Sizing Up Size-Exclusion Chromatography

André Striegel spoke to LCGC Europe about innovations in size-exclusion chromatography (SEC) in polymer analysis, including the benefits of hyphenating SEC with on-line multi-angle static light scattering (MALS) and differential refractometry (DRI) detection, the non-trivial nature of a “trivial” solution, the complementary value of “interaction” polymer liquid chromatography (LC) techniques, and the role of quintuple detection in practice.

Q. What are the benefits of using SEC–MALS–DRI in polymer analysis and where is this technique being used?A: Size-exclusion chromatography (SEC) with on-line multi-angle static light scattering (MALS) and differential refractometry (DRI) detection is a very powerful technique. The most common application is for obtaining the “absolute” molar mass (M) averages and molar mass distributions (MMDs) of polymers, which is more accurate than reporting values that are relative to a calibration standard with little architectural and/or chemical relation to your analyte.

SEC–MALS–DRI can also be used to glean information about macromolecular architecture, including polymer topology to assess whether a polymer is linear or branched; dilute solution conformation to confirm whether a linear polymer adopts a “rod-like” or “random-coil-like” conformation at a given set of solvent and temperature conditions; and dilute solution thermodynamics to identify if a dilute polymer solution is at good, poor, or theta conditions.

SEC–MALS–DRI can also be used to measure the size distribution of macromolecules and the statistical averages of this distribution, and a host of other polymeric and dilute solution properties.

Q. What are the limitations of SEC–MALS–DRI?

A: First, it is important to remember that, because separation in SEC is based on the size of the analytes in solution (their so-called hydrodynamic volume) (1), analytes with different architectures and chemistries can coelute if they occupy the same hydrodynamic volume as each other. I will talk about how to deal with this in more detail when I discuss interaction polymer chromatography.

Second, and again because SEC is a solution-phase technique, the solvent (or carrier liquid, in the case of colloidal suspensions) must be compatible with the SEC column packing material and should not cause appreciable (because there will always be some) chemical or, more appropriately, enthalpic interactions between the analyte and the column packing.

The solvent should also allow for analyte solutions to have good optical contrast with the neat solvent, so that the detectors can generate peaks with adequate signal-to-noise ratios (S/N).

Another limitation of the technique is that most non-destructive chromatographic detectors, including refractometers, light scattering photometers, and viscometers, are differential in nature, that is, they measure the response difference between the analyte solution and the solvent. Non-differential detectors are usually both destructive and display a non-linear, empirically-governed response.

For example, a differential refractometer measures the difference between the refractive index of the solution being analyzed and that of the solvent used to prepare the solution. This tends to preclude the use of mixed solvents for accurate determination of molar mass averages and distributions, inter alia, not only in SEC but also in any macromolecular liquid chromatography (LC) or fractionation method. Let me elaborate on this. When using mixed solvents, we encounter what is known as the preferential solvation problem: the polymer prefers to be solvated more by one of the solvents in the mix compared to the other solvent. For example, if the polymer is dissolved in a 50:50 mix of solvents A and B, within the hydrodynamic volume of the polymer the solvent ratio may be 80:20, or 35:65, or, basically, something not 50:50.

Given the aforementioned differential nature of most macromolecular LC detectors, in the case of preferential solvation the solvent baseline will no longer accurately represent the solvent mixture’s contribution to the detectors’ analyte peaks. This will result in the erroneous calculation of many parameters, most notably in erroneous molar mass averages and distributions.

Q. You attempted to combat the preferential solvation problem mentioned previously using an isorefractive solvent pair as a mixed solvent (2). Can you explain the concept behind this approach, the challenges you encountered, and how you overcame them?

A: Well, with an SEC–MALS–DRI approach one has two detectors, the response of which depends on the refractive index n₀ of the solvent (for the DRI it’s a first-power dependence on n₀, whereas for MALS it’s a squared dependence). Consequently, if one can find an isorefractive solvent pair, that is, a pair of solvents with the same refractive index as each other, then the problem of preferential solvation is obviated. In this case, for our generic polymer dissolved in a 50:50 mix of generic solvents A and B, it won’t matter if, within the hydrodynamic volume of the polymer in solution, the solvent ratio is or isn’t 50:50: In either case, the refractive index of the solvent mix within the hydrodynamic volume of the polymer will be the same as that outside this volume. This means that the solvent baseline now represents an accurate contribution of the solvent mix to both the MALS and DRI detector peaks. I refer to this as the “trivial” solution to the problem.

