A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation
Jeffrey A. Fagan
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the scan file time-stamps of elapsed time since the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of 4.304 ± 0.188 S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the precision of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the instrument-to-instrument variation in the determined fraction of BSA oligomers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation, Analytical and Bioanalytical Chemistry
(Accessed December 4, 2023)