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A method for removing time- and radially invariant noise from sedimentation velocity and sedimentation equilibrium experiments performed in an analytical ultracentrifuge is presented. The method averages repeat radial incident light measurements as a function of the photomultiplier response at different wavelengths to remove the majority of the time-invariant noise contributions from intensity data measurements. The results of this method are compared to traditional absorbance data generated with a buffer reference and the Beckman Optima AUC data acquisition program, and with the standard UltraScan refinement workflow. The method avoids the amplification of stochastic noise inherent in the absorbance scan subtraction traditionally employed in sedimentation velocity and equilibrium data. In addition, the collection of intensity data frees up the reference channel for additional samples, doubling the capacity of the instrument. In comparison to absorbance data, the residual mean square deviation of a fitted sedimentation velocity experiment without additional noise correction by UltraScan was improved by a factor of 4.5 when using the new method. This improvement benefits sedimentation equilibrium experiments as well as analytical buoyant density equilibrium experiments where routine time-invariant noise correction calculations cannot be performed., (© 2023. European Biophysical Societies' Association.)

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recently developed technique that has proven to be a promising tool to investigate mixtures of molecules containing multiple chromophores. It provides an orthogonal separation approach by distinguishing molecules based on their spectral and hydrodynamic properties. Existing software implementations do not permit the user to assess the integrity of the spectral decomposition. To address this shortcoming, we developed a new spectral decomposition residual visualization module, which monitors the accuracy of the spectral decomposition. This module assists the user by providing visual and statistical feedback from the decomposition. The software has been integrated into the UltraScan software suite and an example of a mixture containing thyroglobulin and DNA is presented for illustration purposes., (© 2023. European Biophysical Societies' Association.)