As to the challenges behind this seemingly trivial approach and how we went about overcoming them, first one must find a pair of solvents isorefractive at a particular temperature and wavelength (remember that, in most tabulations, refractive indices are usually given as n_D²⁰ values, meaning that they have been determined at 20 ºC at a wavelength of 589.29 nm corresponding the average wavelength of the sodium D-line doublet). Unfortunately, in the case of wavelength, we are pretty much limited to that of the light sources in the MALS and DRI detectors (which, it should be mentioned, if not identical should match each other pretty closely to avoid introducing an additional source of error). There are tables of isorefractive solvents in the literature, but it must be kept in mind that isorefractivity does not imply miscibility. So, we first had to find solvent pairs with fairly similar n_D²⁰ values, for which we used the published tables. Second, we minimized the difference between the refractive indices of the two solvents by slowly changing the temperature, in 1 ºC increments, until the refractive index difference was less than 0.001; this took advantage of the fact that our differential refractometer also has the ability to measure absolute refractive indices. Third, we had to make sure the two solvents were miscible with one another in all proportions, for which we initially relied on both published solvent miscibility charts and our own experience when making choices, followed by miscibility experiments in the laboratory at various solvent ratios. Fourth, we had to make sure that the individual solvents, as well as mixes thereof, would actually dissolve our intended analytes. For the latter, we chose polystyrene (PS) and poly(methyl methacrylate) (PMMA) because there are well-characterized narrow‑MMD standards for both of these spanning a wide molar mass range.

 Our particular experiments covered a fourfold range in M, and we could do a “quick-and-dirty” evaluation of solubility using an ultrahigh M sample of each. If a standard with M > 106 g mol-1 dissolved, it would be natural to assume that samples with M lower than this would also dissolve. This assumption was turned on its head by one of our supposedly atactic PMMA samples, which led to our discovery that it contained a significant percentage of isotactic triads. That is a story for another day, however!

We also had to choose a column packing material that did not interact chemically with the polymer solutions, which ensures elution by a predominantly entropically-controlled, size-exclusion-based mechanism. Lastly, we needed solvents with a refractive index different enough from that of the polymer so that the dilute solutions would show good optical contrast with respect to the solvents to generate large S/N MALS and DRI peaks. As you can see, the challenge really was anything but “trivial”!

Q. Are you planning to explore the use of other isorefractive solvent pairs, or develop this research further in any other way?

A: We will probably leave more applied exploration of isorefractive pairs to those more heavily involved than we are in interactive polymer LC. At the moment, my focus in this area in on trying to solve the “non-trivial” problem, that is, on how to obtain accurate M averages and MMDs by SEC when using a mix of non-isorefractive solvents. We are just starting to scratch the surface of this problem. All I can tell you right now is that calling it “non-trivial” is definitely appropriate!

Q. What are “interaction” polymer LC techniques and how do they complement a more popular technique such as SEC?

A: Interaction polymer LC refers to techniques that separate predominantly on the basis of an enthalpic difference (chemical interaction) between the solution and the stationary phase (as opposed to SEC and hydrodynamic chromatography (HDC), where the separation is based on entropic differences). This includes methods such as gradient polymer elution chromatography (GPEC)-which some people have recently taken to calling solvent gradient interaction chromatography (SGIC)-and temperature gradient interaction chromatography (TGIC), among others.

The main advantage of these techniques is their ability to separate polymers based on chemical differences. As such, they are most advantageous for separating the different chemistries present in a macromolecular sample (for example, separating the methyl methacrylate from the styrenic component in a PS-PMMA copolymer or blend). One can find wonderful applications of these techniques in the literature (3). One major challenge to their widespread use, however, has been the difficulty in laying out a first‑principles-based, non-empirical approach to method development. This is another area in which we are working here at the National Institute of Standards and Technology (NIST).

Interaction LC methods can truly complement size-based separations such as SEC, HDC, and flow field-flow fractionation (flow FFF) for the so-called comprehensive characterization of complex samples, which include most polymers either employed by, or produced by, industry. One can separate the components of a sample based on its chemical constituents by interaction LC in the first dimension of a two‑dimensional separation (2D-LC), then separate by size utilizing, for example, SEC in the second dimension. Employing both physical (MALS) and chemical (IR, NMR) detectors, this 2D-LC experiment will map the physicochemical phase‑space of the material, showing the co-dependence of polymer chemistry on molar mass, both as continuous functions of each other. This is a very powerful approach to understanding and solving macromolecular problems, though still a challenging one from a method-development standpoint

Q. You also developed a method to characterize copolymers and blends by “quintuple detector” SEC (4). What detectors did you incorporate and why did you embark on this research?

A: This method included on-line MALS, quasi-elastic light scattering (QELS, also sometimes referred to as dynamic LS), viscometry (VISC), DRI, and UV detection. Each detector brings its own individual advantages into the mix-and its own challenges. Equally, or more importantly, is how the detectors can combine with each other synergistically to better inform our understanding of complex polymers and of blends. What this particular SEC–MALS–QELS–VISC–DRI–UV set-up allowed us to do, when studying a random copolymer of acrylamide and N,N-dimethylacrylamide as well as blends of the individual homopolymers, was to see how both the monomeric ratio and the polymer architecture changed continuously across the MMD, providing a window into the underlying chemical causes behind the architectural changes. One reason for embarking upon this research was to more fully apply our available suite of detectors to obtain as comprehensive as possible (within the framework of the instrumentation in our laboratory at the time) a macromolecular-level picture of a polymer, so that others could later take our approach and apply it to their analytes.

