ChromGraph REPORT can rapidly process an almost unlimited number of Data files in the fully AUTOMATIC MODE. A slower interactive approach is useful, however, when optimizing a Method for a particular analysis, and when learning to use the software.
INTERACTIVE DATA PROCESSING includes three main functions: peak detection, integration and quantitation. During peak detection the software determines where peaks start and stop. During integration, which can only occur after peak detection, the areas under the peaks are determined. These peak areas are compared to the areas of known standards during quantitation, which estimates the amount of each analyte in the injection.
Begin the interactive processing mode by loading the Data files to be processed. Take the LOAD DATA option from the Data Menu:
The SELECT CHROMGRAPH DATA FILES dialog box appears:
In interactive mode only four Data files (runs) can reside in RAM simultaneously. The Files Selected section of this window gives the name and slot number for each Data set that is already present in RAM. If you wish to keep these in RAM, do nothing. If you wish to replace these with other Data files, click the Remove button.
Click on the Data files you wish to load and their names will be transferred to the Files Selected section. To follow along in this section, load the files indicated in the figure above. These will be present on your disk if you followed the installation instructions (link to software installation). Click on OK and the files will be sequentially loaded into the designated slots. Also load the appropriate Method for these Data files, CATS.MET (if necessary, review the LOAD METHOD procedure).
Data or Method files can be loaded from other locations by using the mouse to select other drives or subdirectories in the Look in: section of this window. Such a selection automatically becomes the new DEFAULT DATA LOCATION. The default location will remain in effect throughout the session, unless changed by a LOAD or SAVE operation. When you finish your session and QUIT the software, saving the REPORT.INI file will save the new default data location for subsequent sessions. If REPORT.INI is not saved, the new data location will be forgotten; the original default location will be used for subsequent sessions.
When you load multiple Data files, the last-loaded file automatically becomes the focus for subsequent operations. You can change this focus with the SELECT DATA SET option under the Data Menu, or with some of the options under the Process Menu.
The first step in interactive processing is to identify the peaks that are present. In this step the software determines what a normal baseline is (a consecutive set of data points whose values don't change significantly) and then determines where peaks start and stop (consecutive sets of data points whose values change markedly). The Processing Options involved in these decisions (INITIAL SLOPE THRESHOLD, SMOOTHING WIDTH, PREBUNCH, PREFILTER, and PROCESSING EVENTS) are discussed in the Method section.
To begin peak detection, first turn on the List Peaks option in Processing Options. Then select the DETECT PEAKS option from the Process Menu:
Select the screen as LIST DEVICE when prompted. As peak start and stop points are detected, a list of times and baseline values will be posted to the output window:
These can be reviewed by using the scrolling feature of the Output Window.
If you wish to see the chromatogram at this point, take the REVIEW BASELINE option from the Process Menu:
The graph shows the chromatogram, with up- and down-arrows indicating the start and stop points of peaks. All the interactive features of ChromGraph graphics are available in this screen.
At this point you can save the graph by taking the SAVE BASELINE option from the Process Menu. The file will be saved with the data name and run number, and a .BLN extension. Baseline files take up a lot of disk space, and can easily be recreated from the raw data using the REVIEW BASELINE option. Therefore, we recommend that they only be saved if you are required by regulation to archive the original.
You can also LOAD a BASELINE from the Process Menu, which brings up a choice of previously saved baseline files for review.
It's likely that the first attempt at peak detection for an unknown Data file will not provide entirely satisfactory results. There may be peaks that have not been detected. Or every little blip in the baseline may have been interpreted as a peak. If this is the case, return to the PROCESSING OPTIONS and PROCESSING EVENTS sections of the Method, adjust the relevant parameters, and detect the peaks again. Feel free to enter extreme values to see what will happen; it's a good way to gain familiarity with the software. Or use the OPTIMIZE VARIABLES procedure to let Report suggest some values.
Integration can proceed only after the peaks have been detected. The integration function determines the area under each peak between the start and stop points. To integrate the peaks, select INTEGRATE from the Process Menu and choose the screen as LIST DEVICE when prompted:
A list of sequentially numbered peaks, with retention times, heights and areas, will be posted to the Output Window. These may be reviewed with the scrolling feature of the Output Window:
Small peaks of little interest may be deleted from the list of peaks by raising the MINIMUM PEAK AREA. This change requires that you re-detect the peaks.
Quantitation is the comparison of the integrated data to a pre-defined set of integrated standards, called a STANDARDS FILE. The Standards file must be created as described in the STANDARDS section.
In order to quantitate in the interactive mode, you must have integrated the current Data file and have a Standards file in RAM. If a Standards file is specified in the Method, as is the case with CATS.MET, it will automatically be loaded when the Method is loaded. Otherwise, Standards files may be loaded through the LOAD STANDARDS option of the Standards Menu.
To quantitate, select QUANTITATE from the Process Menu, and choose the screen as LIST DEVICE when prompted. A sequential list of peaks, and the results of the quantitation, will appear on the Output Window:
The items that appear in the list are those checked in the REPORT OPTIONS and STANDARDS OPTIONS sections of the Method The column under the asterisk (*) is a comment column: a question mark (?) here indicates that the calculated amount was extrapolated more than 10% from the standard curve, and may be questionable. A pound sign (#) indicates that a peak specified in the standards file was not found in this sample.
A complete report, including a descriptive header and all items (including the graph) selected in the Method, can be obtained by selecting the REPORT option in the Process Menu:
Once the data analysis and reporting options are properly set, be sure to SAVE the updated version of the Method.
From one to four runs can be processed fairly rapidly, once a satisfactory Method has been developed. Select PROCESS DATA SET from the Process Menu. The Process Data Set dialog box will appear, allowing the selection of any (or all) Data files in RAM for processing:
Click on the Data files to be processed, and then on OK. The Data files will be processed sequentially according to the options specified in the Method.
**** This is a special topic that most users need not read. ****
The digitizer in epsilon and DA-5 systems from BASi divides a full-scale signal into 1,000,000 subdivisions, informally called counts. Peak heights are reported in counts, and areas are reported as the summed counts for all the data-collection times under the peaks.
This is an excellent procedure for most purposes, including quantification ” counts of an unknown are compared to counts of a known, and the units of 'counts' don't matter.
An occasional user who's well-versed in electrochemistry might want to convert counts to amperes and area to coulombs. Here's how it's done:
COUNTS TO AMPERES
Simply divide counts by 1,000,000 to get the proportion of full scale that the peak represents, then multiply by the Range setting:
Be sure to use the correct units of Range (e.g., nA, µA).
AREA TO COULOMBS
Use the formula:
where pps = points per second = data-collection rate, in seconds.
Again, be sure to use the correct units of Range.