Editing Absorbance Equilibrium Data:

All experimental equilibrium data aquired on the XL-A requires substantial editing and preliminary diagnostics before the data can be successfully analyzed with various analysis methods. UltraScan assists you in this process by handling most essential diagnostics and editing steps automatically, but permits you to intervene, where necessary. Editing is started by selecting

"Edit Equilibrium Data": "Absorbance Data"

from the main menu of UltraScan. This will bring up the editing window with the plot area and the control panel.

At the bottom of the control panel is a "Wizard" that provides step-by-step instructions to guide you through the multi-step process of equilibrium data editing. Refer to it to determine which action is required next.

Step 1: Select a data directory that contains the data files acquired by the Beckman data acquisition program. Such a directory can generally be found as a subdirectory of the Beckman data acquisition software. The default installation for the Beckman software is C:\XLAWIN (under Windows). Under Unix, the drive with Windows will generally be mounted under the /dosc or /dos directory. You can always set the desired default location in the configuration panel so you can avoid lengthy directory traversals to find the desired location quickly.

The experimental data will then be located in a subdirectory of the form mmddyy/hhmmss (month/day/year/hour/min/sec), identifying the date and time on which data acquisition commenced for a particular run. A file dialog will allow you to specify the desired directory. If the directory contains files of the type 00001.ra1, 00002.ra1, ..., etc., then it is a proper data directory. If the program cannot find enough scans in the selected directory, a warning message will be printed on the screen.

Data directories retrieved from the database contain an additional file called "db_info.dat" which contains database index information that is automatically included in the edited data. The file is parsed during editing and parameters such as run identification, celltype, centerpiece type, rotor, buffer corrections, etc. are included in the edited data and later available during analysis.

Please note that UltraScan needs at least 3 scan files for each cell in order to identify the wavelengths used for the run. Once you have navigated through the directory tree to the desired data directory, simply press the OK button. Next, the program will load the data in this directory and determine a number of important diagnostics from the scan files present in this directory:

  1. The number and position of cells that were scanned in this run. If not enough datapoints are found in the selected directory, a warning message will be printed. If the sequence of the scan files is out of order, a warning message will be shown. If this message shows up, and you acquired the data at a single wavelengths, you can rescue your data by rebuilding the data sequence with the Scanfile Reordering Utility.
  2. The number of wavelengths and their settings that each cell was scanned with
  3. The number of scans that were taken for each cell and each wavelength
  4. The average temperature over the entire run
  5. The temperature of each individual scan
  6. The program will determine if the temperature varied for more than a pre-set tolerance value over the course of the run and warn the user if the tolerance was exceeded. In such a case, a warning message is printed forcing the user to acknowledge the diagnostic before editing can resume. Depending on the method of analysis of equilibrium runs, a temperature variation during equilibrium experiments may actually be of little consequence, since the software will compensate automatically for variations in temperature-dependent density changes. When measuring thermodynamic parameters such as association constants it is important to realize that temperature variations may significantly affect the results and should be avoided if possible. Also, for velocity experiments a constant temperature is required throughout the experiment in order to avoid systematic errors introduced through density and viscosity changes, as well as variations in the thermodynamic properties of the sample.
  7. The length (duration) of the entire run in hours and minutes
  8. The rotor speed at which each measurement was made
  9. The identifications for each cell that were entered at the begin of the run in the Beckman data acquisition program.
The status bar will keep the user informed about the progress of the diagnostics. The length of time required to complete these diagnostics will depend on the number of files in the directory that have to be analyzed. Once the diagnostics are completed, the diagnostics window will be displayed that shows the details of the selected run. The top panel of the "Run Detail" Window will provide you with a profile of the temperature variation over the course of the run. Shown is the temperature value of each scan in the experiment. Ideally, the temperature should not vary more than a few tenth of a degree over the course of the run. If the temperature varies more than a pre-set tolerance value, the "Temperature Check" field will show a red flashing LCD, otherwise a continuous green LCD.

Clicking on the "Time Plot" button will present you with a plot of the time differences between scans. The "Time Plot" button will then change to a "Speed Plot" button. Clicking on the "Speed Plot" button will then show you a plot of the speed at which each scan was recorded. Finally, you can recall the temperature plot by clicking on the "Temp. Plot" button, and cycle through the different plots by repeatedly clicking on the same button.

The purpose of this window is to facilitate the identification of experiments from the rather cryptic information provided by the file- and directory names created by the Beckman data acquisition software. Information about various cells available for a particular run can be obtained by selecting the appropriate cells in the listbox (UltraScan allows for up to 7 cells to accommodate experiments performed with the 8-hole rotor AN-50 Ti, and allows for an unlimited number of scans to be analyzed for each dataset. The cell description will be updated for each cell that is selected in the listbox. If the description for a particular cell was not sufficient, it can now be updated and edited to accommodate changes.

At the lower left corner of the diagnostics window you will find a list of speeds for this run. If you want to know how many scans and wavelengths were collected for a particular speed, you can click on any of the speeds to obtain a window with the listing for that speed.

