Inositols, paper chromatography

Paper chromatography of inositol phosphates obtained from soil extracts is described. ... 

The linkage of the mj/o-inositol to the phosphatidic acid has not yet been determined with certainty the inositolphosphoric acid, isolated after hydrolysis of the intact phosphatide (or of the water-soluble portion, obtained by alkaline hydrolysis), gives, on paper chromatography, a mixture of inositol 1(3)-phosphate and a small proportion of inositol 2-phosphate (separated on paper by the method of Pizer and Ballou ). This indicates that, in the phospholipid, the m /o-inositol is probably linked by its C-1- or C-3-hydroxyl group to phosphoric acid. 

Paper Chromatography.—Solvent systems have been developed for the separation of trichloromethyl- phenyl-, and pentafiuorophenyl-phosphonic and -phosphinic acids. Phosphoramidates and their hydrolysis products have been chromatographed, using triethylamine as a buffer. Silica gel on glass-fibre sheets may be used to separate inositol from its phosphate and to analyse phosphatide glyceryl ethers. ... 

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. 

The water that separated (23 ml.) was considerably more than 1 mole, and the monocyclohexylidene derivative (3) obtained by extraction with water was only a minor product. Most of the inositol had been converted into the three diketols (2), which were separated by chromatography and crystallization. The structures were established by tests with sodium periodate and methylation experiments and by the observation that all three di-derivatives are converted on very mild hydrolysis into the 1,2-mono-derivative (3), which is cis. Note that the three di-derivatives all contain one group bridging rra/is-hydroxyl groups (3,4-, 4,5-, and 5,6-). A later paper reports improvements in the preparative procedure and several new derivatives, including 1,2,3,4,5,6-tri-O-cyclohexylideneinositol. 

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