How to create a quantitation table

Introduction

The quantitation table contains all the necessary data, such as the calibration curves, that are needed to quantitate one or several components in a sample. This section describes how the quantitation table is created.

How quantitation tables are created

Quantitation tables are created in the same way for both external standard quantitation and for recovery calculations. They both use absolute values of standard peak data.

For quantitation with internal standard, the peak sizes relative to the size of the internal standard peak are used to create a calibration curve.

Four process steps

The creation of the quantitation table can be divided into four steps:

  1. Standard data input

  2. Component selection and definition

  3. Peak identification

  4. Calibration curve and quantitation table creation

Step 1 - How to input the standard data

The table below describes how to input the standard data in the Evaluation module.

Step

Action

1

Select Quantitate:Edit Quantitation Table:New on the menu bar.

Result: The New Quantitation Table dialog box opens with the name of the active chromatogram displayed in the Source chromatogram field.

2

  • Double-click a result file in the Select peak table list if you want to select a source chromatogram from another result file.

If desired, the standard can be expressed in Concentration instead of in Amount.

  • Click the Concentration checkbox and edit the injection volume in the Inj. volume field.

Note: The software will always calculate both amount and concentration for the sample.

  • Highlight the standard peak table of level 1 on the Peak table(s) list and click the Select button.

Note: This should be the table for the highest or lowest concentration of the standard.

Result: The peak table is added to the Level/Peak table(s) list.

3

  • The level is automatically copied onto the list if it already was set in the method. If so, continue with step 4.

  • If a level has not been set, the Select Level dialog box opens. Select 1 on the Level menu and click OK.

4

  • Click another result file in the Results field and select the new source chromatogram.

Result: The peak tables associated with this chromatogram are displayed on the Peak table(s) list.

5

  • Repeat steps 3 and 4 until all the standard peak tables have been selected.

Note: Increase the level number for each new standard concentration in consecutive order of decreasing or increasing concentration.

  • Click the Current button at any time to return to the chromatogram that was active before you activated Quantitate.

  • Highlight unwanted tables on the list and click Remove.

  • Click OK to finish the selection.

Result: The Define Component(s) dialog box opens.

Continue to "Step 2, How to select and define components" below this table.

Standard concentration levels

It is useful to think of each level as an alias for a specific concentration of the standard. You can incorporate up to 10 peak tables at each level, prepared from runs repeated at the same concentration. Quantitate will later allocate each with an incrementing suffix, e.g. 1:1, 1:2 etc.

The Define Component(s) dialog box

The components that will be used to produce the calibration curves are selected in the Define Component(s) dialog box. Quantitate must be able to identify these components on all levels. This dialog box is used to set the criteria by which peaks are identified.

The illustration below shows the Define Component(s) dialog box.

Examine the components

The Define Component(s) dialog box initially displays the components from level 1:1, that is the peak table from the highest or lowest concentration of the standard. The Show curve for level list is used to examine the curve for each standard run. The size of the components are reduced or increased progressively as you select levels further down on the list, which reflects the decreasing or increasing concentration of the standard.

If an internal standard has been incorporated, its peak remains about the same size on each level.

Peaks detected during the peak integration

Each component peak that was detected during the peak integration, i.e. that is present in the peak table, is identified by a lower triangle (black in level 1:1, green in other levels). There may be different peaks detected for different levels. Upper triangles will later identify the peaks that are selected for quantitation.

Step 2 - How to select and define components

The table below describes how to select and define the components.

Step

Action

1

Select level 1:1 in the Show curve for level list and click a peak.

Result: The peak is highlighted in the table.

2

  • Double-click the peak.

or

  • Click the Include button.

Result: The peak is selected for quantitation, marked with an upper triangle and "component no." is listed as the Component name. The selected peak is affected on all levels.

Note: More than one peak can be selected to produce calibration curves for several components.

3

Highlight the component name and type a new name.