Step 2: Before editing of the data can proceed, a unique run identification needs to be entered in the box labeled "Run Identification". This run identification should not contain any spaces and should help the user in identifying the run by name. Spaces can be replaced by underscores. Please note that unlike under Microsoft Windows, file names will follow Unix name conventions (case sensitive). For example, a run identification such as "Chromatin_pH8" is distinct from "chromatin_ph8". A practical way of naming runs is to use the logbook number of the run, and to append a "v" for equilibrium-, or an "e" for equilibrium runs. If the data was retrieved from the database, the run identification will be filled in automatically.

If you change your mind and do not want to edit this data, you can cancel the editing process at this point by clicking on "Cancel" and you can then select a different directory instead. However, if you do want to proceed, click on "Accept". If you forgot to enter a unique run identification, you will be reminded by an error message.

Step 3: Make sure that the cell descriptions are correct and adequate to identify the contents of each cell that contains data. If not, you can now edit the description for each cell by modifying the string in the editable text box. After entering a unique run identification, the window will close and you will return to the main editing window. The control panel will now show all fields updated with the proper information obtained during the diagnostics. If the data directory was retrieved from the database, the run identification is automatically set and all cell and centerpiece parameters should be automatically set to their proper values. Otherwise, adjust the centerpiece, cell and rotor settings now. Some data analysis methods rely on the geometry information included in these definitions.

The number of editable datasets will be displayed in the "Run Detail" box. A dataset is the collection of scans acquired for a particular wavelength and a single cell. For example, if the cell in position 3 has been measured at 260 nm, all scans measured at this wavelength for cell 3 are considered to be a single dataset. To load the first dataset for editing, click on "Start Editing". All scans will then load into the editing plot window, and the status bar indicates the progress of data loading.

Step 4: After loading is completed, the data will be displayed in the editing window. Review the settings for rotor selection and centerpiece type, and adjust if needed. If you used a 6-channel centerpiece to collect your data, make sure to select the 6-channel centerpiece. Also verify that the rotor is correctly selected.

The selection of these items determines the method of editing as well as the cell dimensions needed for integration of the modelled functions. If the data was loaded from a directory that was retrieved from the database, these settings should already be adjusted to their appropriate values.

In case the sample and reference channels were reversed during loading, you can click on the "Invert Sign" button to flip the data back into a standard display mode.

Step 5: Now you need to define the meniscus limits. Those are the limits between which the data contains the meniscus for all displayed scans. These menisci may differ, because of rotor stretching, which causes the meniscus to shift at different speeds. The program will assign a unique meniscus for each scan by searching for the highest absorbance of each scan between the limits you have entered. First, click with the mouse on the left limit, then on the right. If you are using 6-channel centerpieces, each channel needs to be treated separately.

Step 6: In the next step, you have to defined the limits for the useful data range for all scans. This range defines the data used in the data analysis. This region should exclude the meniscus and the bottom of the cell. First, the left limit is defined by dragging the mouse to a position near the right of the meniscus, and then clicking on it. If in doubt, rather include a little more data, it can be excluded later in the global analysis by fine tuning the editing limits.

Step 7: Next, the right limit is chosen in a similar fashion. The data will be rescaled to the new limits, and the extraneous datapoints will be discarded. If you have data from a 6-channel centerpiece, a six- channel centerpiece should be selected, which will prompt you 3 times to repeat the process for each channel.

Step 8 (optional): Now you will have the opportunity to exclude and edit scans. To exclude one or more scans, select the scan with the counter and then click on "Exclude Single Scan". To exclude a range of scans, click on "Exclude Scan Range", after setting the scan number in the counter. ">>" and "<<" buttons move in steps of 10, while ">" and "<" move in single step mode. To select a range of scans for exclusion, select the scan at the beginning of the range with the counter of "Exclude Single Scan". Then use the counter from the "Exclude Scan Range" button to define the last scan of the range to be excluded. As you select scans, the about to be excluded scans will be highlighted in red. After clicking on one of the "Exclude..." buttons, the highlighted scans will be deleted from the dataset.

To edit a spike, click on the ">" - button until you reach the desired scan. The selected scan will be highlighted in magenta to assist you in finding the appropriate scan (if you click on the ">>" and "<<" buttons, you can move around in steps of 10, which may be useful if you have a large number of scans in your dataset). Once you found the appropriate scan, click on "Edit Single Scan". This will bring up the scan editing dialogue.

Step 9: Once you are satisfied with the subrange of edited data you can save the subset by clicking on the "Save" button.

Now the data for the first dataset is edited and the program will automatically cycle to the next dataset by returning to the main editing window and updating the progress bar to indicate the status of file loading. Repeat Steps 4 - 9 for all datasets in the run. If you have selected the 6-channel centerpiece setting, the you will be able to select the next data range for the same cell and wavelength before the program cycles to the next dataset. This way you are creating 3 sub-datasets for each dataset, for each channel one. When all datasets are edited, the program will return with a message window indicating that all scans have been edited and the data has been written to a binary file.

www contact: Borries Demeler

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