Multi-detector GPC/SEC responses to a series of standards
Multi-detector GPC/SEC data can be complex! With two, three, or more detector responses to consider, understanding the collected data can be a challenge. Fortunately, each detector shares part of the characterization puzzle, and when combined, a few conclusions can be made before you even set up the calculations!
To illustrate this, I’m going to present some examples of a series of eleven polystyrene standards analyzed with a multi-detector OMNISEC system in THF, with molecular weights ranging from 1200 Da up to 4,200,000 Da. We’ll look at the refractive index (RI), right angle and low angle light scattering (RALS & LALS), and viscometer signals, with an emphasis on the RI and light scattering responses.
Refractive index response
The RI overlay of the series of standards is shown below. The various molecular weight samples are separated nicely, with good resolution between the peaks. It should be noted that these standards were run individually to maximize clarity. Remember, separation is based on molecular size and larger molecules elute first!
The feature of this data that draws my attention is the decreasing peak size with increasing molecular size, or as you move from right to left. I specifically wrote “size” and not “height” because the third peak from the right, shown in green, possesses the highest peak, but it’s the peak in red on the far right representing the smallest, lowest molecular weight standard that presents the largest peak area.
The RI detector responds directly to a sample’s concentration; therefore, we can quickly conclude: the concentration of the lowest molecular weight standard is highest, and the concentrations then decrease with increasing molecular weight. As the standards get larger, their concentration is lower.
When we look at the concentrations of each standard, we find that assertion is correct:
Light scattering responses
We’ll look at the right angle and low angle light scattering detector responses individually first, and then look at them together in a comparison with the RI response.
Right angle light scattering
If we view the right angle light scattering response of the polystyrene series we see the opposite trend. The largest standard with the highest molecular weight produces the strongest detector response. The peak intensities then trend downward as the standards decrease in size and molecular weight.
The right angle light scattering response trends with the standard’s molecular weight. This is because higher molecular weight materials scatter more light. The right angle detector is best for observing the intensity of the smaller standards, but above a certain size threshold, angular dependence and interference occurs, obscuring the scattered light detected for the largest standards. To avoid this, it’s best to observe the intensity of scattered light as close to the zero angle as possible. For more details, please see my previous post on the topic.
Low angle light scattering
To ensure the scattered light is observed as accurately as possible, OMNISEC pairs a low angle detector with a right angle detector. A look at the low angle light scattering detector responses for the series of standards shows that the observed signal is even more biased toward the highest molecular weight standards.
This illustrates how the low angle detector is critical for accurately measuring the intensity of scattered light of high molecular weight materials and thus correctly calculating their molecular weight.
Light scattering vs. refractive index
I wanted to use the standards on the extremes of the series to highlight the difference between RI and light scattering detector responses. A light scattering detector responds most strongly to a sample’s molecular weight, even more than concentration. This data series highlights that fact because the standard with the lowest concentration, the highest molecular weight polystyrene, provides the strongest light scattering responses.
The two following figures show the RI and light scattering responses for the highest molecular weight standard and the lower molecular weight standard, respectively.
In the image above, the highest molecular weight polystyrene standard provides a good RI response to go along with the strong light scattering response (similar to the profile of an aggregate). In contrast, in the image below, the light scattering response of the lowest molecular weight polystyrene is almost imperceptible, and pales in comparison to its RI response. Not only does this highlight what molecular features RI and light scattering detectors respond to, but it demonstrates how obtaining reliable light scattering data for low molecular weight materials can be a challenge.
Viscometer response
The viscometer responses for the series of polystyrene standards behave like a less-intense version of the right angle light scattering signals. The strength of a viscometer response is based on how much the sample increases the solution viscosity compared to the mobile phase. Therefore, higher molecular weight samples comprised of longer polymer chains have the most significant effect.
Viewing the viscometer signals of a series like this illustrates how the delay peak is an inverse of the sample’s peak that elutes at a consistent volume after the sample.
Final thoughts
I hope that viewing the series of polystyrene standards one detector at a time has helped you understand the differences in each detector’s response. 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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