FAST PROTEIN SEPARATION AND CHARACTERIZATION BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) AND LOW-ANGLE LASER-LIGHT SCATTERING PHOTOMETRY (LALLS)

Fast gradient HIC has been interfaced with low angle laser light scattering photometry (LALLS) to separate and characterize proteins. Molecular weights (MWs) have been determined on-line for a set of four proteins. A fast HIC column has been used to elute proteins under fast (5 min) and conventional (10 min) gradients. Certain variables used in the calculation of MW, such as refractive index of the mobile phase (RI) and differential refractive index (dn/dc) have been calculated off-line. The MWs for three of the four proteins studied matched literature values at any given gradient time. One of the proteins, beta-lactoglobulin-A (beta-LACT), was a dimer when injected. Our results have indicated that fast gradient HIC can be easily interfaced with LALLS in spite of a significant change in RI across the gradient.