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Identifying Peaks

Peaks are identified with the use of the Peak Table in the Data Handling section

of the Method. The main purpose of the Peak Table is to list the names of your

compounds and their expected retention times. Since retention times may vary

slightly from run to run, you can specify a window of time during which the

Workstation will try to identify a particular peak. The peak window is the actual

span of time on the chromatogram that the software searches for the peak,

centered around the retention time you entered for that peak. If the peak falls

within that time window, it will be identified, otherwise it will not be identified. You

may choose to report unidentified peaks, but these peaks cannot be accurately

quantified (refer to the on-line help in Interactive Graphics for a description of the

unidentified peak factor).

If your peaks tend to drift, you can automatically modify their peak windows by

marking a peak as a reference peak. Non-reference peaks will have their peak

windows adjusted by the same percentage that the reference peaks have

deviated from their expected retention times, accounting for the direction of

change.

Interactive Graphics provides an easy way to build a Peak Table using your pilot

run. You select the Edit MethodFill Peak Table menu item, and one by one

click on the peaks in your pilot run which you want to identify in your production

runs. As you click on a peak, a new entry is added to the Peak Table with the

retention time and a default name. When you have added all the peaks of

interest, you can change the default names to real compound names.

Building a Calibration Curve

In order to determine absolute amounts of an analyte, you need to compare the

peak area from a run in which the amount is unknown to a run in which you have

injected a known amount of the compound. A sample that contains known

amounts of the compounds to be analyzed (analytes) is called a calibration

standard. When you inject a calibration standard, you designate the run as a

Calibration Run. You enter amounts (for each analyte in the calibration standard)

into the Peak Table. Note that this is the same table used for identifying your

analytes.

Sometimes you will want to do several calibration runs, and in each successive

calibration run you inject different calibration standards with different amounts of

each analyte. This is called multipoint or multilevel calibration. The Varian MS

Workstation allows you to calibrate with up to ten levels. Each level represents

the amount of each analyte injected for each of the different calibration

standards. Multilevel calibration can be more accurate than single level

calibration, because you calibrate over large range of analyte amounts. For more

accuracy, you may want to repeat injections at a given level. This is a way of

averaging out variations from injection to injection.

Regardless of the number of calibration runs you perform, the result of the

calibration runs is a calibration curve. The calibration curve for an analyte is

generated by plotting the peak size on the y axis and the injected amount of the

analyte on the x axis for each calibration run you have performed, and then

calculating the best line through the points. There are options to do a linear,

quadratic or cubic fit through the points.

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