Macromolecular Separation Science—Trends and Developments: An Interview with André Striegel

Why did you feel the time was right to publish the textbook Macromolecular Separation Science,¹ and who is the book primarily aimed at?

Macromolecules, synthetic and natural polymers, are ubiquitous, in the objects we use and build and in the building blocks of our bodies. While complex polymers form the basis of increasingly important products (flexible electronics, micromotors, self-healing coatings), we also continue to discover fundamental roles and properties of proteins, peptides, and saccharides in processes underlying the living state. That is why the first sentence of the Introduction to the first chapter of the book is “We live in a macromolecular world and a macromolecular world lives inside us.” It is of paramount importance to accurately characterize these macromolecules, including the distributions of their various physicochemical parameters such as molar mass, branching, copolymeric ratio, and so on. The gateway to determining these distributions is separation science. However, there is an increasing (and worrying) dearth of experts in this area, especially in academia where future generations of experts can be trained. I wanted this book to help fill that educational gap. It is thus aimed equally at experts and at those just entering the field. Hopefully, whatever topic and level of depth a reader is seeking, she/he will find it here.

Who did you collaborate with on this project, and how long did it take from the initial idea to completion?

This was a “solo” project; I did not collaborate with anybody on it. I call it “My two-year project that took five years to complete!” I started it, with my supervisor’s approval, during the first summer of the pandemic (summer of 2020), figuring it would take about two years. However, as I delved deeper into the subject matter of each chapter, I kept adding material. I had everything to the publisher (Springer) by fall of 2024 and they had the first set of proofs to me by next spring. With a project this big (the final book is nearly 700 pages, including front and back matter), we had to go through a couple rounds of proofs. Thankfully, Springer was very responsive to all my comments. The final version appeared electronically last May and, in print, a month later.

What feedback have you received on the book so far?

I am happy to say that response has been very good (so far!). I think the book meets a need, and that is appreciated by the polymer separations community, and beyond. I’ve received very positive feedback from different parts of the world and from scientists in industry, government, and academia. As a result of your question, I looked it up and found that the various chapters of the book have already been accesses nearly 3000 times via the publisher’s website. It is reassuring to see that there does, indeed, seem to be something in the book for everyone.

Are there significant differences in analytical approaches for macromolecules compared with small molecules?

There are several. First, small-molecule separations usually focus on separating from each other as many peaks as possible. Peak capacity is a primary concern. Then, there is the issue of identifying all those peaks. Mass spectrometry is great (though not unique) at this, especially as a detection method; hence, all the liquid chromatography mass spectrometry (LC–MS) instruments in laboratories and LC–MS papers in journals.

By contrast, in macromolecular separations we deal with only a few peaks, sometimes only with one broad one. And, we oftentimes already know what our analyte is (we might have silos or train cars full of the stuff!). It is the physicochemical properties of this material that interest us: Its molar mass distribution (the MMD, which can often span several orders of magnitude) and how other properties, such as branching or copolymeric ratio, depend on molar mass. We are interested in these because of how they affect processing and end-use properties such as elongation, tensile strength, stress crack resistance, crystallinity, and so on. We thus use, in macromolecular separations, a host of on-line detectors, usually in series, such as multi-angle static light scattering (MALS), dynamic light scattering (DLS), differential refractometry, viscometry, UV/vis, and also detectors such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and more exotic ones such as Raman spectroscopy, small angle X-Ray scattering (SAXS), etc. We use these because of the information they can ultimately provide about the analyte. For its own sake we don’t care a whole lot, if at all, about how much a dilute polymer solution scatters light. But, by measuring this quantity, we can determine the molar mass of our macromolecule, which is of great interest. Likewise, for its own sake we don’t care much about how differently a macromolecule scatters light in solution at one angle versus another. But, measuring this angular dissymmetry provides us with a size metric for the polymer, the so-called radius of gyration. We can take this further, by employing multi-detector separations to see how this radius varies with molar mass, which can then inform our knowledge of macromolecular rigidity in solution, of branching status, of fractal dimension, etc.

So, yes, while the front-end hardware in macromolecular separations seems similar (and parts of it, e.g., pumps and autosamplers, are identical) to that used in small-molecule separations, the goals and, hence, the experiments, including separation methods and detectors, are usually quite different.

