1. Standard Data Analysis - Calibration
ChemStation – Level 1 - Training
In this module you can learn the basics about the ChemStation Calibration features.

2. Building a Calibration Table
Inject standards
Set-up appropriate integration events for the low standard.
Begin a new calibration table with level one, the lowest level. Fill in the dialog.
Integrate each additional standard level and add to the calibration table.
Examine the calibration curve and save it to the method.
In order to create a calibration table, first, inject the standards. Load the signals for the low standard and integrate the chromatographic signals using appropriate integration events. Fill in the calibration settings such as amount units and treatment of reference peaks. Create a new calibration table. Fill in the amount for each standard and the name of the standard or internal standard. Repeat this process with each level. Examine the calibration curve and save the calibration table as part of your method.

3. Example Calibration Standard ISTD 3 Levels ng/L ng/L
A series of slides will lead you through a systematic procedure for building a calibration table. The example given is for an internal standard calculation. The calibration curve will be constructed with three levels of standards. The standard and internal standard are labeled in the chromatogram.

4. Signal Details Define signals to be evaluated during a method.
First, set up the Signal Details found in the Calibration menu. The Signal Details dialog box defines which signals will be evaluated during a method run. Signals that are not in the list box will not be integrated or reported if all defined signals can be found. Whenever defined signals cannot be found, the system loads all available signals.
Add to Method
The signal is copied to the method signal table that comprises a list of signals that will be processed by the method. After the signal is copied to the list, you can edit the start and end times. The signal is processed only between these limits.
Available Signals Combo Box
This box displays the signal description of the available signals that may be processed in the method. The list of available signals comprises the currently activated signals for the configured detectors and the acquired signals of the loaded data file. If this list contains two contradictory signal descriptions for the same detector channel, for example
DAD1 A, Sig = 330, 0 Ref = off
DAD1 A, Sig = 330, 0 Ref = 550, 20
a message will be displayed. This is an indication that there is a mismatch between the signals for data acquisition and the currently loaded data file.
Method Signal Table Editor
Displays descriptions of the signals that will be processed in the method. The table editor also displays the start, end, and delay columns. Only signals contained in this table will be evaluated during a method run. If you set the start, end, and delay times to 0.00, all data for the selected signal is processed.
Start and End
You can use the Start and End parameters to set the evaluation range of the signal.
Delay
You can use the Delay option to compensate for the signal delay of the detector. For example, if you acquire signals from two detectors in series there is a delay in one signal. By specifying a delay value, you can compensate for the detector signal delay. This information is used for peak correlation where the ChemStation groups peaks together on several signals for one compound. In peak identification, the software groups peaks with the same retention time to one compound, however, by using a delay it is possible to group peaks with different retention times.
Align
This item is used by the LC/MSD or any detector in series. Align allows the user to align LC chromatographic peaks with MS chromatographic peaks.

