van Holde - Weischet Tutorial |
The first step in distinguishing diffusional boundary spreading from spreading due to heterogeneity in S is to calculate apparent sedimentation coefficients, s*, along the entire boundary. First, the boundary is divided into equally spaced horizontal boundary fractions between the baseline and the plateau. The intercepts of these divisions are located and mapped on the boundary as shown below:
Figure 4: In this plot, 11 equally spaced boundary fractions between the plateau concentration and the baseline absorbance have been mapped onto a single scan; the corresponding radial positions in the boundary (r_{b}) are identified r_{1} through r_{11}. For clarity, only 11 divisions are shown, in practice, the number of divisions is often larger (50-100).
The known time of the scan and the radial positions associated with the intercepts (r_{1} - r_{11}) are then used to calculate an apparent S-value, s_{b}*, for each boundary position:
Here, r_{b} refers to the radial position of the boundary fraction intercept, r_{m} refers to the meniscus position, t is the corrected time of the scan, and t_{0} is the start time of the experiment, corrected for rotor acceleration. The rotor acceleration time correction can be determined by taking the omega-square-t integral times from scans taken at constant velocity and plotting them against the actual time of the scan. Extrapolating to zero omega-square-t will reveal the rotor acceleration time difference, which needs to be subtracted from the times recorded for all scans.
This procedure is repeated for each scan in the experiment. Because of radial dilution, the absolute spacing between the boundary fractions will decrease over time, since the plateau absorbance decreases. This gives rise to the same number of apparent S-values, s*, for each scan in the experiment. It is clear from this algorithm that only scans that span the entire range from baseline to plateau concentration can be included in a global fit.
This document is part of the UltraScan Software Documentation distribution.
Copyright 1998, 1999, The University of Texas Health Science Center at San Antonio.
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Last modified on June 12, 1999.