If you are adding a large number of components whose parameters are the same, you might want to customize the component defaults before starting this procedure. For more information, refer to “Setting Component Defaults” on page 8-20.
¾ To prepare to add a new component:
• Do one of the following:
• Choose New Component from the Components menu to open the Components dialog box in add mode.
• Choose the New Component command button on the Identification tab of the Components dialog box.
Entering Component Identification Information
When adding components, the first step is to complete the component identification information. You can add the following types of components: • Single peak components
• Named group components • Timed group components
Note the following about component types:
• Any peak component you add to the component list in a method can also act as a reference component or internal standard.
• You must have identified the single peak or timed group components before you can create a named group. The members of a named group do not need to be located contiguously in the chromatogram: they can appear anywhere. • Peaks need not be identified to be included in a timed group.
¾ To add a peak component to the component list:
1. Select the Identification tab of the Components dialog box and select Peak as the component type.
2. In the Name text box, enter a name for the component.
3. In the Retention Time text box, enter the expected retention time for the component.
4. In the Absolute Window text box, enter the size of the absolute search window you want to use on either side of the expected retention time. 5. In the Relative Window text box, enter a percentage of the expected
retention time you want to add onto either side of the absolute search window.
6. Select Find Largest Peak In Window if you want TotalChrom to identify the largest peak (based on height, not area) as the component rather than the one closest to the expected retention time.
7. Do one of the following:
• To use the current component as a retention reference for other components in the method, select This Component Is A Retention Reference.
• To use another component in the current method as a reference, select a retention reference from the Reference list.
• Select <no reference> in the Reference list if this component needs no retention reference.
8. If the method specifies internal standard calibration, do one of the following:
• To use the current component as an internal standard for other components in the method, select This Component Is An Internal Standard.
9. To use this component as the reference for calculating RRT, select the corresponding check box.
You will be prompted to confirm any change that you make.
The state of this check box for each component reflects what you entered for RRT reference in the Global Information dialog box (or what you set here last time):
• If you designated this method to use the first peak as the RRT reference, then this check box will not be selected for any component.
If you designated this method to use a specific component as the RRT reference, then this check box will be selected when you select that component.
¾ To add a named group to the component list:
1. Select the Identification tab of the Components dialog box and select Named Group as the Component Type.
2. In the Name text box, enter a name for this group.
3. Select the components you want to include in the named group from the Group Members box.
The list includes all peak and timed group components contained in the open method. To select multiple components, hold down the Ctrl key and then click on each name.
4. From the Internal Standard list, select the component you want to use as the internal standard.
This list consists of all components in the open method that have already been designated as internal standards.
¾ To add a timed group to the component list:
1. Select the Identification tab of the Components dialog box and select Timed Group as the Component Type.
2. In the Name text box, enter the name you want to use for this timed group.
3. In the Start Time text box, enter the starting time you want to use. TotalChrom only includes in the group those peaks whose crests occur after this time.
4. In the End Time text box, enter the ending time you want to use. TotalChrom only includes in the group those peaks whose crests occur before this time.
5. From the Reference list, select the component you want to use as a reference component.
If you specify a reference component, TotalChrom adjusts the actual start and end times used to define the group based on the actual retention time of the reference peak in each run.
6. If necessary, select the name of a component you want to use as an internal standard from the Internal Standard list.
Setting Calibration Parameters
Calibration values affect how TotalChrom calculates component amounts. You can choose among the following types of calibration for components:
• Using a constant calibration factor • Calculating an average calibration factor
• Using the calibration curve of another component (calibration reference) • Solving the component's calibration curve
The procedures for setting up each calibration type follow. This section also provides instructions for working with calibration levels.
If you select Use Calibration Factor, TotalChrom divides the component's response by the calibration factor to obtain an amount.
¾ To calibrate using a constant calibration factor:
1. Select the Calibration tab of the Components dialog box. 2. Select Use Calibration Factor as the Calibration Type.
3. Enter a calibration factor value in the Cal Factor text box. 4. Under Response, select whether to use Area or Height.
If you select Average Calibration Factor as the calibration type, TotalChrom averages the slope at each calibration point on the curve and uses this average as a single calibration factor. TotalChrom does not perform a linear regression on the data. Because this calculation requires that the intercept of the curve pass through the origin, the Origin options are not available when you add new components or edit existing components.
The slope at each calibration point referred to above is either a response-to- amount ratio (for external standards), or response ratio-to-amount ratio (for internal standards) for each replicate.
