Procedures for Group Quantitation Global Application Development Center Analytical & Measuring Instruments Division Shimadzu Corporation Group Quantitation In anionic surfactant analyses based on inspection methods specified by the water quality standards, five components are targeted: alkyl chains with 10 to 14 carbon atoms. Alkyl chains with the same number of carbon atoms come in two structural isomers: straight-chain and branched-chain. These form multiple peaks, which are then separated and detected. Group quantitation treats alkyl chains with the same number of carbon atoms as a single group. In this method, multiple peak area values are summed for quantitation. This article introduces the method s operating procedures when using the Shimadzu HPLC LabSolutions workstation. Differences from General Quantitation Methods General quantitation methods and group quantitation differ with respect to whether the results are grouped after peak detection. Flowcharts for the respective methods are shown below. General Quantitation Methods Peak detection Group Quantitation Method Peak detection Grouping Compound registration Compound group registration Calibration Calibration Fig. 1: General Quantitation Method and the Group Quantitation Method
Quantitation Processing In the [Method View Quantitative Parameters] pane in the [Data Analysis] window, select [Group Calibration] in the [Grouping Type] area. Fig. 2: Selecting Group Quantitation
Group Name Registration Enter the group names ("C10" for example) in the [Method View Grouping Table] pane. Enter the calibration point concentrations in the [Conc. (n)] areas. (1) Register the group names. (2) Register the calibration point concentrations. Fig. 3: Registering the Group Names and Calibration Point Concentrations Compound Group Registration Enter the compound names in the [Method View Compound Table] pane. Enter the group numbers in the [Group#] column. (Example C10: "1"; C11: "2",...) (1) Register the compound names. (2) Register the group numbers. Note: Compound names are displayed at the peak tops in the analysis report. Fig. 4: Registering the Compound Names and Group Numbers
Creating the Group Calibration Curves Create the calibration curves in the same way as for general quantitation. Select [Group] in the [Calibration Curve View] pane in the [Calibration Curve] window. Click the arrow buttons beside the group name to check the calibration curves for each group. (1) Select [Group]. (2) Click the arrow buttons to switch groups. Fig. 5: Creating the Group Calibration Curves
Creating Analysis Reports Click the folder icon in the Data Explorer pane in the [Data Report] window. The [Select Folder] pane will be displayed to the right of the Data Explorer pane. Select the "C: Labsolutions Sample LC" folder. The report format file will be displayed in the [Data Explorer] pane. Click [Close] to close the folder selection pane. Click the folder icon. 1 2 Fig. 6: Folder Selection (1)
3 4 Fig. 7: Folder Selection (2) Drag & drop "GroupResults_1.lsr" in the Data Explorer pane to the report pane. The "GroupResults_1" report format will be applied. Place the cursor in the grouping results table and right-click the mouse to display the [Grouping Results Properties] window. Place the cursor in the table and right-click the mouse. Fig. 8: Applying the Group Quantitation Report Format
Select the [Grouping Results] tab, and specify the items to be displayed. Configure any other necessary settings, click [Apply] and then [OK]. Set the items to be displayed. Fig. 9: Grouping Results Properties
Creating Group Calibration Curve Reports In the [Creating Analysis Reports] window, drag & drop "CalibrationCurveReport_1.lsr" in the Data Explorer pane to the report pane. The "CalibrationCurveReport _1" report format will be applied. (For a description of how to display report format files, see the "Creating Analysis Reports" section.) Fig. 10: Applying the Calibration Curve Report Format
Right-click in the frame titled "Calibration Curve" in the calibration curve report, and select [Properties]. Place the cursor in the frame and right-click the mouse. Fig. 11: Select [Properties] The [Calibration Curve Properties] window is displayed. Select the [Position] tab. Select [Groups] in the [Calibration Curve] area. Select [Groups]. Fig. 12: Selecting Group Calibration Curves
Select the [File] tab, and click [Browse]. Fig. 13: Method File Selection (1) The [Open] window is displayed. Select the method file used for calibration curve creation. Select [LC Method File] in the [Files of type] area. The method file used will be displayed in the [Name] area. Then click [Open]. Fig. 14: Method File Selection (2)
The selected method file name will be displayed in the [File] area. Configure any other necessary settings, click [Apply] and then [OK] to complete the calibration curve report. Calibration curve data for each group appears on the calibration curve report. Also, the group number (ID #) and group name are displayed. Group number Group Name Fig. 15: Group Calibration Curve Report
Report of repeatability for n measurements Click the [Summary (Standard)] icon on the toolbar in the [Report] window. In the report window, drag the summary display range to specify it. The property window for the summary (standard) is displayed. (1) Click the [Summary (Standard)] icon. (2) Drag the display range to specify it. Fig. 16: Summary (Standard) Application
Click on the [Display] tab, and select [Each Detector] in the [Display chromatogram] area. Fig. 17: Selecting the Chromatogram Display Method Select the [Chromatogram] tab, and set a baseline shift value. Fig. 18: Setting a Chromatogram Baseline Shift Value
Click on the [Summa] tab, and select [Area] in the [Data] area. Set the items to be displayed. Set the items to be displayed. Fig. 19: Summary Data Settings Select the [File] tab, and click [Add]. Fig. 20: Data File Selection (1)
Select the data files for n measurements, and click [Open]. Select the data files. The selected data files are displayed. Fig. 21: Data File Selection (2) The selected data file names will be displayed in the [File] area shown in Fig. 20. Configure any other necessary settings, click [Apply] and then [OK] to complete the summary report.
The sum of the area values for each group in each chromatogram will be displayed below the chromatograms for n measurements in the summary report. Sum of the area values for each group Fig. 22: Sum of Area Values for Each Group in Each Chromatogram