ISOLATION OF PROLINE 43 



(a) Proline. 



Proline is the only product contained in these fractions which is 

 soluble in alcohol ; it is also much more easily soluble in water than 

 the other products. 



The dry residues are therefore extracted several times with boiling 

 absolute alcohol ; these extracts on cooling frequently become turbid, 

 and on standing deposit other amino acids which, though insoluble in 

 alcohol, are dissolved when proline is present. They are filtered off 

 and returned to the portion insoluble in alcohol. 



The combined alcoholic extracts of the three fractions are again 

 evaporated to dryness in vacuo and the residue is treated several times 

 with cold absolute alcohol. A considerable amount is undissolved ; 

 as before this is returned to the insoluble portion. 



The alcoholic solution is again evaporated to dryness in vacuo and 

 extracted with cold absolute alcohol, and this operation is continued 

 until all the insoluble amino acids are removed. 



The final alcoholic solution is evaporated to dryness and the residue 

 weighed. As thus obtained, the proline is a mixture of the optically 

 active and the racemic forms. These are separated by conversion into 

 their copper salts by boiling with freshly precipitated copper oxide. 

 The resulting dark-blue solution is evaporated to dryness, and the re- 

 sidue is treated with absolute alcohol which dissolves the copper salt 

 of the optically active proline. This solution on concentration yields 

 the greater part of the compound in a crystalline state, but the remain- 

 der is amorphous. The copper salt of the racemic proline, which is 

 insoluble in alcohol, is purified by crystallisation from water. 



The identity of the compounds is established by a determination 

 of the water of crystallisation and of the copper. The racemic copper 

 salt contains 2 molecules of water of crystallisation, and in this state 

 it is dark blue in colour ; in the anhydrous state its colour is violet. 

 Further characterisation is obtained by preparing proline from it. The 

 copper salt is dissolved in water and decomposed with hydrogen sul- 

 phide. The filtrate is concentrated to a small volume and precipitated 

 with alcohol. The product, crystallised from alcohol, is obtained in flat 

 needles, /-proline has a sweet taste, melts at 206-209 and has a 

 rotation of [~ a l D = - 77*4. The phenylhydantoin of /-proline is the 



L J 20 



most suitable derivative for still further characterisation. 



The amount of proline in the protein is given by the yield of the 

 two copper salts obtained in a pure state. The actual proline content 



