t-RNA (transfer ribonucleic acid) from Escherichia coli may be resolved on partition columns composed of Sephadex G-25 fine beads and a biphasic solvent mixture. The mixture is composed of potassium phosphate pH 6.88 buffer, ethoxyethanol, butoxyethanol, mercaptoethanol, and triethylamine. Linear variation in triethylamine concentration results in an exponential variation of the partition coefficient of t-RNA in the mixture, allowing t-RNA dissolved in aqueous phase immobilized on the Sephadex beads to be extracted by mobile phase containing a gradient of triethylamine. Resolution is sufficient to provide individual amino acid specific t-RNA's of 25-40% purity, as much as 24-fold enriched, in one passage over the column. Of 13 specific t-RNA's examined, at least 9 are heterogeneous, and 5 separable varieties of t-RNA accept leucine. Results are reproducible for columns of varying diameter, and at least 3.4 g of t-RNA may be fractionated with no decrease in resolution. Except for mechanical losses, recoveries of t-RNA and acceptor activities are complete.
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