5. Calibration Settings
The Calibration Settings dialog box adjusts various parameters related to the current Calibration Table. Default values are provided for all items and should not be changed unless you thoroughly understand how calibration works.
Title
Allows you to enter a title that will appear at the top of all Calibration Table listings, for example, Default Calibration.
Use Sample Data
The sample data can be obtained from one of the following two sources:
From Sample Defaults Below
These values are displayed and set in the Sample Defaults section of this dialog box.
From Data File
When you select the From Data File option, you cannot set the sample defaults in this dialog box. You will use the information provided with each sample in the Sample Info panel or the Sequence Table.
Amount
The amount is necessary to calculate an ESTD% or ISTD% report. In these types of reports, the results are calculated as a percentage of the original sample.
Amount Units
The units in which all amounts of the sample are measured, for example, ng/ul.
Multiplier
A number by which all calculated results are multiplied before the report is printed. The multiplier can be used to change the scale of the results or correct for changes in sample composition during pre-analysis work. The multiplier can also be used for any other purposes, which requires the use of a constant factor.
Sample ISTD
Specifies the known internal standard amounts in the unknown sample. Up to 99 ISTD's can be used. I#
Identifies the internal standard compound. This I# corresponds to the I# in the Calibration Table (1-99).
ISTD Amount
The amount for each Internal Standard compound. The amount that is entered here is used as default if no sample information exits.
Default RT Windows
The Retention Time windows are used by the ChemStation software to identify peaks in the integration results. By specifying a window rather than a specific retention time, the ChemStation can compensate for slight shifts of retention times from one run to the next. The used windows are a sum of an absolute part in minutes and a relative part given in percent of the expected retention time. The percentage part compensates for variations in retention time that are proportional to the expected retention time. The absolute part compensates for variations that are independent from retention time.
Reference Peaks
Reference peaks are used to correct for retention time shifts that apply to a complete chromatogram. To do this the ChemStation software identifies all reference peaks, finds the reference peak shift, and applies this shift to the other peaks
NOTE: When a reference peak is identified in the calibration table, then the reference peak window is centered on the expected retention time of that peak. The biggest peak that appears in that window is identified as the actual reference peak.
Other Peaks
The retention time shift of the reference peaks is first applied to the expected retention times of the other peaks in the calibration table. The other peak window is centered on the expected retention time. For ISTD peaks, the biggest peak that appears in the peak window is chosen. For the rest of the peaks, the peak closest to the center of this retention time window is chosen.
Calculate Uncalibrated Peaks
This option allows you to define the response factor for unidentified compound peaks in each individual signal.
Default Calibration Curve
This feature is used for multiple level calibrations. A regression curve is calculated based on the given calibration points and the Fit and Origin parameters. Note that you may specify a fit and origin treatment for each calibration curve.
Curve Type
There are various possible curve-fit calculations for use with multiple-level calibration.
* Piecewise
* Linear
* Quadratic
* Cubic
* Exponential
* Logarithmic
* Power
* Average Rsp/Amt
Origin
There are various ways to treat the origin when the response curve is plotted, you can ignore origin, include origin, force origin or connect origin. The default values are linear and include origin.
Weight
When the calibration curve is generated, you can specify the relative weighting, (or importance), of the various calibration points. The weighting options are as follows:
Equal
All calibration points have equal weight in the curve.
Linear (Amt)
A calibration point with amount x has the weighting 1/x normalized to the smallest amount.
Quadratic (Amt)
A calibration point with amount x has the weighting 1/x^2 normalized to the smallest amount.
Linear (Resp)
A calibration point with amount x has the weighting 1/y normalized to the smallest response.
Quadratic (Resp)
A calibration point with amount x has the weighting 1/y^2 normalized to the smallest response.
# Calibrations
A calibration point has a weighting according to the number of calibrations for this point. For example, if level 1 has been calibrated twice, then the points on level 1 have a weighting of two. No normalization is done.

6. Load the Signal(s) for the Low Level Standard and Integrate
To create the calibration table, first load the low-level standard and integrate. The low-level standard is often the most difficult to integrate, therefore, the integration events for this standard may be suitable for more concentrated standards. Make certain that all necessary peaks are integrated and that tic marks are placed appropriately.

7. Create a Calibration Table: Level One
Once you have integrated the signal for the lowest level, go to the Calibration menu and select New Calibration Table.... This menu item will create a calibration table from the currently loaded data file. An existing calibration table will be removed. Specify the starting calibration level. The Default is one. You can specify a default amount to use for all peaks.
Select Calculate Signals Separately to ensure that, in the calculation of Norm% reports, the amount percent of separately reported signals adds up to 100% for each signal. When this options is deselected, the amount % of all signals adds up to 100%.
Calculate Signals Separately is a prerequisite for sorting by signal in the calibration table.

