ESTERIFICATION 35 



cipitate ; alcohol is added so long as precipitation occurs, about i litre being required. 

 The precipitate is filtered off and washed thoroughly with alcohol. If a small precipitate 

 forms as the wash alcohol comes in contact with the filtrate, it is neglected : it does not 

 contain glutamic acid and aspartic acid, but may consist of the calcium salt of tyrosine. 

 The precipitate is dissolved in about 300 c.c. of water and treated with oxalic acid to re- 

 move the calcium completely. The filtrate from calcium oxalate is treated with silver 

 sulphate solution to remove traces of chloride and pigmented substance ; the pigmented 

 substance dissolves if the solution be heated, but is removed later. Excess of silver is re- 

 moved from the solution with hydrogen sulphide and the volume reduced to about one 

 half. By now adding a solution containing 5-10 grams of phosphotungstic acid the 

 colouring matter is precipitated, and the excess of this reagent is thrown down by baryta, 

 which is added until the blue colour, if formed, disappears ; excess of baryta is removed by 

 sulphuric acid and the filtrate evaporated to about 50 c.c. The solution is transferred to a 

 weighed dish with a stirring rod, further evaporated to 20 or 25 c.c. for 40 grams of protein 

 and allowed to cool. The acids crystallise out, are stirred and dried further in a desiccator 

 over calcium chloride. 



The mass does not consist of a mixture of the pure amino acids ; it is therefore stirred 

 up with cold glacial acetic acid ; the insoluble residue is filtered off, again stirred with 

 glacial acetic acid, filtered off and washed ; the residue is dried and weighed. The acetic 

 acid solution dries in vacuo over potash to a gum which contains pyrrolidone carboxylic 

 acid, which may be estimated by determining the amino nitrogen before and after hydro- 

 lysis with concentrated hydrochloric acid. 



The residue of glutamic acid and aspartic acid is separated by preparing the copper 

 salts and crystallising out copper aspartate. The glutamic 'acid is isolated from the filtrate 

 as hydrochloride ; or glutamic acid may be isolated first as hydrochloride and the aspartic 

 acid from the filtrate. By determining the carbon content of the mixture of the acids, the 

 proportion of aspartic acid in the mixture is given by multiplying the yield by 



40*82 - per cent, of carbon 



40-82 - 36-09 

 the remainder is glutamic acid. 



The method has only been carried out with caseinogen ; the yield of aspartic acid was 

 1*8 per cent, and of glutamic acid about 16-2 per cent. On adding the amount of glutamic 

 acid corresponding to the amino nitrogen determination of the pyrrolidone carboxylic acid 

 the yield was 2i'8 in one experiment and 24-1 in another. This is considerably higher than 

 the yield of 15-6 by the hydrochloride method ; the yield of glutamic acid actually isolated 

 was in good agreement with the above. 



(2) Esterification. 



The filtrate from the glutamic acid hydrochloride, to which the 

 mother liquors from the recrystallisations are added, is concentrated 

 in vacuo at a low temperature to a thick syrup ; this is dissolved in 

 absolute alcohol (3 litres to I kilo, protein), and the amino acids are 

 esterified by saturating the alcohol with dry gaseous hydrochloric acid 

 at the ordinary temperature and then warming on the water-bath for 

 half an hour. In the process of esterification a large amount of water 

 is formed, which prevents its completion ; the alcohol is therefore 

 evaporated off in vacuo at a temperature below 50 and the result- 

 ing syrup again dissolved in absolute alcohol and saturated with 



3* 



