Reverse salt gradient chromatography of tRNA on unsubstituted agarose. III. Physical and chemical properties of different batches of Sepharose 4B.

Four batches of Sepharose 4B, which gave differing elution profiles in reverse salt gradient chromatography of tRNA, were analysed by a number of techniques. Charged groups were estimated from infrared spectra, and gave 0.04-0.05 sulphates and 0.18-0.27 carboxyls per disaccharide. Nuclear magnetic resonance showed the presence of 0.21-0.23 6-O-methyl substituents per disaccharide. The mass concentrations of the beads in aqueous suspension (including agarose-bound water) ranged from 4.7 to 5.0%. High-resolution scanning electron microscopy demonstrated a three-dimensional network of threads with diameters between 2 and 20 nm. None of these properties varied appreciably between batches. There was, however, a variation in the size distribution of beads. In two batches which induced a high degree of adsorptive retardation during chromatography, very few beads exceeded 100 micrometers in diameter; two other batches which gave inferior retardation had a preponderance of somewhat larger beads. The significance of this observation remains unclear.