Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection

Abstract

The determination of molecular weights for certain proteins haas been performed. This was involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-μm, non-porous, Monosphere RP-C 8 column has been used in order to perform fast and conventional RP-HPLC gradients (5–45 min). Traditional specific refractive index increment (d n/d c) and refractive index ( n) measurements have been performed in order to derive absolute weight-average molecular weight ( M w) information for ribonuclease A, lysozyme, and bovine serume albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate M w data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5–10 min of fast gradient elution, each protein appears to exhibit unusual M w values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC—LALLS—UV approaches.