Light scattering detection in aqueous high-performance gel permeation chromatography (HPGPC): Relative molecular mass distribution of eye lens proteins

Summary

Light scattering techniques have been greatly improved since laser light sources, enabling dynamic light scattering and measurements at low angles have been available. Relative molecular mass determinations can be performed at low solute concentration, extrapolations to zero-angle are no longer required and, because of the low scattering volume, a flow-through cell construction has become possible where dust particles only interfere as spikes [1]. For HPLC detection the low-angle laser light scattering (LALLS) signal corresponds to the product of relative molecular mass and concentration; combination with a concentration detector (for example, differential refractometer, UV absorbance) enables calculation of relative molecular mass [2]. The sensitivities of LALLS and the usual concentration detectors are comparable for (bio)polymers with relative molecular masses near 100,000. The minimum relative molecular mass to be determined in this way is near 10,000 and, therefore, the only mode of HPLC using the LALLS detector to date is gel permeation (size exclusion) chromatography.