Peak Detection, Identification, and Quantification

This video will demonstrate peak detection, identification, and quantification in GC Image.

Note

Demos 1, 2, and 3 should be completed in sequence.

The public dataset used in this demo can be downloaded here:

Supporting files to help in following along with this quick start demo can be downloaded here:

1 Detect Blobs

First, we will detect 2D compound peaks, or Blobs, using the Interactive Blob Detection tool.

  1. From the menu, select Processing > Interactive Blob Detection.
  2. Click Detect once to see the default detection results.
  3. Select Outline from the Outline dropdown list to change how blobs are displayed.
  4. Use the chromatogram view at the top of the dialog to zoom into regions of the chromatogram and review the detection.
  5. Click Back once to return to the previous detection controls.
  6. For this demo, adjust the following parameters:
    • Set the Col II Smoothing to 2.0.
    • Uncheck Detect 1D Shoulders.
    • Check the with Peak Filter option, using the default filter.
  7. Click Detect again to apply any detection parameter changes.
    • If desired, filters can be applied to the blobs via the Advanced Filter area.
  8. Click Apply to apply the blob detection.

2 Calibrate RI

We will identify the Alkane peaks and use these to calibrate Retention Indices.

We can quickly identify Alkane peaks by performing a library search against a custom library consisting of the Alkanes.

  1. Add the library to NIST by copying the custom library folder to your NIST install folder using File Explorer.
    • The Alkanes library is included in the Fragrances Demo Processing Files linked above (AlkanesLibrary/).
    • The NIST install location may vary, but defaults to C:\NIST##\MSSEARCH.
  2. From the menu, select Configure > Settings.
  3. Select Search Library from the list on the left.
  4. On the NIST Search Settings tab:
    • Set Hitlist size to 100
    • Set Included Libraries to only your custom NIST library
  5. On the NIST RI Setting tab:
    • Enable Use Retention Indices(RI) comparison
  6. On the Post Search Settings tab:
    • Enable Apply post-search constraints when selecting the top hit
    • Set Match Factor Threshold to 700
    • Set Reverse Match Factor Threshold to 700
  7. Click OK to save the settings.

2.2 Calibrate RI from Alkane Peaks

  1. From the menu, select Processing > Search Library for Selected Blobs.
  2. From the menu, select View > Blob Table.
  3. Select all of the Alkane peaks from the Blob Table dialog.
  4. Right click on the selection to open the Multiple Blob Properties dialog.
  5. Enter Alkanes into the Group Name field and select the Include checkbox.
  6. Click OK to apply.
  7. From the menu, select Configure > RI Table.
  8. Select Retention Index (Col I) from the list on the left.
  9. Click Populate.
  10. Select Compound Property and Library RI then click Append.
    • All Included blobs will be added to the table, along with the RI from each blob's library result if available.
  11. Manually enter the RI values for any rows with 0 or incorrect Retention Index values.
  12. Click OK to apply the RI Configuration.

The compounds present in this sample are known (see the standard's product page). We can quickly identify peaks corresponding to these known compounds by performing a library search against a custom library consisting of the known compounds.

  1. Follow the steps from Configure Targeted Library Search above but use the fragrance standards library included in the Fragrances Demo Processing Files linked above (FragrancesStandardLibrary/).
  2. From the menu, select View > Blob Table.
  3. From the blob table, select all unnamed blobs (i.e., leave the previously identified Alkanes unselected).
  4. From the menu, select Processing > Search Library for Selected Blobs.
  5. From the blob table, select all named blobs not part of the Alkanes group (i.e., the blobs newly identified by the library search).
  6. Right click on the selection to open the Multiple Blob Properties dialog.
  7. Enter Fragrances into the Group Name field and select the Include checkbox.
  8. Click OK to apply.

4 Create a Template

We will add the identified compounds to a template for easy automated identification in other chromatograms. The template created from this step is included in the Fragrances Demo Processing Files linked above (Fragrances.bt).

  1. From the menu, select View > Blob Table.
  2. From the blob table, select all blobs from the Alkanes group.
  3. Right click on the selection to open the Multiple Blob Properties dialog.
  4. Select the Un-include checkbox and click OK to apply.
  5. From the blob table, select all blobs from both the Alkanes and Fragrances groups.
  6. Click the Add to Template button on the blob table toolbar. Close the blob table.
  7. From the toolbar, select the Template mode to view the newly added template peaks.
  8. From the menu, select View > Template Table.
  9. Click the Save Template button on the template table toolbar. Browse to a desired save location and click Save.

