Limits & Baselines with OMNISEC: Part 1
When teaching new users how to use their OMNISEC system and the corresponding OMNISEC software, a frequent question I receive is “how do I know where to set my limits and baselines?” While it might sound simple, this is an important topic, as the placement of limits and baselines have a direct effect on a sample’s calculated data. In this post I’ll discuss why you should pay attention when setting limits and baselines and highlight some of the different ways to do so in the OMNISEC software.
What are limits and baselines?
Limits and baselines allow you to define the detector response that corresponds to your sample. One set of limits consists of two individual limits that bracket a sample peak. The limits in the image above are the blue vertical lines with the flags at the top. The [1] indicates that pair of limits corresponds to peak 1.
A baseline is set to indicate where the detector signal would have been if the sample peak had not eluted. At least two points are used to define a baseline. The square icons just before 7 mL and 29 mL in the image above are the points used to define the baseline for each detector.
Why are limits and baselines important?
Limits and baselines matter because they define the peak area the software will use to calculate data for your sample. You can have beautiful data but if your limits and baselines don’t accurately capture it, the results will not be reliable.
How do I set limits and baselines?
The OMNISEC System User Guide (section 4.3) and Interactive Help are excellent resources for detailed instructions on the different options available to define limits and baselines. Both are conveniently accessible from the Documentation section on the Start Page of the OMNISEC software, as shown in the image below. I encourage you to explore the Interactive Help, as it makes searching for information easy and groups related topics (and also includes videos such as this one).
Where should I set my limits?
To analyze the entire sample peak, the limits should be placed so that they encompass the entire peak without clipping its edges on any detector. Since the detectors respond to different characteristics of the sample, they do not all rise and return to baseline at the same retention volumes.
In the image at the top of the post, the left limit is placed close to the light scattering signals’ ascension. The light scattering detector responds most strongly to high molecular weight material and therefore is often the first detector signal to rise from the baseline. The right limit is set where the refractive index response returns to baseline. Since the refractive index detector responds to concentration, even a small amount of low molecular weight material will generate a response even though the other detector signals have returned to baseline.
A handy feature of the OMNISEC software is the shading between the limits. The different colors correspond to usable signals in different detector combinations, indicating various results can be calculated. A light blue shading represents the area between the limits; I recommend making sure your limits are set in such a way that you see this light blue shading between any other shading and the limits themselves. This way, you can ensure you are using all of the peak area that can offer calculated results.
If you use the Find limits function in the software, I recommend reviewing the limits to check that they are appropriate for your peak(s) of interest.
What if I have multiple peaks?
If there are multiple components within your sample and you want to analyze each, then simply place a set of limits around each component. If there is not complete resolution down to the baseline between two peaks, I recommend splitting the peaks at the minimum of the refractive index response. Even if you have a lot of oligomers you want to analyze, that’s ok! There are seven defined peaks in the image below and OMNISEC calculated data for all of them.
Where should I set my baselines?
If you are setting your baselines manually, I recommend using areas of the chromatogram that offer a flat baseline in all detector responses. In the image at the top of this post, those points are before the peak at just before 7 mL and then after the solvent peak and viscometer inverse peak at around 29 mL. You don’t need to spend time trying to click a point on an actual detector baseline; the baseline points will snap down to the baseline.
What’s the best way to set limits and baselines?
In my opinion, as long as the limits and baselines accurately indicate your sample peak, then you should use whichever strategy works for you and your samples and allows you to be consistent from sample to sample. If you’re seeking a recommendation, then I suggest clicking the Find limits button to allow the software to place the limits and baselines, and then modify the limits if necessary. This way your baselines will be set the same way each time and your limits and can be easily adjusted.
Is there a way to automate setting limits and baselines?
I’m glad you asked! With the current and future versions of the OMNISEC software, the answer is yes! I’ll describe the available options and how to use them in a limits and baselines part 2 follow-up post, so stay tuned!
Final thoughts
In conclusion, I hope this offers a deeper understanding of limits and baselines. They are an important part of GPC/SEC data analysis, so knowing how to use them correctly will improve the quality of your data. 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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