Limits & Baselines with OMNISEC: Part 2 – Automation!
Last month I wrote about limits and baselines, with some tips on where to manually place them when analyzing GPC/SEC data. And if you paid close attention, you might remember that I mentioned there is a way to automate the process in the OMNISEC software. Sounds convenient, doesn’t it? I’ll describe the options available and how to do so in the sections below.
General process
When setting up an analysis and working your way through the Calculation method window, shown below, you might have noticed the last section is called Peak detection (1). On this page, you have three options from the drop-down menu (2). The first option, No Detection – Manually Set Limits and Baseline, is what you should select when you plan to use the Find limits button or hold Shift while using the mouse to set limits and baselines.
The other two options, OmniSearch and Automatic Detection – Fixed BL (baselines) and Limits, allow limits and baselines to be added as soon as the corresponding method is applied to the data. In order to fully capitalize on this feature, it helps (but isn’t necessary) if the method has been set up and exists prior to data collection.
To have an analysis method automatically applied to an injection, you can select the appropriate Calculation method when setting up your sample sequence, as indicated by the arrow in the image below. To do so, click the Add Calculation Method field in the sequence and make the appropriate selection.
If you’ve done this, then the first time you open the acquired data a window will pop up confirming that you want to apply your method to the new data.
Once you click Ok, the software will apply the limits and baselines and calculate results for all available samples in the sequence. At that point, all you have to do is review the data!
Batch re-processing
If you don’t have your method with automatic limits and baselines already created before you run your first sequence, you can still create the method with your narrow standard and then apply it to the remaining samples in your sequence. To make this as easy as possible, you can use the batch re-processing option.
To start, open your sequence, select your narrow standard, set limits and baselines on the standard, and begin setting up your calculation method as you typically would. When you get to Peak detection, choose whichever option you prefer (more details on each below), then click Save. Since you have limits and baselines on your standard in place, you will receive the following message:
When you click Yes, the software will perform the calibration. Once complete, click the Save button in the main toolbar. Then click (and hold) and drag the mouse pointer to highlight the remaining, non-narrow standard samples, so that sequence looks similar to the one shown below on the left. At this point, you can choose your newly created method from the drop-down menu, as shown below on the right (even though it appears that the method is already selected).
At this point, you will receive a message like the one shown above in which the software informs you that the auto peak detection method will be applied to your selected samples. Once you click Ok, the analyses will occur, and then you can review your data.
OmniSearch
The first of the two automated limits and baselines methods, OmniSearch allows you to let the software determine the most appropriate limits and baselines for your samples. When you choose this option, you will see the following screen:
The Baseline Detection Parameters allow you to set the baseline order for each detector signal. The default is a second order fit for all, just like what the software provides when you click Find baselines (or Find limits). You also have the option to adjust the sensitivity for each detector or the choice to have the software automatically determine what will work best.
The Peak Qualification section contains the one field that has to be changed. You can leave the Start Detection at 0 mL, but the End Detection must be changed from 0.0001 mL otherwise you won’t capture any peaks (highlighted by arrow above). I recommend setting the End Detection to the retention volume of the solvent peaks to ensure the software is able to identify any/all of the sample peaks.
This section also lets you set minimum peak areas and identify the Master Channel the software will follow for recognizing peaks. I recommend leaving this on the default choice of Refractive index, unless you are using the UV-Vis PDA as the concentration detector, in which case you should use that.
If your sample has multiple components, you have the ability to adjust the settings for how their corresponding limits are set in the Peak Separation section. I encourage you to explore the drop down menus and see how the preview updates to get a feel for what is possible.
Automatic Detection – Fixed BL and Limits
The second of the two automated limits and baselines methods, Fixed BL and Limits, allows you to set the retention volumes at which you want your limits of integration and baseline points placed. These locations will be the same for all samples analyzed with this method. This type of method works well in QC environments where the same type of sample is analyzed repeatedly. It’s worth pointing out that if you have three different sample types, A, B, and C, you can create three different Fixed BL and Limits methods that correspond to each type of sample and assign the appropriate analysis method in the sequence.
When the Fixed BL and Limits option is selected, the adjustable parameters are presented as shown below.
There are two main items to specify in this method: the start and end retention volume of the integration limits (1) and the retention volumes of the first and last baseline points (2). In both cases, I do not recommend using any of the default values. You’ll want to make sure the Peak Start retention volume is early enough to capture the ascent of the light scattering response, which often rises from the baseline first. And you’ll want to ensure that the Peak End allows for the refractive index signal to return to baseline on the later eluting side of the peak.
As for the Baseline Start and End points, I suggest using the same points for all to keep it simple. However, if you are unable to locate a point in your chromatogram that provides a flat, stable baseline in all detector signals, you have the option to set each individually. The Baseline Styles are described at the bottom of the window. My preference is to use the Point to Point option, as both the Horizonal and Best Line run more of a risk of not being representative of the actual detector baseline.
Final thoughts
In conclusion, I hope this post helps you understand what options are available to make your analyses easier and more automated. As described in my previous post, limits and baselines are a critical part of every GPC/SEC analysis. With these automated options you can improve your consistency while simultaneously minimizing the time required. If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.
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