Correcting Ultraviolet-Visible Spectra for Baseline Artifacts

Rayleigh and Mie light scattering from particulates, soluble protein aggregates, or large proteins can lead to inaccuracy of concentration measurements using ultraviolet (UV) spectroscopy and Beer's Law. While a number of light scattering correction equations have been proposed in the literature, they can also lead to incorrect values if samples vary in particulate and/or soluble aggregate levels or depart in other ways from which the equations were developed. We propose a curve-fitting baseline subtraction approach based on fundamental Rayleigh and Mie scattering equations which also factors in instrument baseline artifacts. We validated this Rayleigh-Mie correction against a wide variety of positive and negative controls, including protein size standards, protein aggregates induced by forced degradation, lentivirus and polystyrene nanospheres.