How sample concentration can affect peak shape in OMNISEC data
There are many factors that can affect the quality of your OMNISEC GPC/SEC data. These include things like analysis methods, mobile phase, and column set, but can also include less obvious parameters such as concentration and injection volume.
I mention concentration and injection volume together because they both influence the mass of sample introduced into the system. A 100 µL injection of a 2 mg/mL sample loads the same amount of sample as a 50 µL injection of a 4 mg/mL sample. This is important to remember if you are trying to optimize analysis conditions, because you can use the same prepared sample and simply adjust the injection volume instead of preparing multiple samples at varying concentrations.
What is the ideal concentration for my sample?
Like many things with GPC/SEC, the answer is dependent on your sample and system. A general sample concentration range is 3-5 mg/mL; that’s what I aim for when I’m analyzing samples for the first time. If you have a column set with a lot of columns (more than three), then you might need to inject more sample than expected, since each column dilutes the amount of sample passing through the detectors at any given time. However, if you are analyzing a sample, you know has a very high molecular weight, then you will likely need to use a concentration lower than the 1-5 mg/mL suggested range.
You want to inject enough sample to obtain good detector signals but also make sure you don’t overload the column set, which can result in unusual chromatography. To me, an ideal sample concentration loads the minimum amount of sample required to obtain reliable responses in all detectors.
How can I tell if I’m overloading the column set?
That’s a great question! The most noticeable clue is the shape of your sample peak.
In the image below, I’ve included a multi-detector chromatogram of a sample displaying a sample peak with a shoulder. That by itself is not necessarily a reason for concern; that shape could accurately represent the sample’s molecular weight distribution.
However, if you expected your sample to possess a more Gaussian distribution, then you might suspect something isn’t quite right. The first thing I would do in this situation is inspect the magnitude of each peak, especially the refractive index (RI) signal (which directly corresponds to sample concentration).
In the example above, the RI peak height is greater than 400 mV. Like the peak shape, this by itself isn’t necessarily a problem. But it does indicate that there is room for the sample concentration (or injection volume) to be reduced without becoming undetectable. This holds for the other detectors, as well.
Therefore, to determine if the sample is overloading the column set, a dilution series of the sample was prepared to analyze the sample at lower concentrations and monitor any changes in the peak shape.
Dilution series
In addition to the original analysis, three analyses at lower concentrations were performed. The RI chromatograms of those injections are overlaid below.
As expected, the magnitude of the sample peak decreases with each dilution. And along with the first dilution is a change in the peak shape – the shoulder disappears! This suggests that the concentration of the first injection was too high for the column set, and once it was reduced, the sample could be properly separated, and the expected Gaussian distribution was observed.
Mark-Houwink plot
To confirm the sample’s structure wasn’t changing with the concentration, the Mark-Houwink plot of all four injections, shown below, was examined.
The majority of all the plots overlay nicely, indicating a consistent structure. The linearity of the plots is as expected for synthetic, linear polymers where the molecular weight (chain length) and molecular size increase at constant rates relative to each other.
The plot of the first injection with the shoulder, shown in red, presents an elbow in the lower molecular weight region, most likely a result of the compromised chromatography.
Ideal concentration
As I stated previously, I think the ideal concentration is the minimum concentration required to obtain reliable responses in all detectors. The multi-detector chromatogram of the last injection in the dilution series, shown below, provides strong signals for all detectors without any of the shoulder. This concentration is about one fourth that of the original concentration.
Final thoughts
In conclusion, I hope this examination of concentration and its effects on peak shape help you become a more proficient OMNISEC user. 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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