ADVANCED PHYSIOLOGICAL CHEMISTRY 425 



mixed in a flask with 100 c.cm. 60 per cent. KOH (Pfliiger recom- 

 mends la Merck). After vigorous shaking for a few minutes the 

 ilask is placed on a boiling water bath for two hours. By heating 

 ivith this strength of KOH (viz., 30 per cent.) all the glycogen 

 both that which is free and that which is combined with proteid 

 is extracted, and, on the other hand, there is no hydrolysis of 

 glycogen, because strong KOH attracts water and thus prevents its 

 being added to the glycogen molecule. Another advantage of strong 

 KOH is that it destroys proteid to such an extent that this no longer 

 is precipitated by alcohol. The use of proteid precipitants at a later 

 stage is thus obviated. 



(While this is heating the estimation of sugar as described below 

 (B II.) should be proceeded with.) 



The brown opaque contents of the flask after heating for two 

 hours are made up to 400 c.cm. by the addition of water, and 

 filtered through asbestos or glass wool. (Such a filter can be made 

 by placing a small cone of wire gauze in the apex of the funnel 

 .arid then the asbestos.) If the filtrate comes through opaque refilter 

 it till it is clear ; 100 c.cm. of the filtrate are then mixed in a beaker 

 with 100 c.cm. 96 per cent, alcohol, and the mixture briskly stirred. 

 By this means the glycogen is precipitated. The precipitate, after 

 standing a few minutes, is collected on a small filter paper (15 cm. 

 diam.). As filtration at this stage is slow the filter paper may be 

 folded as described in the Appendix (Fig. 278). After all the fluid 

 has drained away the precipitate is washed twice with alcoholic potash 

 of the following strength: 1 vol. 15 per cent. KOH + 2 vol. 96 per 

 cent, alcohol, then twice with 96 per cent, alcohol and once with 

 ether. A pure glycogen precipitate is thus obtained. To ascertain 

 its amount it is hydrolysed into sugar, which is then estimated as 

 described on p. 274. The technique of the method is as follows : 

 the stem of the filter funnel is closed by a piece of indiarubber 

 tubing and a clip, and the funnel is filled with distilled water. The 

 glycogen soon dissolves, and then the clip is removed and the solution 

 run into a large flask (500 c.cm.). This process is repeated till the 

 last trace of glycogen has disappeared. A piece of litmus paper is 

 then thrown into the glycogen solution and HC1 (con.) added, drop 

 by drop, to neutralisation. The volume of the solution is then made 

 up to 500 c.cm. and 25 c.cm. HC1 (con.) added to it. This gives 

 .a solution containing 2-2 per cent. HC1, which is the most suitable 

 .strength for producing hydrolysis. The flask is ^now placed on a 

 boiling water bath for 4 hours, after which it is cooled, neutralised 

 with KOH and the sugar estimated by titration, etc. (p. 274). 



