168 Appendix: analytical methods 



suitable for work with radioactive phosphate and require supple- 

 mentation by chromatographic techniques to avoid the errors 

 introduced by the overlapping of various fractions and contamina- 

 tion with radioactive inorganic phosphate. 



Chromatographic Methods 



Application of chromatographic methods to the separation of 

 the acid-soluble phosphates of the brain has developed over the 

 past 4-5 years. Since the individual investigators have been 

 concerned with specific problems no general scheme resolving all 

 the major phosphates has been described. After a preliminary 

 fractionation by the barium procedure followed by the removal of 

 barium with anion exchange resins, electrophoresis on paper of the 

 free phosphates or their ammonium salts yields phosphates free 

 from contamination with inorganic phosphate (Heald, 1956a). 

 Under these conditions adenosine nucleotides were separable from 

 inorganic phosphate and phosphocreatine, while phosphocreatine, 

 adenylic acid, the hexose monophosphates and inorganic phosphate 

 were separable from one another and from the phosphopyridine 

 nucleotides. Cross contamination with inorganic phosphate was 

 negligible. The method provides an unequivocal separation and 

 identification of phosphocreatine, which is separated at an alkaline 

 pH and does not decompose, and has proved valuable for work 

 involving radioactive phosphate (Heald, 19566). The method 

 appears capable of extension by elution of the phosphates from the 

 paper followed by further separation by diiTerent chromatographic 

 techniques. The fraction containing adenosine triphosphate is now 

 known to contain guanosine nucleotides and separation of the two 

 groups and inorganic phosphate may be achieved first by chromato- 

 graphing on paper in the acid solvent of Eggleston and Hems 

 (1952), which remove inorganic phosphate from the mixture, 

 followed by an alkaline propanol solvent to separate the adenosine 

 from the guanine nucleotides (Heald, 19576). The nucleotide 

 di- and triphosphates are not separated by this method. A more 

 complete separation of nucleotides upon paper has been described 

 (Doring and Gerlach, 1957; Gerlach et al., 1958), adenosine 

 mono-, di- and triphosphates being separated from one another and 

 from guanosine di- and triphosphates. These latter were also separ- 

 ated from one another but the guanosine phosphate diphosphate 

 fraction contained some uridine triphosphate. The separations 



