Approaching Effective Differential Centrifugal Fractionation by Combining Image Analysis with Analytical Ultracentrifugation

Centrifugation is one of the most commonly used methods for separation in biology and chemistry. However, effective fractionation is not always easy to obtain, as preparative centrifuge experiments are mostly conducted in an empirical way, even when it is guided by the quantitative results from analytical ultracentrifuge (AUC). Very few works have been performed to enhance the fractionation resolution of the differential centrifugation method in a swing-out rotor. This is primarily due to the absence of a characterization tool for sedimentation in the preparative centrifuge. In this study, we utilized image analysis to map the particle concentration distribution throughout the preparative centrifuge tube, revealing an unexpected and abnormal sedimentation process. By characterizing the sedimentation coefficient distributions of the fractionated product via AUC, we demonstrated that the overall sedimentation efficiency in a swing-out preparative centrifuge was significantly reduced. Furthermore, effective fractionation was confined to the intermediate phase of the entire sedimentation process. We propose that the mechanism here is a combination of the inverse Boycott effect and droplet sedimentation. The actual sedimentation process within a preparative centrifuge can be described by modifying the Lamm equation phenomenologically, which simply results in an effective sedimentation coefficient. Our work builds a foundation for determining the optimal preparative centrifugation conditions for various systems.