Setting up a Chromatographic Data Analysis Method using the Optional Chromatographic Software Module

New features in MassHunter 4.2 make creating and calibrating a chromatographic data analysis method easier than ever.

As the first step, you need to acquire a set of calibration standards and ensure that the resolution, sensitivity, peak shape, baseline etc. are acceptable. In general, this can be verified visually.

If you are working with a previously acquired batch of samples, you can open the batch using Offline Data Analysis by clicking [Open Folder] from the [Batch] group on the [Home] tab, and selecting the batch from the list. The list of samples in the batch is displayed in the Batch at a Glance table.

Ensure that the calibration standards are designated as sample type “Calstd” in Data Analysis. If they were originally acquired as sample type “Sample” during acquisition, change the sample type in the Batch View Grid. You do not need to include a Calblk in chromatographic analysis. The lowest concentration standard should contain recognizable peaks for all analytes.

CH_BatchTableCalStd.png 

  1. Click [Edit] from the [Method] group on the [Home] tab.

    EditIcon.png 

    The [Method Editor] window is displayed.

  2. In the Method Development Tasks Pane, click [Open Data File…], and then select a calibration standard data file with good quality peaks (a mid-range standard is a good choice).

    MethodDevelopmentTasksPane.png 

    The total ion chromatogram (TIC) for the selected calibration standard data file is displayed as shown below. If your acquisition includes more than one element or isotope and internal standards, the baseline may look noisy and the peaks may appear smaller than expected because you are seeing a composite of all signals.

  3. Pull down the list box that currently shows TIC (circled) and select the mass for the first group of analytes that you want to enter into your method.

    For example, if you are looking for arsenic compounds, select mass 75.

    CH_TIC.png 

    The integrated extracted ion chromatogram (EIC) for the selected mass is displayed.

    CH_75.png 

    If your method includes compounds or species of different elements, then select <All> from the TIC/EIC menu to display all acquired EICs and the TIC simultaneously. This allows you to add all your selected analyte peaks from different masses to the peak list table graphically. You may need to enlarge the Chromatogram window to see all of the EIC traces clearly if there are more than 2-3. In the example below, the only analyte is As at m/z = 75. However, m/z 35 was included to indicate the presence of interferences from ArCl due to Cl containing peaks.

    CH_All.png 

  4. If all of the peaks are not correctly integrated using the default integration parameters, as shown below, click manual_integration_tool.png on the manual integration tool and draw in the correct baseline.

    The manually integrated peaks are red.

    CH_ManualInteg1.png 

    Before manual integration above; after manual integration below.

    CH_ManualInteg2.png 

  5. After all peaks are correctly integrated, you can add these compounds to the Peak List by using the “Add All Peaks” tool.

    CH_AddAllPeaks.png 

    The integrated peaks are added to the Peak list in order of retention time.

    CH_PeakList.png 

    Alternatively, you can choose individual peaks from each EIC to add to the Peak Table by using the “Add Peak Mode” tool Add_Peak_Mode_tool.png.

    At this point, you can edit the peak table to change the compound names, designation as analyte, or internal standard and target or qualifier ion. If the auto-integration process did not correctly, integrate a peak (in this example peak 5 was incorrectly integrated due to a noisy apex), select the Smoothing column for the noisy peaks, and then turn on Gaussian smoothing.

  6. Once the peaks are added, you can step through them one by one by clicking on them in the table and view the default integration windows in the chromatogram display.

    The windows should be wide enough to easily capture the peak start and end, but not so wide as to contain several peaks (if possible). Adjust the integration windows as needed by changing the “Integ Start” and “Integ End” values in the Peak Table. The graphic below on the left shows a correctly set integration window for peak 1. The graphic on the right shows an integration window for peak 2 that is too wide.

    CH_Peak1.png          CH_Peak2.png 

  7. Once all desired peaks are correctly integrated and added to the peak list with the correct information filled in, Click on “FullQuant” under the Method Development Tasks pane on the left of the screen.
  8. Fill in the calibration and QC information as shown below. You may or may not have spike samples or QC samples. If not, these columns can be left blank. For more information about completing various QC samples information, refer to the training module “Understanding QC Error Action Functions”.

    CH_FullQuant.png 

  9. Click [Validate] near the bottom of the Method Development Tasks Pane to check for any errors and correct them if necessary.
  10. Click [Return to Batch-at-a-Glance] at the bottom of the pane, to return to the Batch View. Click [Yes] to the “Update Data Analysis Method?” question.

    CH_DAWindow.png 

    Results are displayed for the entire batch via the Batch at a Glance Table at the top, and graphically for each sample at the bottom. Click individual samples and/or peaks from the table to select them. The Chromatogram pane shows the EIC for the entire run on top, and the selected analyte peak on the bottom. If the baseline on the target ion (bottom chromatogram trace) indicates incorrect integration, use the manual integration tool manual_integration_tool00574.png to correct the integration. The [Process Batch] icon changes to ProcessBatchIconRed.png. Click this icon to requant the batch including your manually integrated peaks.

  11. When you are satisfied with your results, click [Save Batch Results] from the [Batch] group on the [Home] tab.