ioo THE CHEMICAL CONSTITUTION OF THE PROTEINS 



bases thrown down by adding 15 c.c. of concentrated hydrochloric 

 acid and 5 grams of phosphotungstic acid, the final volume being 

 made up to 200 c.c. as in the original precipitation. Filtration and 

 washing is carried out as above. 



6. Treatment of the Phosphotungstate Precipitate. The precipitate 

 is removed with a spatula, as completely as possible, to a beaker of 

 over I litre capacity. The filter paper is then spread out on the 

 bottom of a basin and washed with water made just alkaline with a 

 few drops of 20 per cent, sodium hydrate. The small folded paper 

 is similarly treated. The particles of precipitate are dissolved by 

 the soda, and any granules remaining in the original flask are dis- 

 solved in the same way. The alkaline solutions and washings are 

 poured into the beaker containing the main bulk of the precipitate. 

 The whole is then carefully dissolved in soda by adding 50 per cent, 

 alkali, drop by drop, with continual stirring. Phenolphthalein is 

 added as indicator ; as soon as the solution becomes red, addition of 

 the alkali must be stopped ; if the colour disappears, more alkali, but 

 only to the excess of three or four drops, must be added. A red 

 solution must finally result. 



The solution is diluted to 800 c.c. and the phosphotungstic acid is 

 removed by slowly adding, in portions of a few c.c., a 20 per cent, 

 solution of barium chloride, until a test portion gives an immediate 

 granular precipitate with neutral sodium sulphate solution. If the red 

 colour disappears in the process two or three more drops of alkali are 

 added. A large excess of barium chloride should be avoided. 



The barium phosphotungstate is filtered off using the same funnel, 

 paper and precautions as before, except that larger portions of wash 

 water may be used. The final washing must give no reaction for 

 chlorides. 



The filtrate and washings are concentrated in vacua, until they are 

 reduced in volume to 50 c.c. The barium phosphotungstate, which 

 separates out, is filtered off, and the filtrate and washings received in 

 a 200 c.c. double-necked distilling flask ; the volume is then again 

 reduced and made up to 50 c.c. in a measuring flask. 



Van Slyke [1915, i] found that the following procedure was 

 preferable to the original as described above : 



The precipitate is removed from the filter by a spatula and by 

 washing with water and transferred to a 500 c.c. separating funnel, 

 using 200-300 c.c. of water. 5 or 10 c.c. of concentrated hydrochloric 

 acid are added, and the mixture shaken with about 100 c.c. of a 



