Reverse salt gradient chromatography of tRNA on unsubstituted agarose. I. Variations in elution profile and evidence for two fractionation mechanisms.

E. coli tRNA was fractionated by the application of ammonium sulphate reverse gradients to Sepharose 4B. Variations in elution profile were partly attributable to differences between batches of Sepharose. The profile also varied with column length and gradient parameters. This suggests the existence of two distinct mechanisms which do not separate different tRNAs in the same sequence. The first mechanisms, believed to be interfacial precipitation, releases tRNAs progressively as the salt concentration is reduced. A second mechanism introduces adsorptive retardation in which molecules lag behind the solvent. This process, widely believed not to be important in the chromatography of macromolecules with multiple binding sites, is in the present case mainly responsible for the improved resolution of peaks on passage down a long column. Isocratic (constant-salt) fractionation is also feasible. The Sepharose batch variation affects the second mechanism more than the first.