Paper chromatography deionization

The radioactive material is separated from the enzyme by dialysis against 20 volumes of deionized water. Salts are removed from the dialyzate by filtration over Dowex 50 X 8 (H form) and neutralization of the acid in the filtrate with Dowex 1X8 (OH form). If denatura-tion is necessary, it is advised to remove the denaturant by dialysis before the hydroxylamine treatment. The desalted solution containing the released label is concentrated to dryness, and the reaction product is identified by descending paper chromatography in acetone/water 6 1 and n-butanol/acetic acid/water 4 1 P and cocrystallization with authentic reference substances. Three reaction products can be expected from racemic conduritol B epoxide, provided the reacting group on the enzyme is a carboxylate ion (-j-)-cliiro-inositol, (—)-cAiro-inositol, and scyllo-inositol. 

These treatments convert to ionic substances, and remove, nearly all constituents of natural materials the acid treatments release any inositol present as phosphate, or combined in phospholipids, glycosides, etc. Glycerol remains in the deionized sample, but it can be oxidized separately, or be removed by heat decomposition or by repeatedly evaporating the solution to dryness. Such polyhydric alcohols of greater chain length as erythritol and mannitol, when present, would still interfere. However, corrections can be made for these compounds by determining the formaldehyde which they form on periodate oxidation, or they may be removed by chromatography on filter paper. The micro-periodate method is well suited to the analysis of samples eluted from filter paper, provided that care is exercised to remove the tiny particles of cellulose which are usually found in such eluates. 

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