Getting Started with Peak Integration

This is a tutorial for PEAXACT software for quantitative spectroscopy from S-PACT. Its main objective is to get you familiar with the most widely known analysis method for spectra and chromatograms: Peak Integration (PI). The tutorial addresses to PEAXACT users and persons interested in PEAXACT.

In this tutorial you learn how to:

  1. Create a new Preferences Profile
  2. Create an Integration Model
  3. Use the model to calculate peak areas of chromatograms

Data files used during the tutorial can be downloaded for free. If you have PEAXACT installed on your computer you may try this tutorial right away. If you don't have PEAXACT yet, get a free trial now.

Note that this tutorial was created for PEAXACT 3 and may look different in the newest version!

Preparations

  • Download demonstration samples (zip-file containing gas chromatograms) and unpack it to any directory. Your directory will be referred to as DATA-directory in this tutorial.
  • Start PEAXACT.
  • Choose File > Preferences from the menu which opens the application settings window.
  • From the pulldown menu in the top-left corner select Chromatography, then click OK.

Integration Model

The Integration Model calculates peak areas by integrating the measured signal within specified ranges. These integration ranges we need to set up in an interactive modelling step.

  • First, load data which should be modelled. In this case, choose File > Load..., browse to the DATA-directory, and open file GC.csv.
  • Click on the first sample in the Data Sets Panel (labelled GC.csv#1) to get displayed the gas chromatogramm in the Plot Panel.
  • Create a new model: File > New Model. The new model is displayed in the Model Tree Panel. In the Model Tree Panel, click on the + sign to expand the tree structure. The model tree contains 4 sub-models.
  • Normally, modelling starts by choosing data pretreatments, but for peak integration this often can be skipped. We come back to it later.
  • We now add the first integration range for the peak at x = 15.75 minuts. Choose Edit Model > Integration Model > Add Integration Range... from the menu. Each range is defined by a (unique) name, lower and upper bounds (limits of integration), and a baseline function. Copy values from the following dialog and click OK.
  • The new integration range is added to the model tree. Also, the integrated signal is displayed in the Plot Panel as colored patch. Zoom in on the peak to have a closer look. Use the zoom tool from the toolbar and draw a rectangle around the peak.
  • Apparently, baseline option "Offset" was not a good choice, so we are going to change it. The fastest way is by right-clicking on the center of the patch in order to invoke the context menu; then choose Baseline Type > Straight Line.
  • Note that the zoom tool must not be enabled if you wish to invoke the context menu. Also note that if you do not hit the patch correctly, a different context menu might be shown.
  • Have a look at the next figure which displays the different kinds of baselines. Option "Straight Line" works well for peaks on a non-constant underground, but you have set the bounds carefully in case of overlapping peaks to not end up with negative peak areas.
  • Double-click in the Plot Panel to zoom out. Then zoom in on peaks between 11.5 and 13.5 minutes.
  • A fast way to add more integration ranges is by right-clicking on the center position of a peak in the Plot Panel and choosing Add Integration Range... from the context menu. The next dialog automatically contains predefined lower and upper bounds centered around the clicked position. Pick a baseline and click OK. If the range is not positioned as you expected, drag the dashed lines (representing lower and upper bounds) to a new position. Try it out on some peaks...
  • Up to now, peaks weren't overlapping. The problem with overlapping peaks is, that the integrated area partially belongs to two or more peaks. We need to split up the area by a reasonable selection of bounds and baselines. Best baseline choice for overlapping peaks is Linear Fit. The baseline will be best fitted to the signal within the bounds, and therefore, bounds can be chosen a bit more freely. Try it out with peaks between 11.5 and 12.5 minutes. You may want to zoom in on this region for a better view.
  • This concludes basic integration modelling. Choose File > Save Model and save the model to your DATA-directory. The model can now be used for analysis, i.e. for the calculation of signal areas.

Analysis

  • For a quick preview of integrated areas of the active sample select the Integration item in the Model Tree Panel. A list of all integration ranges, including their parameters and areas, is displayed in the Model Properties Panel.
  • For customizable graphical and tabular reports of multiple samples select those samples in the Data Sets Panel and choose Analysis > Integration from menu.
  • Confirm the dialog to start the analysis. When finished, integration results are displayed in a new Report Generator Window.
  • Toggle between different reports by using the upper right pull-down control in the Report Generator Window. Note that for this tutorial you only get results for two samples (which are virtually identical). However, the same procedure can be used to analyse hundreds or thousands of data files, not only chromatographic but spectroscopic samples as well.
  • Click the Export... button to save the visible report. Graphical reports are saved as image files, tabular reports as spreadsheet files.

Advanced Modelling

  • In some cases it may be desirable to handle overlapping peaks with baseline option Offset, but then bounds need to be chosen very carefully. Try it out by changing the baseline to Offset for all overlapping peaks between 11.5 and 12.5 minutes. Adjust bounds appropriately.
  • If you want the peak at 12 minutes to be integrated using a similar baseline level as the adjecent peaks, you need to remove the baseline differently:
    • Select the Pretreatment item in the Model Tree Panel.
    • From the Baseline correction control in the Model Properties Panel choose Rubber band subtraction.
  • Select integration range Overlapping B2 and change the baseline type to None. "None" corresponds to 0, which is OK now because the baseline has been subtracted using data pretreatments.

This concludes the tutorial on Peak Integration. Please note that peak areas can be calibrated if reference concentrations are available. Calibration is not part of this tutorial, but you may contact Technical Support if you need help with calibration.

 

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