¾ To calibrate using an average calibration factor:
1. Select the Calibration tab of the Components dialog box. 2. Select Avg Calibration Factor as the Calibration Type.
3. Under Response, select whether to use Area or Height.
The last column of the table changes to Area or Height, depending on the option you choose.
4. Edit the Level, Amt, and Area or Height values as necessary. For more information on levels, refer to “Working with Calibration Levels” on page 8-37.
You can perform a calibration by using the calibration of another component. TotalChrom calculates the amount for the component based on the calibration curve of the other component. This type of calibration is called calibrating by reference. The calibration reference component, in this case, can be any component: it does not have to be one previously defined as a reference peak.
¾ To calibrate using a reference component:
1. Select the Calibration tab of the Components dialog box. 2. Select Calibrate By Reference as the Calibration Type.
¾ To calibrate using a calibration curve:
1. Select the Calibration tab of the Components dialog box. 2. Select Use Curve as the Calibration Type.
3. Under Response, select whether to use Area or Height.
The last column of the table changes to Area or Height depending on the option you choose.
4. Select the Curve Fit Type you want to use from the list.
Depending on the calibration curve type, the minimum number of levels required are as follows:
• Point-To-Point: 1
• 1st Order Polynomial: 2 (or 1 if you include the origin) • 2nd Order Polynomial: 3 (or 2 if you include the origin) • 3rd Order Polynomial: 4 (or 3 if you include the origin) 5. Select the Scaling factor you want to use from the list.
For definitions of the options, refer to “Selecting Fit Parameters” on page 8-16.
6. Select the Weighting factor you want to use from the list.
For definitions of the options, refer to “Selecting Fit Parameters” on page 8-16.
7. Under Origin Treatment, select Include or Force as the origin option if required.
Include origin adds point 0,0 to the calibration (zero amount to zero response). Force origin prevents any offset, forcing the curve to go through the point of origin.
Leave both options deselected to omit the origin from the calibration. 8. Edit the Level, Amount, and Area/Height values as necessary.
For more information on how to create new calibration levels, refer to “Working with Calibration Levels” on page 8-37.
Working with Calibration Levels
If you did not build calibration levels in the Component Defaults dialog box, or if you need additional levels, you can add new levels in the Components dialog box. To determine unknown component amounts, TotalChrom compares the
component's response (peak area or height) to the response of a known amount of the same component in a single standard sample. To improve the accuracy, you can choose to inject more than one standard sample, where each sample contains the same component but in different amounts. These varying samples are called
calibration levels.
The response produced by the component at each calibration level, plotted against the level amount, defines the points on which the component's calibration curve is based.
Some notes about calibration levels:
• You can add calibration levels only with the Avg Calibration Factor and Use Curve calibration types.
• A component can have up to 100 calibration levels, but the level names for all components in a standard sample must be the same.
• Use unique level names for different levels.
• Ensure that two different levels have different amounts unless the component is an internal standard component.
You can also revise an existing calibration level, or delete it from the list if it no longer applies to a specific method or component. If you are using named or timed groups, these actions apply to a group of components.
¾ To add a calibration level:
4. Enter a value in the first empty cell in the Amt. column.
TotalChrom adds a new row to the table after you complete the columns for the current level.
The value displayed in the Area/Height column is grayed-out because it is not relevant for default components.
5. Repeat Steps 2 to 4 to add additional levels.
¾ To change an existing calibration level:
1. In the table showing the levels, select the cell that contains the parameter you want to change
2. Edit the parameters as necessary.
If you change level entries that contain any replicates, TotalChrom will delete the replicates.
¾ To delete a calibration level:
1. Click inside the row you want to delete. 2. Press the Delete key.
Setting User Values and LIMS Options
You can use the User Values/LIMS tab to:
• Specify a number of constants associated with the component
• Set up SQL*LIMS reporting for the component (if you have Connect) You can specify constants and then use these values in custom expressions, which you create in the Report Format Editor. For example, you can multiply the peak area or the peak concentration (or other value) by a constant factor for each individual component.
If you are using Connect, you can also use this tab to specify whether to send the results for the component to LIMS.
¾ To set user values for the component:
1. Select the User Values/LIMS tab in the Components dialog box.
2. In the User Label text box, type a label that associates the values with the component.
3. In the User Values text boxes, type the constants that you want to use. 4. Select Send Results For This Component To LIMS if you want to send