ether, precipitating as phosphotungstate, and repeating the 

 above process for obtaining lysine picrate. 



Colorimetric Estimation of Histidine. 



Weiss and Ssobolew [1913] attempted to estimate histidine colori- 

 metrically by its reaction with diazobenzene sulphonic acid. With 

 pure solutions of histidine their attempts were successful ; the esti- 

 mation was apparently not so successful in solutions containing other 

 amino acids, as no data were given. In all cases, tyrosine which gives 

 a very similar reaction, must be absent from the solution. 



In performing the reaction a fresh solution of diazobenzene sul- 

 phonic acid is necessary. The authors prepared a standard reagent 

 made up by mixing I part of solution A with 2 parts of solution B. 



The composition of these is : 



A = 4 grams of sulphanilic acid, 40 c.c. concentrated hydrochloric 

 acid, water to 400 c.c. At room temperature solution takes place in 

 twenty-four hours. 



B = o'5 per cent, solution of sodium nitrite in water. 



The solution is made alkaline with a 10 per cent, solution of 

 anhydrous sodium carbonate. 



A standard solution of I in 10,000 of histidine monochloride is 

 also required 



The standard colour for comparison is made by thoroughly mixing 

 I '5 c.c. of the diazo reagent (lA + 2B) with 10 c.c. of the standard 

 histidine solution and adding 3 c.c. of the soda solution. The colour 

 produced is in five to ten minutes of a pure red shade, optimal, and 

 stable. 



The unknown solution is successively diluted say I in 5, I in 20, 

 i in 40, i in 80, I in 160. To 10 c.c. are added 1*5 c.c. of diazo 

 reagent and 3 c.c. of soda as above. 1 



The dilution at which a colour corresponding with the standard 

 colour is noted. As soon as the colour intensity decreases, the optimal 

 dilution is passed. For example the matching of colours may be be- 

 tween i in 80 and I in 160. Intermediate dilutions of I in 100 and 



1 No colour appears with very concentrated solutions of histidine, so that absence of 

 colour does not imply absence of histidine, unless no colour appears on further dilution. 



