Equilibrium: Ln(C) vs. r 2 Analysis:

The Ln(C) vs.r2 analysis can be used as a first order check on the quality of equilibrium data. The analysis doesn't require any non-linear least squares fitting, and provides some rudimentary statistics, such as standard deviation, correlation coefficient and runs analysis. This analysis should not be used for a detailed description of the sample, since at most it can only verify the presence of homogeneity or heterogeneity. More sophisticated methods such as non-linear global fitting methods should be employed for a more detailed analysis of equilibrium data.

The Ln(C) vs.r2 analysis is started from the equilibrium menu in the main menu of UltraScan. This will display the the Ln(C) vs. r 2 data analysis window.

The first step in the analysis requires that you load an UltraScan equilibrium dataset that has previously been edited with the UltraScan editing module. UltraScan sedimentation equilibrium datasets always have the suffix ".us.e". Click on the "Load Dataset" button in the left upper corner of the control panel. Simply select the desired run from the selection of available runs in the dataset loading dialog. Once loaded, the Run Details will be shown. Click on the "Accept" or "Cancel" button, and you will be returned to the analysis window, which will show the first available dataset of the selected run in the edited data window on the lower right panel, and the Ln(C) vs. r 2 analysis will be shown in the upper right panel of the analysis window.

Next, you can use the baseline adjustment tool to adjust the baseline to minimize residuals visually/manually. Note that not all scans in the same cell will necessarily show best residuals with the same baseline:

Uncorrected Baseline

Corrected Baseline

Analysis Functions:

Click on these buttons to control the Ln(C) vs. r 2 analysis:
  • Load Data: Load edited data sets (with the *.us.e suffix). A file dialogue will allow you to select a previously edited and saved velocity experiment. If the data was edited with a version of UltraScan less than 4.0, an error message will be displayed.
  • Run Details: View the diagnostic details for a particular run.
  • Save Data: Write out a copy of all results to an ASCII formatted data file suitable for import into a spreadsheet plotting program. See "File Structures and Formats" for details. Note: These files are overwritten each time this button is clicked. Only the last version of the analysis will be saved!
  • Print Data: Load the printer control panel for printing of plot graphics
  • View Data Report: Review the data report for the last analysis setting. Note: This file is re-written each time it is accessed. Only the current analysis result is available. This file will provide information about molecular weight as well as fitting statistics.
  • Help: This help file
  • Close: Close the Ln(C) vs. r 2 analysis window.

Run Information:
  • Run ID: The name of the run given during editing
  • Temperature: The average temperature calculated from the entire run
  • Available Cells: The numbers the cells that contain analyzable data
Clicking on a cell, wavelength and channel selection will bring up the cell contents description for that cell and wavelength. Scroll through the cell listing to bring up information for cells > 3. If there is no data available for the selected cell, the program will list "No Data available". Selecting a cell/wavelength/channel combination will automatically bring up the corresponding dataset and present the analysis. Note: Not all experimental data contains multiple channels or wavelengths. Only data edited with the 6-channel centerpiece will provide multi-channel data, otherwise the channel selection dialogue will be disabled.

Experimental Parameters:
Here you can enter the corrections for density and partial specific volume of your sample. As you change the information in these fields, the program will automatically update the analysis to correct the results according to the specified conditions.
  • Density: Click to calculate the density based on the composition of your buffer.
  • vbar(20o): Click to calculate the partial specific volume for a peptide based on its primary amino acid sequence and correct the vbar value for the current temperature.
  • Reset Data: Reset all parameters to the default values, which includes restoring all scans that have been excluded, resetting the smoothing and baseline value to zero, and resetting the boundary to 100%.

Analysis Controls:
  1. Adjust Baseline: Since inclusion of the baseline as a parameter into the fit would cause the fit to be nonlinear and require an iterative fitting procedure, the baseline can in this analysis routine only be adjusted by manual intervention. You can use this counter to fine-tune the baseline offset for all scans and attempt to improve the fitting statistics by finding an optimal value for the baseline. Alternatively, you can overspeed the experiment at the end of the run and pellet all material in order to obtain a baseline value. You can use this counter to dial in the experimentally determined baseline value.
  2. Data Smoothing: Use this feature to smooth the experimental data. For noisy data, increasing this parameter can improve the clarity and appearance of the results considerably. Please note: Whenever possible, especially for steep gradients, try to avoid using too much smoothing to prevent artificial modification of the boundary shape. Smoothing is performed based on a frame average. The number shown represents the size of the smoothing kernel used (the number of datapoints averaged for a single point). The algorithm used is non-destructive of the original data and hence the smoothing is reversible. Also, point smoothing is independent, where the smoothing of one point does not have any effect on the smoothing of its neighbors. Only unsmoothened points are used for the calculation of the smoothed value. Each time you click on the counter, the current dataset will be reset to the full dataset.
  3. % of Boundary: This is the portion of the boundary used for the analysis. 100 % refers to the entire boundary, reaching from the baseline concentration value to the plateau concentration value. This portion is shown in yellow in the experimental data plot at the lower right panel of the Second Moment analysis window. Excluded data is shown in blue. Changing this number will automatically reset the position of the analyzable portion of the boundary in the center of concentration between the baseline and the plateau concentration.
  4. Boundary Position (%): For percent-boundary values less than 100 %, this number refers to the percentage of total concentration by which the remainder (=un-analyzed portion of the boundary) is shifted away from the baseline. A value of 0% refers to a data analysis start at the baseline. This number is always less than or equal to 100 % - (% of Boundary). It allows you to control the position of the analyzed portion relative to the baseline. The blue colored portion of a scan is excluded from the analysis, and the yellow portion is analyzed.
  5. Exlude Single Scan: When setting this counter to a non-zero value, the respective scan will be highlighted in red. Clicking on "Excl. Single Scan" while a scan is highlighted in red will delete this scan from the analysis. Deleting scans from the analysis is irreversible and can only be reset by clicking on the "Reset" button or by reloading the data (when smoothing, the data is always automatically reloaded).
  6. Exclude Scan Range: Same as "Exclude Single Scan", except for multiple scans. To use this feature, select first the start scan of the range by using "Exclude Single Scan", then complete the scan range by using "Exclude Scan Range". Use this feature to exclude a range of scans.
  7. Status: The status bar is not used in this analysis, since calculations are very fast.

www contact: Borries Demeler

This document is part of the UltraScan Software Documentation distribution.
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Last modified on January 12, 2003.