5 Create a Method

Next, we will create a method, which defines a sequence of processing steps and parameters. The method created from this step is included in the Fragrances Demo Processing Files linked above (Fragrances.method/).

5.1 Update Configuration

We will first adjust the configuration settings which will be saved with the method.

  1. From the menu, select Configure > Settings.
  2. Select Baseline Correction from the list on the left.
    • Click the Defaults button.
    • From the Data Channel dropdown list, select TIC.
  3. Select Blob Detection from the list on the left.
    • Click the Defaults button.
    • Set the Col II Smoothing to 2.0.
    • Uncheck Detect 1D Shoulders.
    • Check the with Peak Filter option, using the default filter.
  4. Select Template Matching from the list on the left.
    • Click the Defaults button.
    • Set the Col I (pixels) Distance Threshold to 4.
    • Enable both Match Factor and Reverse Match Factor Spectral Constraints and set both values to 700.
  5. Click OK to save the settings.

5.2 Method Editor

We will next create the method by specifying a series of processing operations to perform.

  1. From the menu, select Method > New Method.
  2. From the Method Settings pane of the Method Editor:
    • Enable Set Configuration with the value <From Current Configuration>.
    • Set Use Color Map to the value <From Current Image>.
  3. From the Quick Add menu on the right of the Method Editor, select Shift Phase
    • Set Shift Amount to the value 2.2.
  4. From the Quick Add menu, select Correct Baseline
  5. From the Quick Add menu, select Subtract Spectrum from Image
    • For now, set the Spectrum field to 1,1. We will fill in the correct value later.
  6. From the Quick Add menu, select Detect Blobs
  7. From the Quick Add menu, select Apply Template
    • Set the Template File field to the template you saved in Step 4.
  8. From the Quick Add menu, select Apply RI Table (Col I)
    • Set the RI Table Path to <From Current Image>.
  9. Click Save. Browse to a desired save location and click Save.
  10. Next, we will update the Spectrum field with the correct value.
    • Close the Method Editor and open the Journal from the menu at View > Journal
    • Scroll until you reach the Spectral Arithmetic Operation on Image entry.
    • Find the row with the Spectrum parameter. This should be 9 rows down.
    • Copy the full spectrum string value from the Spectrum parameter row.
    • Return to the Method Editor to update the previously created method and select the Subtract Spectrum from Image
    • Past the copied spectrum string into the Spectrum field.
    • Click Save and close the Method Editor.

6 Specify Compound Amounts

The amount tables used in this step are included in the Fragrances Demo Processing Files linked above (AmountTables/).

  1. From the menu, select Quantification > Amount Table.
  2. Click Populate to automatically add all included blobs to the table.
  3. Manually enter the amounts for each compound.
    • Alternatively, the amounts can be imported directly from the provided CSV files. Click Browse, browse to the appropriate CSV file, click Open and then Copy to import the amount table values.
  4. Click OK to save the Amount Table.

7 Create Calibration Table

In order to create a calibration, we must process additional data files to include in the calibration. Save and close the current image with File > Save Image and File > Close Image.

7.1 Processing with a Method

We have already processed the file CalCurve_2ppb_A.cdf. We will process the following files using the following steps: CalCurve_02ppb_A.cdf, CalCurve_04ppb_A.cdf, and CalCurve_1ppb_A.cdf.

  1. From the menu, select File > Import Image.
  2. Browse for the data file and click Open.
  3. Enter a Modulation Period of 4.0 seconds in the Import dialog.
  4. From the Automated Actions tab, enable Use Method and browse to the method you saved in Step 5.
  5. Click OK to import the file and run the method.
  6. Repeat Step 6 to specify the amounts for each image.
  7. Save and close the image with File > Save Image and File > Close Image.

7.2 Calibration

  1. From the menu, select Quantification > New Calibration....
  2. Browse to your previously saved processed images and select all 4.
  3. Set the Calibration Type: to External Standard.
  4. Click Open.
  5. Click Next on the 4 Extract Calibrants from... dialogs. If any Split class... warnings appear, Click OK.
  6. Examine the resulting calibration by selecting an entry from the Calibrants table and checking the Calibration Curve tab.
  7. Click Save. Browse to a desired save location and click Save.