164 



Appendix: analytical methods 



wet v/t. (Chance and Yaxley, 1950; Le Baron, 1955). A more 

 probable source of error lies in the large amounts of phospholipid 

 which is present in fine suspension in extracts made with trichlor- 

 acetic acid. This is not removed by centrifuging in fields of up to 

 3000^, the field normally obtainable in average centrifuges. For 

 complete removal a field of 12-20,000^ for 10-15 min is necessary 

 (Heald, 1956^). Failure to remove the phospholipids can lead to 

 difficulties in constructing a balance sheet for the total phosphates 

 present in the extracts. In this respect perchloric acid appears to 

 possess a distinct advantage for it has been found (Dr. G. H. 



Table 25. — Effect upon Labile Phosphates of Thawing Frozen 

 Brain Tissue in Trichloracetic Acid before Grinding 



(Quantities are in /amoles/g wet wt. di the S.E.M.) 



Data from Lin et al. (1958). 



Sloane-Stanley, personal communication) with this extractant that 

 centrifuging at low speeds yields a supernatant free from phospho- 

 lipid. A further advantage is that the potassium salt is insoluble at 

 0° enabling the extractant to be easily removed by centrifuging 

 after neutralization. This is important when enzymic methods of 

 analysis are subsequently used. However, the major analytical 

 procedures still widely adopted depend upon preliminary frac- 

 tionation of the extracts. Such fractionation methods were 

 developed specifically for use with extracts made in trichloracetic 

 acid and similar extensive studies have not been reported with 

 extracts made in perchloric acid. Trichloracetic acid may be 

 removed by extraction with ethyl ether without any apparent loss 

 of phosphorus. Whichever extractant is used its removal is 

 essential before analysis of the phosphates either by enzymic, 

 chromatographic or ion exchange techniques. 



