Look again: 3 examples of quality GPC/SEC data
If you’ve worked with GPC/SEC data, then you’ve likely seen your share of odd-looking chromatograms. At times, they can be confusing, but that’s what keeps it fun! Imagine if every sample produced data that looked like a standard. Sure, it would be easy, but it wouldn’t be very interesting!
The good news is that quality data doesn’t have to look like a perfect peak. And in fact, when it doesn’t, or if there are other features in the chromatogram, the information available from a multi-detector GPC/SEC (like OMNISEC!) can tell you quite a bit about your sample. In this post I’ll highlight three examples of this type of data and discuss why each looks good.
Example data 1
At first glance, it might seem like something is missing from this chromatogram. There is a large light scattering peak from 4-6 mL but no corresponding peak in the RI or UV detectors. But upon closer inspection, it is clear the later eluting peak between 8-9 mL is the main sample peak. All three detectors respond well to this peak, and their signals smoothly ascend and return to the baseline.
As for the other features of the chromatogram: the unaccompanied light scattering peak from 4-6 mL is likely responding to a small concentration (no RI or UV) of a large (eluting early) and high molecular weight material (strong light scattering response, even at low concentration). This combination is common in aggregates. The good news is that it is well-resolved and will not affect the calculated data.
The strong RI peak between 11-12 mL is a result of the sample preparation solvent being different than the mobile phase. Just like with the aggregate, the resolution between the main sample peak and the solvent peak means the calculated results will remain unaffected.
Example data 2
There’s a lot going on in this chromatogram! But if we break down the data and look at each feature individually things will start to make more sense.
First, let’s identify the main sample peak. As in the previous example, the peak of interest should produce good responses in all detectors. Therefore, the peak between 20-30 mL is the main sample. Its shape is fairly Gaussian, as well, making it a bit easier to recognize in this example. And even though the remainder of the chromatogram is busy, the sample peak is well-resolved, rising from and descending to stable baselines. Even amidst the other features of this chromatogram, placing limits around this sample peak will be easy!
Second, the other features present in the chromatogram offer information on the analysis conditions. The negative peak in the viscometer between 0-15 mL doesn’t even belong to this sample. In fact, it is the inverse delay peak from the previous sample! The fact that it is eluting here simply means the run time is not long enough for it to elute during its own chromatogram.
Third, the negative RI peak from 34-36 mL is the solvent peak. In this case the preparation solvent has a lower refractive index than the mobile phase. This is often the case when samples are prepared in plain water and analyzed in an aqueous buffer containing salt (which raises the solution’s refractive index).
Example data 3
Here’s another example of a great chromatogram. There are not many features to distract from the main sample peak between 14-20 mL; the viscometer inverse delay peak is situated as expected at the end of the run.
However, the peak shape is unusual. There are clearly multiple species present within this distribution – but that’s ok! The fact that the peak smoothly rises from and descends to baselines that are clean and stable suggests that the interesting peak shape is real and representative of the sample. That the sample presents itself as multiple peaks indicates the variance of molecular size (and potentially molecular weight) of each component. Maybe with additional columns the resolution can be optimized to provide further separation of each population.
Final thoughts
In conclusion, I hope this post helps you understand the process of assessing GPC/SEC data to determine its quality and encourages you to be undeterred by features of the chromatogram that are not related to the sample. None of the three examples here look like a typical GPC/SEC sample peak, but after close inspection it’s clear they all look pretty good. After all, beauty is in the eye of the beholder! If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.
Related content