Tip #26: Performing Cross Channel Internal Standard Calibration and Quantitation

In the last Empower tip-of-the-week blog for Empower Software, we explored how to perform internal standard calibration and quantitation. This tip will address how to perform cross-channel internal standard calibration and quantitation.

Here we go!

Now that we understand how internal standard quantitation works, let’s explore a case where our internal standard peak is in a data channel different from the channel with the peaks we are trying to quantitate. This could be the case when using a Photo Diode Array detector (PDA) or a mass detector (MS). This sounds like it will be a chore, however, it is easier than you think! (This data was collected with an ACQUITY QDa mass detector and the channels of interest are QDa1, QDa2 and QDa3.)

Step 1

Bring raw data (Injections or Sample Set) into Review and open the processing method. Click the Processing Method tool (Figure 1).

Figure 1

Step 2

Click the Components tab and scroll to the right so that you can see the Channel field. Click in the Channel field for each component and select the Channel (Figure 2).

Figure 2

Step 3

Scroll to the right so that you can see the Internal Std field. Click in the Internal Std field for the peaks 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.

Figure 3

Step 4

In the project window, right-click on the Injections and select Alter Sample (figure 4).

Figure 4

Step 5

Enter Sample Weights and Dilutions for your samples and then click the Amount tool to enter the amounts for the standards (Figure 5).

Figure 5

Step 6

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).

Figure 6

Step 7

Highlight the Processing Method and click Open, which copies the component names from the Processing Method into the Component Editor (Figure 7).

Figure 7

Step 8

Select STANDARDS & UNKNOWNS from the SampleSet Type list. Enter the amount of internal standard for all standards and samples. Then enter the amounts for the other components in the standards (Figure 8). Click OK to the Component Editor, save all changes made in Alter Sample, and exit out of Alter Sample.

Figure 8

Step 9

Back in the project window, right-click on the Injections and select Process. Select the Method Set and click OK (Figure 9).

Figure 9

Since we’ve collected multiple channels per injection we are going to process with a method set that defines the channels and their processing methods (Figure 10).

Figure 10

Step 10

Go to the Result tab, right-click on the Results and select Review (Figure 11).

Figure 11

Step 11

Select one of the sample results and note the Amount field displays 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 on the 2D Channels tab in the lower left corner (Figure 12).

Figure 12

Step 12

We can see the multiple channels of data that were collected and their associated sub-channels, which are part of the data. Highlight the next channel with the check mark in the Visible field (Figure 13).

Figure 13

Step 13

Click the Peaks tab to view the quantitative results (Figure 14).

Figure 14

Step 14

Repeat steps 12 and 13 to look at the results for the next channel (Figure 15).

Figure 15

Step 15

Click the Calibration Curve tool. From the Component list select the component of interest. The y-axis of the calibration curve is the Area Ratio. 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 16).

Figure 16

It’s that easy!

Final notes:

1. We are collecting multiple channels of data per injection; therefore, it is easier to work with injections when creating the processing method.
2. While working in Review, I recommend adding the Sample Name and Channel to the legend so that as you click through the channels of data, you can easily see which is which.
3. This procedure can be followed for both the QuickStart and Pro Interfaces.

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Next week in Empower Tip #27: Using System Suitability to Flag Failing Results

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