There is clearly a need to update techniques used in macromolecular analysis. What major trends have emerged in this field over the past ten years?

Hardware is always being tweaked by equipment manufacturers, as is column chemistry. I don’t think there are any completely new techniques which have appeared over the last decade. However, there is an increased realization that there is more to macromolecules than their MMD, other distributions of equal or, depending on the process or application, greater importance. These other distributions are usually determined employing some type of interaction polymer chromatography (IPC) technique, such as gradient polymer elution chromatography (GPEC) or temperature gradient interaction chromatography (TGIC). The big problem with IPC is that most interested parties, save for a few experts around the world, don’t know how to even begin developing a method. So, one major trend that has emerged, and to which I’ve hopefully been able to contribute, has been to improve our understanding of method development in the various IPC methods, so as to “democratize” these a bit more than is currently the case.

What role does LC play in macromolecule analysis?

The overwhelming majority of macromolecular separation methods are LC methods. This includes, most prominently, size-exclusion chromatography (SEC), which I address in the next question, but also techniques such as hydrodynamic chromatography and the aforementioned GPEC and TGIC. As was also mentioned earlier, LC hardware plays a central part in macromolecular separations. As will be mentioned later, so does an integral knowledge of LC fundamentals.

SEC/GPC remains central to macromolecular analysis. What major advances have been made in this technique in recent years?

There has been an increased realization, by some column manufacturers, of the need for silica-based packing particle chemistries that will minimize enthalpic interactions between the packing material and biopolymers and related biopharmaceutical products, when working in aqueous eluents. This minimization can ensure that analytes elute by something closer to a truly entropic size-exclusion mechanism, thus not compromising experimental accuracy.

We are also realizing that, when trying to understand what makes complex polymers and blends tick, molar mass is only one variable in the equation. Other variables may also be physical, such as branching or tacticity, or they may be chemical, such as comonomer content and copolymeric ratio. To determine these other variables, we usually need to employ one of the family of IPC methods. More important, however, is to determine the mutual, continuous interdependence of molar mass and one of these other parameters. For this, we need two-dimensional liquid chromatography (2D-LC) and here SEC has also proven itself crucial, usually as the second separation dimension. We are beginning to better grasp the need to understand macromolecular complexity, not simply as an academic exercise but for how it informs our knowledge of processing, end-use properties, and circularity of materials. Along with this realization, I hope, comes more fully embracing the power of macromolecular 2D-LC.

Is there renewed interest in field flow fractionation (FFF) in macromolecule analysis, and if so, why?

I don’t know if I’d use the word “renewed” so much as “increased.” We have long recognized some of the shortcomings of size-exclusion chromatography, especially with respect to characterizing ultra-high molar mass macromolecules and colloids. However, it is only in the last couple decades that FFF instruments, especially (though not exclusively) flow FFF instruments, have become available to the non-specialist. This, in conjunction with the coupling of FFF separations to on-line MALS and dynamic light scattering (DLS) detection, has permitted determination of the MMD and size(s) of ultra-large analytes and of the aggregation state of analytes under certain experimental conditions or as a function of time. Actually measuring these parameters is definitely far superior to trying to calculate them from first principles and from the assumption of “ideal” FFF behavior by the analyte and the system.

Also, while in the past equipment for other, non-flow, FFF methods, e.g., thermal or centrifugal, was
mostly home built, this equipment is now commercially available. As was originally the case with flow FFF, though, at present this equipment is being used only sparingly, by a very few groups of experts around the world.

By the way, while writing the book I did find that one noticeable exception to what I just mentioned regarding home-built equipment is dielectrophoretic (DEP) FFF, which has proven to be a very gentle technique for the separation of large particles, including cells. No commercial supplier for DEP-FFF channels seems to exist, so those wishing to perform experiments have taken to building their own channels.

One chapter focuses specifically on polyolefins. Why does this class of materials require special attention?