4

Double-click the internal standard peak (if applicable) and type a new name.

5

Continue to "Step 3, How to identify the peaks" below this table.

The Define component(s) peak table columns

The peak table within the Define Component(s) dialog box has three columns:

  • The (absolute) Retention value of the component in level 1:1.

  • The width of each component’s window. If you change the width of the window by adjusting the cursor lines, this is reflected in the Window column.

  • The Component name, with the currently selected component highlighted.

Step 3 - How to identify the peaks

Description

When a component is selected, vertical cursor lines show the current identification window. The software uses this window to search for peaks on other levels and in the sample runs. A peak found in the window is assumed to be the component of interest. You can change the limits by dragging a limit cursor line. Both cursor lines move symmetrically so that the limits center on the component peak.

The window should be set wide enough to include peaks on the other levels despite minor variations in retention volumes. However, the window should also be narrow enough to exclude unwanted peaks that will interfere with the quantitation.

Instruction

The table below describes how to adjust the window width for the best results.

Step

Action

1

Drag the cursor lines to set the window to a suitable width.

2

  • Use the Show curve for level menu to display all levels and check that the width is suitable (the window width is the same on all levels).

  • Click the lower green or black triangle to display the actual retention for a peak.

3

Repeat steps 1 and 2 for all selected peaks.

Note: Overlapping windows are not allowed.

4

If necessary, click the Identification settings button to edit the settings. See "How to adjust the identification settings" below this table.

5

  • Click the OK button to accept the default identification settings.

Result: The Quantitation table dialog box opens.

6

Continue to "Step 4, How to create a calibration curve and a quantitation table" below this table.

Identification settings

The criteria by which peaks are identified are set in the Identification Settings dialog box. The criteria are valid for all the selected peaks in the Define Component(s) dialog box. These settings also affect the information provided in the peak table in the dialog box.

How to adjust the identification settings

Description

By default, peaks are identified by their absolute retention values and the highest peak maximum within the window. In most cases, it is not necessary to change these default settings. Peak identification by absolute retention works well when there has been little or no drift in retention between successive runs of the standard. Quantitate will find corresponding peaks in these successive runs providing any drift in retention does not move a peak outside the peak window.

Instruction

If you have drifting retention that makes peak identification difficult you can choose to identify peaks according to their position relative to a reference peak. The table below describes how to adjust the identification settings in the Define Component(s) dialog box.

Step

Action

1

Identify a component peak that can be used as the reference.

Note: Choose a peak that is well separated from any other peaks. This enables the window to be set relatively wide and the system can accommodate a larger drift in retention value.

2

Click Identification Settings.

Result: The Identification Settings dialog box opens.

See "How to identify peaks within a window" below.

3

Select Relative retention on the Identify peaks on droplist. (See "Absolute and Relative window width" below)

4

Scroll down the Component menu and select the component to be used as the reference peak.

5

  • Type the window width for the reference peak (an absolute value).

Note: Set the width fairly wide to accommodate a larger drift in the retention value. Make sure that there are no other large peaks within the window.

  • Click OK.

Result: A column for the relative retention is added in the peak table, Ret/Ref. The column displays the value of each component relative to the retention value of the reference component. This reference component is marked Ref. in the Window% column. The Window% column shows the window width for each peak expressed as a percentage of its relative retention value.

How to identify peaks within a window

Quantitate must be advised of how the peaks are to be identified if any of the windows includes more than one peak. The second droplist in the Peak identification field of the Identification Settings dialog box offers the following options:

  • Highest peak maximum (default).

  • Closest to retention, i.e. closest to the center of the window (see the retention column in the peak table.)

  • Maximum peak area.

Examine the nature of the peaks enclosed by the window and select the option that differs between the wanted and the unwanted peaks. Use Closest to retention if there are large peaks from components that are not going to be quantitated.

Note: The selection applies to all peaks, even the internal standard and reference if used.