8. Calibration Table Overview: Fill in the Dialog
Compound Name
Group
Amount
Check if:
Reference Peak
ISTD
Indicate #
The Calibration Table will appear. At this point, you will fill in information such as the concentrations of the standards and the names of the peaks to be calibrated.
Compound Number #
The ChemStation assigns a unique compound number to each compound in the calibration run in order of elution. Compound numbers are reassigned whenever insertions or deletions are made. If you add parallel signals to the calibration data at the same time, using the New Table or Add Peaks options in the Calibrate/Recalibrate dialog box, parallel peaks are automatically grouped to one compound. To see all peaks of a compound, use the Compound Details dialog box. RT
The retention time of the main peak of the compound in the calibration run appears in the RT column.
Signal
Indicates to which signal the calibrated peak belongs.
Compound
Name of compound
Lvl
The calibration level is one point on a calibration curve. An amount and a response corresponding to that amount define the level. The level number corresponds to a calibration sample containing a particular concentration of components. All peaks that belong to a particular calibration sample (mixture) have the same level number.
Amt
The amount of the compound in the calibration sample. The Amt entries for an internal standard peak are usually the same in all levels.
Area (or Height)
The area of the peak calculated by the integrator. This is a fixed value, and cannot be edited.
Amt/Area (or Amt/Height)
Amount, Area or Height, and the response factor are interdependent, according to the following formula:
Response Factor = Amount/(Area or Height)
Consequently, the response factor is displayed as Amt/Area or Amt/Height. This column is adjusted automatically depending on the Based On option chosen in the Specify Report dialog box. The ChemStation calculates the response factor from the results of the calibration run and the Amount entry. If you modify the response factor manually, the number that you type in will be adjusted slightly according to the area, which has a fixed value. The ratio is calculated to an accuracy of six decimal places.
Ref
Identifies a peak as a time reference peak. Reference peaks correct for run-to-run variations in retention time due to temperature, flow, or injection technique.
ISTD
Identifies a compound that is used as an internal standard. An Internal Standard (ISTD) is assigned to a group of peaks by entering the same I# number for those peaks. I#
Identifier of the ISTD compound. The software expects a number between 1 and 99. The non-reference compounds are linked to the corresponding ISTD by entering the ISTD identifier to the I# column. It is possible to use multiple ISTD's at the same time.

9. Load the Signal - Add Level Two
To add the second level to the calibration table, first load the signal for the second standard and integrate it. Go to the Calibration menu and select Add Level.... Fill in the level number and the default amount if so desired. In this case, the level number is two. Then, OK the panel.
When the calibration table reappears, you need only fill in the amounts for the second level, then OK the table. Repeat the process with all other levels.

10. Add Additional Levels
Repeat the Add Level process until all standards have been added to the table. Note that the calibration curve display automatically updates as each new level is added to the calibration table.

11. Calibration Table Options
Compound, Amt, Area, Rsp Factor,
Ref, ISTD#
Compound, Grp, Amt, Low Limit
High Limit
Compound, Amt, Area, Def, Curve
Type, Origin
Compound, Area, Rsp%, Def, +-,
Pk Usage
The Calibration Table Options menu item contains four different predefined calibration table layouts.
Overview
This default layout contains the retention time, signal, name, level #, amount, area, and ISTD information for each peak. You can change this default layout using the Edit Options.... menu item.
Compound Details
Use this calibration table layout to get an overview on your compound information. The default layout contains the compound name, compound group #, level #, amount, and amount limits. In some revisions, the control samples are specified here.
Peak Details
Use this calibration table layout to get an overview on your peak details. The default layout contains the expected retention time, the signal, compound name, level #, amount, area or height, and calibration curve information.
Identification Details
This table displays the information important to peak identification. The default layout contains the expected retention time, retention time window, signal, compound name, area or height, response%, qualifier tolerance and peak usage. Use this layout if you want to define peak qualifiers.

12. Use Low and High Limits as qualifiers to confirm peak
Compound Details
The Compound Details table displays all peaks and allows you to specify amount limits.
Amount Limits Low and High
Specifies an upper and lower limit for the compound amount. If the calculated amount is outside the limits, a warning appears in the report. The amount limits can be used as qualifiers to confirm the peak identification. Compounds that are out of the amount limits are not used to recalibrate the Calibration Table. The minimum and maximum amounts do not consider a multiplier value that you may have specified. If the check boxes are selected then the limit is applied.
Use Low and High Limits as qualifiers to confirm peak
identification.

