Determination of second virial coefficient of proteins using a dual-detector cell for simultaneous measurement of scattered light intensity and concentration in SEC-HPLC

A method is proposed for the measurement of the B-22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90degrees, after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration ( ultraviolet) detector is converted to Rayleigh's ratio, R-theta, and concentration, c, respectively. The B-22 value is calculated from the slope of the Debye plot (K-c/R-theta versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B-22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature.