Determination of on-line differential refractive index and molecular weight via gradient HPLC interfaced with low-angle laser light scattering, ultraviolet, and refractive index detection

Gradient ion-exchange chromatography (IEC) was interfaced with a low-angle laser light scattering photometer (LALLS) and ultraviolet (UV) and refractive index (RI) detectors connected in series for on-line determination of the differential refractive index (dn/dc) of proteins and eventually their molecular weights (Mw's). Interfacing of gradient HPLC with a RI detector was made possible by using two isorefractive buffers, which helped generate stable baselines for the LALLS and RI detectors. An optically modified, laser based RI detector was used for determination of dn/dc. On-line determinations of dn/dc required smaller amounts of sample compared to off-line determinations (3-4 mg vs 200 mg). Also, dn/dc could be measured by making one repetitive injection of the sample, if the molar absorptivity of the sample was known. dn/dc values determined on-line were in close agreement with those determined off-line. Additionally, percent recovery of the sample was calculated on-line, and this provided true concentration detected at the UV detector, which was then used for determination of Mw.