Q. What were the main analytical challenges you overcame developing quintuple detection?

A: Part of the challenge involved trying to find the proper solvent conditions to minimize the polyelectrolytic character of the solutions, which can influence both the separation as well as the data quantitation. We then had to make sure that, for each chromatographic slice eluting from our SEC columns, all five different detectors “spoke to each other” and did so for the same slice. Lastly, after quantitating the chemical changes across the MMD as well as the architectural changes, we had to try and discern the causal relationship between the former and the latter. The project thus involved, at a fairly deep level, both analytical chemistry and polymer science, which has been a hallmark of most of my career. Fortunately, I had in my group at the time (this was a project that began during my time in academia, and finished at NIST) a brilliant graduate student, Steven Rowland, now at the National Renewable Energy Laboratory In Colorado, who was not only up to the challenge but also took this project way beyond what I had originally foreseen. In the end, I’m certain I learned more from Steven on this project than he did from me!

Q. Can you illustrate the benefits of quintuple detection and how it benefits polymer analysts in practice?

A: The type of chemical and physical information obtained by this approach can be used to better understand both processing and end-use properties of the materials examined. For example, chemical heterogeneity is directly related to properties such as conductivity, adsorptivity, and interfacial strength, while molar mass and architecture can affect elongation, tensile strength, encapsulation ability, and diffusion, among many other properties.

So, as I mentioned previously, one of our prime motivators for embarking upon this research was to provide others in the field with a method, or an example of a method, that would give them a deeper, more comprehensive understanding of complex polymers. We have recently seen the same quintuple-detector approach we employed with SEC used with FFF-also to study complex polymers (5)-so our work does already appear to be helping polymer analysts gain a better understanding of different types of macromolecules.

Q. Do you have any general comments on the evolution of SEC? Are there any recent developments that you find particularly innovative or new application areas where it is being more commonly used?

A: At 55 (6), SEC is decidedly a “middle-aged’ technique. Back in 1972, after SEC had been around for only eight years, Gus Ouano wrote that “With the introduction of gel‑permeation chromatography [size‑exclusion chromatography] (GPC) by Moore, molecular weight distribution data for polymers took a sudden turn from near nonexistence to ready availability” (7)-such was the immediate impact of the technique. In those days, and for a couple decades thereafter, most molar mass information was calibrant‑relative and, thus, had to be taken with more than a hint of caution. With the introduction of on-line MALS and viscometry in the 1980s and their popularization in the 1990s, the ability to determine absolute molar masses was realized (in the case of viscometry, by applying Benoit’s universal calibration principle [8]). Since then, most practitioners have fully embraced or, at least, understand the need for multiple detection, especially for the combination of physical and chemical detectors within a single analysis. Nowadays, I think the most exciting developments have been in incorporating seemingly unusual analytical techniques as SEC detection methods (for example, small-angle X-ray scattering, dynamic surface tension, depolarized light scattering), thus expanding our characterization toolbox, and in the integral role SEC plays in most 2D-LC macromolecular separations. While the abundance of unreliable SEC molar mass data in the literature is alarming, I am heartened by the growing realization among polymer scientists that this needs to change and that the twin pillars of analytical chemistry, precision and accuracy, form the foundation upon which our materials knowledge can build and flourish.

References

1. A.M. Striegel, W.W. Yau, J.J. Kirkland, and D.D. Bly, Modern Size-Exclusion Liquid Chromatography, 2nd ed., (Wiley, 2009).
2. A.M. Striegel and P. Sinha, Anal. Chim. Acta1053, 186–195 (2019).
3. H. Pasch and B. Trathnigg, Multidimensional HPLC of Polymers (Springer-Verlag, 2013).
4. S.M. Rowland and A.M. Striegel, Anal. Chem. 84, 4812–4820 (2012).
5. U.L. Muza and H. Pasch, Anal. Chem. (in press). DOI: 10.1021/acs.analchem.9b01384
6. J.C. Moore, J. Polym. Sci. A2, 835–843 (1964).
7. A.C. Ouano, J. Polym. Sci. A-110, 2169–2180 (1972).
8. Z. Grubisic, P. Rempp, and H. Benoit, J. Polym. Sci. C Polym. Lett.5, 753–759 (1967).

André Striegel is a Research Chemist in the Chemical Sciences Division of the Material Measurement Laboratory. He received his Ph.D. in analytical chemistry in 1996 and his B.S. in chemistry in 1991, both from the University of New Orleans. From 1996 to 1998 he performed postdoctoral research for the U.S. Department of Agriculture, at the National Center for Agricultural Utilization Research. For the next six years he worked for Solutia (now Eastman Chemical), achieving the rank of Research Specialist.

From 2004 to 2011 he was assistant professor of both analytical and materials chemistry in the Department of Chemistry and Biochemistry at Florida State University (FSU). In September 2011 he joined the National Institute of Standards & Technology (NIST), where he is a Research Chemist in the Chemical Sciences Division of the Material Measurement Laboratory. His research interests are in the area of polymer characterization, in particular applying separation science to determining structure-property relations of complex macromolecules, and in the fundamental aspects of separation and detection methods.