3 ways to analyze GPC/SEC data
Acquiring quality GPC/SEC data is only half the battle. Once the data is in hand, you need a way to turn those chromatograms into meaningful numerical values, such as molecular weight. There are multiple ways to do this, depending on which combination of detectors you have available. This post will discuss the three most common GPC/SEC data analysis methods (all available with OMNISEC!): conventional calibration, universal calibration, and multi-detection with light scattering.
For a video presentation that includes the following material (and more!) please see my Introduction to GPC/SEC in 30 minutes video.
Conventional calibration
The simplest GPC/SEC configuration is a single-detector system, using a pump, column set, and either a refractive index (RI) or UV detector. One of these detectors is chosen because they respond directly to the sample’s concentration, which is important for calculating molecular weight moments, such as Mw and Mn. To obtain molecular weight values from a conventional calibration (sometimes referred to as column calibration), a series of well-characterized standards must be run. These standards are used to generate a calibration curve that maps out which molecular weight (peak molecular weight or Mp) corresponds to each retention volume, as illustrated in the figure below.
When an unknown sample is analyzed, the molecular weight of each data slice is determined based on its retention volume and the corresponding molecular weight on the calibration curve in use. Since the molecular weight values obtained using conventional calibration are dependent on the standards used to generate the calibration curve, the results for a sample are described as relative molecular weight values.
The advantage of conventional calibration is that it’s the most economical GPC/SEC setup, featuring the fewest components and simplest calculations. The disadvantage is that the molecular weight values obtained might not accurately reflect your samples if the molecular structure, and thus shape and size, of your samples is different from that of your standards. And with a single detector system the calculated data might not represent the real features of your sample and may even misdirect your research. In other words, you might be in a situation where you don’t know what you don’t know!
See conventional calibration in action with OMNISEC!
Universal calibration
The addition of a viscometer detector to a concentration detector (most often an RI detector) allows for a universal calibration curve to be applied to an unknown sample. In addition to molecular weight moments, other molecular parameters such as intrinsic viscosity (IV), hydrodynamic radius (Rh), and Mark-Houwink parameters can be determined.
The viscometer detector’s response is affected by the sample’s molecular structure; more specifically its shape in solution. Therefore, the viscometer’s presence accounts for structural differences between the standards used to generate the calibration curve and the samples under analysis, ultimately providing more accurate data than that obtained from conventional calibration.
In 1967, Benoit et. al. used the relationship between hydrodynamic volume, molecular weight, and intrinsic viscosity (above equation) to show that that the calibration curve for polymers of different types can merge into a single calibration curve. Remember that GPC/SEC separates based on molecular size, or hydrodynamic volume. Therefore, if we think of elution volume as essentially being hydrodynamic volume, we can plot molecular weight times intrinsic viscosity against hydrodynamic volume. This means that all polymers, regardless of structure and shape, will fall along the same, universal, calibration curve, as shown in the image below.
The advantage of universal calibration is that you can obtain accurate molecular weight values for your samples regardless of the standards you have available. Additionally, the inclusion of a viscometer detector offers more characterization data, including IV, Rh, and Mark-Houwink parameters.
However, even though the identity of the standards doesn’t matter, a calibration curve is still required and therefore every GPC/SEC system will have a unique calibration curve. Parameters such as columns, mobile phase, flow rate, and temperature will still affect the resulting calibration curve.
See universal calibration in action with OMNISEC!
Multi-detector analysis with light scattering
Sometimes called Triple Detection, Advanced Detection, SEC-MALS, among others, this method utilizes a light scattering detector in combination with a concentration detector, at a minimum, to calculate absolute molecular weight. This works because the intensity of scattered light is related to the molecular weight of a sample. Viscometer and/or UV-Vis detectors are frequently added to create a powerful multi-detector analytical tool.
As described in detail in this post on multi-detector GPC/SEC, each detector responds to a different aspect of the sample, which allows for the calculation of absolute molecular weight (accurate and not dependent on a calibration curve), IV, Rh, Rg, Mark-Houwink parameters, concentration, and potentially branching and compositional analysis data. The equations governing each detector’s response are shown below.
In addition to providing absolute molecular weight, multi-detection with a light scattering detector is advantageous because it does not rely on a calibration curve. The simple calibration process involves running a single narrow standard, as often as you see fit. We recommend checking the calibration with a verification standard…but that’s it!
Once you have a calibrated method, calculating data for your samples is easy – check it out with OMNISEC in the video below!
Final thoughts
In conclusion, I hope this post helps you differentiate between the three common analysis methods available for GPC/SEC data. There are advantages to each of them, but the accuracy and convenience of multi-detection with light scattering sets it apart from the others as the ideal method for analyzing GPC/SEC data. If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.
Related content
- GPC/SEC standards: How often do I need to run them?
- Easy ways to create custom figures from GPC/SEC data
- A picture is worth 1000 words: creating great GPC/SEC figures
- Malvern Panalytical’s GPC/SEC Expert Advice YouTube playlist
- Color inside the lines: informative shading between the limits in the OMNISEC software