13. Peak Details Check Def to apply the default curve type and
origin from the
Calibration Settings
dialog box.
Curve Types
Piece wise Exponent
Linear Quadratic
Log Cubic
Power Average Rsp/Amt
Origin
Ignore
Include
Force
Connect straight segment
The Peak Details calibration table option contains information on the calibration curve. You can choose from various curve-fit calculations for use with multiple- level calibration including: piecewise, linear, log, power, exponent, quadratic, cubic, and average rsp/amt. If the Def column is selected, then the default calibration curve type and origin treatment from the Calibration Settings dialog box will be utilized. When the default calibration curve is changed in the calibration settings, the calibration table is automatically updated with the new values.
There are four ways to treat the origin when the calibration curve is plotted. You can ignore the origin, include the origin, force the origin, or connect a straight segment to the origin. The default values are linear and include origin.

14. Edit Calibration Table Options
Define the columns included in your Calibration Table
The Calibration Table Options screen allows you to define the columns included in your calibration table dialog. The options dialog comprises two lists: Columns not shown lists the columns that can be added to the calibration table display. Columns shown lists the columns displayed in the calibration table. To add a column to the calibration table simply highlight the column name. Click the right arrow button. Similarly, to remove a column, highlight it and click the left arrow.
For revision A and >, you may add Control Sample Limits to your calibration table by selecting Are Ctrl. Smpl. Limits set to the columns shown list. Once added, you may double click on the peak of interest and define the control sample limits. Control samples can be evaluated during Sequence operation. For earlier revisions, see the Peak Details view.

15. Quantification Tools Recalibrate with Current Chromatogram
Calibration Task
Tool
New Calibration
Table
Signal Details
Add New Level
Add New Peaks
When the Calibration Task Tool is selected, the tool bar contains many tools to help you build your calibration table easily without accessing the cascading menus.
Edit Calibration
Table Options
Select Calibrated peak(s) from
Chromatogram and delete from
calibration table.
Select peak(s) from
chromatogram and add
to calibration table
Select compounds
from chromatogram
and recalibrate

16. To Save the Calibration Table as Part of a Method...
Save Table to Method
When you are satisfied with the calibration table and the treatment of the curve and origin remember to save the table as part of your method.

17. Signal Options Customized display on screen and in reports Include
A chromatogram will be displayed or printed according to the Include options you select. Place a checkmark in the corresponding check box to identify the items you want to include with the report.
Peak Labels Font
You can change the current font attributes (size, style, color, underscore options, and rotation) for the peak labels on the chromatogram by clicking on the Font... button.
Ranges
To print/display the entire chromatogram, select the Full option. The entire range of retention time and response will be plotted. To print or display only a section of the chromatogram, select the Use Ranges option.
The Autoscale option will scale the response range down to the height of the second tallest peak.
Multi-Chromatogram
You may select a Separated or Overlaid format from the drop-down list box to be used when printing/displaying more than one chromatogram. All chromatograms may be displayed using the same y-axis scale, All the Same Scale, or with each chromatogram having its own autoscaling, Each in Full Scale.

18. Annotations
The Annotation sidebar can be activated within all toolsets
Selectable fonts and colors
 Use it for a customized chromatogram in slides or publications
Easy placing and moving of annotations
The annotate function enables you to add annotation to an active window. You can also edit or delete an existing annotation. Before adding an annotation, make sure that a chromatogram is loaded, and that the window is active. Select New Annotation from the Graphics menu, and click the cursor in the active window at the position you want the annotation to start. You will now see the Text Annotation dialog box displayed. Type the new annotation into the field provided. You can use the Options push-button to specify the font, font size, color, and etc. of the annotation.

19. Signal Manipulations Overlay, subtract and align signals
Use the Signal Tool set to graphically work with your chromatogram. This tool set does not have corresponding menu items. You may perform such tasks as aligning the x-axis of multiple signals. This may be useful when you have acquired data on both a diode array detector and a fluorescence detector. Other options include a 3D overlay of signals, mirroring signals and subtracting signals.

20. Lab Time !!!! Lab 7 - Calibration