METHODS OF ESTIMATING CARBOHYDRATES 37 



and also because enzyme actions were in progress in such 

 sap, they abandoned the method. 



More recently Dixon and Atkins showed by physical 

 methods (1913, 1) that sap pressed out never repre- 

 sents the true composition of that in the cells, unless 

 their protoplasm has been made permeable by immersion 

 of the tissue in liquid air. For the application of pressure, 

 whilst rupturing some cells, results in a progressive increase 

 in concentration of the sap solutes in the intact ones, as 

 the liquid pressed out through the cytoplasmic semi-perme- 

 able lining is an approximation to pure water. 



Brown and Morris, abandoning the use of pressed sap, 

 finally adopted the following procedure: The leaves were 

 dried quickly on wire-bottomed trays in a steam-oven. 

 The high temperature soon checked enzyme action, though 

 this had a short period of considerable activity. Also the 

 natural acids of the leaves became greatly concentrated 

 during the desiccation. Rapidity of drying lessened, but 

 did not entirely get rid of, these sources of error. 



Extraction of the dried material with ether removed fat 

 and chlorophyll. About 10 grammes was then extracted 

 with 80 per cent, alcohol for twenty-four hours at 40-45. 

 Further extraction for a like period was followed by wash- 

 ing by decantation. The spirit was then distilled off after 

 neutralization with ammonia to prevent any invertive action 

 of vegetable acids. The residue, after addition of water 

 and evaporation to remove all alcohol, was cleared with 

 basic lead acetate. The latter served to remove tannin, 

 amino-acids, and proteid materials, which would affect the 

 subsequent determinations . 



After nitration, the excess of lead acetate was removed 

 by treatment with hydrogen sulphide and further nitra- 

 tion and washing. The solution thus obtained was ana- 

 lyzed as described in the next section. 