The answer to this lies partly in the sheer volumes of polyolefins produced annually worldwide, nearly 200 million metric tons. More specifically, though, polyolefins merited their own chapter because of the special and specialized techniques employed for their analysis. Because of solubility and precipitation concerns, high temperatures, sometimes as high as 200 ^oC, are needed for polyolefin analysis. As such, the instruments used are, essentially, big ovens, the individual compartments (pump, autosampler, column, detectors) of each needing to be maintained at these high temperatures both accurately and precisely. This type of special high-temperature (HT) equipment is generally unnecessary for analysing other types of macromolecules.

Similarly, while some polyolefin separations techniques, such as HT-SEC, are similar to their room-temperature counterparts, other techniques are particular to polyolefins. This is especially so of crystallization-based methods such as temperature rising elution fractionation, crystallization analysis fractionation, and crystallization elution fractionation. The place to explain how these techniques work and what type of insights they yield into the world of polyolefins was in a chapter of its own.

I think it important to note that this chapter ends with a discussion of how some of the high-temperature chromatographic techniques, HT-SEC in particular, can also be applied to the study of select non-polyolefin macromolecules. I included this because I believe there is great potential for the application of HT separation methods not just to polyolefins but to a host of other polymers, as well, so I wanted to show some examples thereof.

What advice would you give to those considering a career in macromolecular separations (both industry and academia)?

To those considering a career in industry, I would recommend making as many connections around the world as you can. Most likely, you will be the only subject-matter expert at your particular location, maybe even in your whole company. When you run into difficulties and need help, or when you just want to bounce ideas off someone, knowing people around the world at various institutions (companies, universities, government labs) to whom you can turn for advice will prove invaluable.

To those considering an academic career in macromolecular separations, I would advise picking your projects very carefully with respect to “fundability.” While some of the old axiom “publish or perish” remains, the current reality in academic circles, at least at research universities, is “bring in funding or perish.” Over the last few decades, funding bodies (at least in the US) have not been particularly friendly to separation scientists in general, even less so to those in macromolecular separations (hence my abovementioned “dearth” in this area; see also my LCGC Blog “Whence The Next Generation of Macromolecular Separations Scientists?” https://www.chromatographyonline.com/view/the-lcgc-blog-whence-the-next-generation-of-macromolecular-separations-scientists- ). Seek opportunities to speak to program managers at funding agencies to better understand their priorities and grant-grading rubrics. Understand also that, if you remain in academia, you will be chasing after funding for the next several decades. The rewards of teaching and doing research there, however, can be immense and immensely joyful. Try to set yourself up well with fundable projects, so that you can also have the freedom to work on other things which may appear to have a less immediate pecuniary reward. At the end of the day, your contribution to science won’t be how much grant money you brought in.

You recently received the Dal Nogare Award for your work in fundamental chromatography. Why is it important to continue that type of work?

It all comes down to fundamentals! If you don’t understand the basics of retention, band broadening, and resolution, inter alia, you aren’t going to get very far with troubleshooting, with method development, or with establishing useful relations between similarly disparate techniques or concepts. I would add that, for macromolecular separations, there is an added need to understand the fundamentals of polymer science, as well, and of biology, biochemistry, or carbohydrate chemistry if dealing with biopolymers.

I’ve often heard that we already know pretty much all we need to know regarding separations fundamentals. I would say that chromatography is a very large field and “pretty much,” even if true, still leaves plenty of room for discovery, learning, and improvement. Arguably, the most recognizable artwork on the Sistine Chapel ceiling is the Creation of Adam, yet this piece comprises a pretty much negligible area, only about 3 %, of the total ceiling area.

Anything else you would like to add?

Have fun! Science should be fun – it most certainly is for me. Don’t be afraid to try new things, no matter how crazy they may seem. Separation scientists are, mostly, experimentalists. If you are wondering about something, try to design an experiment to prove it. Equally, and probably even more importantly, try to think of how you would disprove your hypothesis. This last will be the most important type of experiment you can conceive.

Also, if you really think you understand something, try explaining it to a child. Having a child, in our case a son, has made me a much better scientist (hopefully, I’m a good dad, as well!). The old adage “If you can’t explain it, you don’t understand it” is most certainly true and children will be your best proving ground in this regard.

Reference
1. Striegel, A. M. Macromolecular Separation Science; Springer; 2025.