Absolute and Relative window width

When the Peak identification is set to Absolute retention, the peak window width can be displayed as Absolute or Relative. Select the appropriate button in the Identification Settings dialog box.

  • Select Absolute to show the window width of each peak in minutes (or the base volume unit).

  • Select Relative to display the width of each component as a percentage of its retention.

If Peak identification is set to Relative retention, Window is set automatically to Relative except for the reference peak.

Step 4 - How to create a calibration curve and a quantitation table

When the component selection and identification settings are completed (see Step 3), the Quantitation table dialog box is opened:

The table below describes how to enter data for the standards and create a quantitation table and a calibration curve.

Step

Action

1

  • Click the IS and Table settings button if you want to use an internal standard or base the calibration curve on peak height (see "How to select an Internal Standard" below this table).

2

  • Verify that the selected components in the Components list are correct.

    If an internal standard is used, the related component is labelled (IS).

    If relative retention has been used, the reference component is labelled (Ref).

  • Click the Define components button to change the components.

3

  • Select the first component at the top of the Components list.

Note: Do not select an internal standard component (if available) as the amount for this has already been entered and does not change between the levels.

  • Highlight the Amount/Concentration for Level 1.

  • Type the amount or concentration of the component in the standard at this level.

Note: This is the amount corresponding to the injected volume, not the total amount used when the standard level was prepared.

  • Repeat this for the other levels for this component.

4

Click the Curve model radio button for the best curve model:

  • Linear (recommended).

  • Linear through origin.

  • Quadratic.

  • Quadratic through origin.

  • Point to point.

Result: The curve is displayed in the Calibration curve window. Each component level is labelled with crosses. If more than one run has been performed for any level, all points in that level will be shown. The average of these points is calculated and this value is used to produce the calibration curve.

5

Repeat steps 3 and 4 for all the remaining components.

Result: The quantitation table is complete with a calibration curve for each component.

6

  • Save the quantitation table and click Close.

or

  • Click the Save as button.

Result: The Save quantitation table dialog box opens.

Note: The Save button is used to save updates in an existing quantitation table. However, this will overwrite the original table. You might prefer to use Save as and create a new name for the edited table to preserve the original.

7

  • Specify if the table is to be globally accessible to any user or restricted to your personal user ID. The default is global.

  • Type a name in the Quantitation table name field.

  • Click the OK button.

How to select an Internal Standard

The table below describes how to select an internal standard in the Quantitation table dialog box.

Step

Action

1

Click the IS and Table settings button.

Result: The IS and Table Settings dialog box opens.

The illustration below shows the IS and Table Settings dialog box with an Internal standard selected.

2

Type the amount and concentration multipliers in the General field.

Note: These values are normally set to 1. See remarks below.

3

Select the internal standard component on the Internal standard peak droplist.

Note: The default option is Not selected, which is used for external standard quantitation and measurements of the recovery factor.

4

Type the injected internal standard amount for the standard and sample runs in the IS amount text box.

5

Select if the quantitation will be based on Area (default) or Height in the Quantitation peaks field.

Note: Select Height if the peaks are not completely separated from those of other components.

6

Click OK.

Note: The amount and concentration of the sample are multiplied by the multiplier values when the calibration curve is applied to a sample. Change the default values if you want to determine the amount or concentration in the starting volume of the sample instead of in the injected volume of the sample.

Quantitation statistics

The Statistics field in the Quantitation table dialog box displays the Correlation and Explained variance values when available.

Click the More button to open the Statistics dialog box for a complete display of available data.

Statistical reference values

  • The correlation (only available for linear models) should be as close as possible to 1.00.

  • The explained variance value should be as close as possible to 100%.

Note that the value is usually rather high even for poor models. A value of 90% indicates a very poor model.

The explained variance is not shown for curve models that are drawn through the origin.

Note: If the point-to-point curve model is selected, no statistics are available.

2005-06-15