Get Empowered: Review Window and the Processing Method | Tip #25
Tip #25: Performing Internal Standard Calibration and Quantitation
In the last Empower tip of the week blog for Empower Software, we explored how to add chemical structures to peaks in either Review or the Report Method. This tip will address how to perform internal standard calibration and quantitation.
Let’s get started.
Internal standard quantitation is commonly used to correct for losses during sample preparation, particularly when working with complex matrices. This technique calls for adding an internal standard to both the calibration standards and samples, often times at the same amount.
The calibration curve for internal standard methods plots the response ratio of the standard component to the internal standard times the amount of internal standard versus the amount of the standard component (Figure 1). That may sound complicated — however it’s easy to do in Empower Software.
Bring raw data (Channel, Injection, or Sample Set) into Review and open the processing method. Click the Processing Method tool (Figure 2).
Click the Components tab and scroll to the right so that you can see the Internal Std field. Click in the Internal Std field for the peak that will be calibrated/quantitated using the internal standard and select the internal standard from the list (Figure 3). Save all changes to the Processing Method and exit out of Review.
In the project window, click on the Sample Sets tab, right-click on the Sample Set, and select Alter Sample (Figure 4).
Enter Sample Weights and Dilutions for your samples and then click the Amount tool to enter the amounts for the standards (Figure 5).
In the Component Editor, the component names must match the names you’ve entered in the processing method. Click the Copy from Process Method tool (Figure 6).
Highlight the Processing Method and click Open, which copies the component names from the Processing Method into the Component Editor (Figure 7).
Select STANDARDS & UNKNOWNS from the SampleSet Type list . If you put the same amount of internal standard into all standards and samples, enter 1 for the internal standard. Then enter the amount for the other components (Figure 8).
If you vary the amount of internal standard in the standards or samples, enter those values accordingly. Click OK to the Component Editor, save all changes made in Alter Sample, and exit out of Alter Sample.
Back in the project window, right-click on the Sample Set and select Process. Select the Method Set or Processing Method and click OK (Figure 9).
Go to the Result Sets tab, right-click on the Result Set and select Review (Figure 10).
Select one of the sample results from the tree on the left and note the Amount field displays 1 for the internal standard and a calculated value for the component of interest. This value is already corrected for the Sample Weight and Dilution, therefore, this is the final amount. Click the Calibration Curve tool (Figure 11).
From the Component list select the component of interest. The y-axis of the calibration curve is the Area Ratio and the Response field in the table displays the ratio of the component peak area to the internal standard peak area for the calibration standards (Figure 12).
It’s that easy!I just learned how to perform internal standard calibration and quantitation in #EmpowerTips! Click To Tweet
This procedure can be followed for both the QuickStart and Pro interfaces.
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Next week in Empower Tip #25: Cross Channel Internal Standard Calibration and Quantitation.
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