Band Sedimentation Experiment in Analytical Ultracentrifugation Revisited
2018-09-04, Schneider, Cornelia M., Haffke, Dirk, Cölfen, Helmut
The band sedimentation experiment in analytical ultracentrifugation (AUC) allows for the performance of a chemical reaction inside the AUC and also offers separation of individual pure components in a sedimentation velocity experiment. Although this experiment offers exciting possibilities for application, it is barely used. This is related to the bad definition of the initial conditions. Both the duration and the time of the solution overlay during rotor acceleration are not known. In this study, we investigate these conditions under the variation of the overlay volume using recording of interference patterns in a continuous mode during the acceleration of the rotor. It was found that the overlay occurs at rotor speeds between 770 and 2000 rpm, which is very low compared to typical experimental rotor speeds from 3 000 to 60 000 rpm and therefore elucidates that the generated reaction products, respectively, overlaid species are subject to the centrifugal force almost from the beginning. Also, the duration of the overlay is less than 1.2 s, which is very fast compared to hours of centrifugation time for an experiment and we demonstrated that the overlay compartment is completely emptied during overlay allowing for the precise calculation of the meniscus using the known sample sector geometry. Our results show that the initial conditions of the experiment are defined and should make an adapted analysis possible if the interdiffusion of the two solvents is taken into account, which lead to a dynamic density gradient.
Next-Generation AUC Adds a Spectral Dimension : Development of Multiwavelength Detectors for the Analytical Ultracentrifuge
2015, Pearson, Joseph, Krause, Frank, Haffke, Dirk, Demeler, Borries, Schilling, Kristian, Cölfen, Helmut
We describe important advances in analytical ultracentrifugation (AUC) hardware, which add new information to the hydrodynamic information observed in traditional AUC instruments. In contrast to the Beckman-Coulter XLA UV/visible detector, multiwavelength (MWL) detection is able to collect sedimentation data not just for one wavelength, but for a large wavelength range in a single experiment. The additional dimension increases the data density by orders of magnitude, significantly improving the statistics of the measurement and adding important information to the experiment since an additional dimension of spectral characterization is now available to complement the hydrodynamic information. The new detector avoids tedious repeats of experiments at different wavelengths and opens up new avenues for the solution-based investigation of complex mixtures. In this chapter, we describe the capabilities, characteristics, and applications of the new detector design with biopolymers as the focus of study. We show data from two different MWL detectors and discuss strengths and weaknesses of differences in the hardware and different data acquisition modes. Also, difficulties with fiber optic applications in the UV are discussed. Data quality is compared across platforms.