An analytical anion-exchange procedure has been developed for the rapid separation of acid-soluble nucleotides (the so-called "free" or tissue nucleotides). It permits assay at low pressures (275-415 kPa; 40-60 psi) in less than 1 h on 10-cm columns of Aminex resins (conventional styrene-type anion exchangers) with alkaline citrate solutions as the eluent. Separation variables have been investigated, to determine optimum conditions for the routine analysis of samples containing tissue nucleotides. Also described here is a simple solvent-extraction procedure for removing HClO4 or CCl3CO2H quantitatively from cell extracts that contain acid-soluble nucleotides: they are removed from aqueous acid solutions with a water-insoluble amine dissolved in a water-immiscible solvent.
March 5 , 1953 SEPARATION OF SUGAR PHOSPHATES BY ION EXCHANGE 1153for the re3ction of ethyl acetate may be caused by a restricted configuration in which there is hydrogen bonding between the 0-hydrogens in the ester grouping and the carbonyl oxygen in the activated state. The same picture applies to the n-propyl ester, but to a lesser degree since only two such 0-hydrogen atoms are available here. This type of H-bonding is, in all probability, found in the isopropyl ester in both reactant and activated state and would therefore not be reflected in the entropy term. It should be noted that a t all temperatures a t which these studies were made ethylene glycol is definitely the best catalyst of the three considered, when comparison is made on the basis of equivalent hydroxyl concentration. Water is somewhat lower in catalytic activity and methanol is almost a power of ten lower than either of the other two catalysts6 It is apparent, as seen in Fig. 1, that, because of the temperature coefficients, a t a higher temperature *water might very well become a better catalyst than ethylene glycol.Separating the various factors affecting catalytic activity of the three catalysts studied presents a difficult problem. The fact that the ammonolysis reaction as catalyzed by water gives the smallest decrease in the entropy of activation is not surprising in view of the small and unhindered water molecule. The high activation energy for water catalysis may very well be caused by the many and complex ways in which water and ammonia can be associated. Catalysis by ethylene glycol gives a low activation energy, possibly because of the (6) R. Baltzly, I M. Berger and A. A. Rothstein (THIS JOURNAL l a , 4149 (1950)) have described methanol as the best catalyst for the aminolysis of a series of esters. These workers, however, used methanol as both solvent and catalyst for their investigation and a fair conlparison of catalysts cannot be made under these circumstances. adjacent hydroxyl groups, both of which may be attached to the same ammonia molecule. The fact that the total energy barrier for the reaction catalyzed by methanol is the same as that for ethylene glycol may possibly indicate that not one but two molecules of methanol bond with one molecule of ammonia to form the amide ion. This would also account for the relative order of decreasing entropies of activation.Because of the necessary modifications of experimental procedure for ammonolysis of the lactates and the relatively large experimental error, quantitative deduction from the data obtained in these reactions is difficult. The reaction is self-catalyzed via the hydroxyl group contained in the lactate ester. The presence of ethylene glycol may be expected to lower the potential energy barrier, because of the increased availability of the hydroxyl groups, but evidently it causes a larger decrease in' the total energy barrier by a large decrease in the entropy of activation. The latter may be caused by hydrogen bonding between the second hydroxyl group of the glycol and th...
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