Separation science of macromolecules

In 1973, Bruno Vollmert wrote “The only completely satisfactory description of the molecular weight (i.e., the degree of polymerization) of a macromolecular compound is the distribution curve...as determined through fractionation” [1]. The accuracy of this statement stems, in large part, from two facts: First, that the molar mass (formerly known as molecular weight) distribution provides information on properties such as the elongation and tensile strength of materials. Second, that while the various molar mass averages (Mn, Mw, Mz, etc.) of a polymer provide important information on their own, it is quite possible for macromolecules with vastly different molar mass distributions to have the exact same molar mass averages and polydispersities. This is true of any statistical distribution and, as such, Vollmert’s statement applies equally well to the distribution of any macromolecular property, of which there are many: Long and short-chain branching, chemical composition, tactility, sequence length, polyelectrolytic charge, etc. Over the last half-century, size-exclusion chromatography (SEC) has established itself as the premier method for obtaining the molar mass distribution (MMD) of natural and synthetic polymers. Enhanced by a multiplicity of physicochemical detection methods, it can also be used to quantify many of the heterogeneities present in macromolecules and increase our knowledge of polymer architecture and dilute solution thermodynamics. The technique is not without its limitations, however. First, it should always be remembered that, by definition, SEC is a technique that separates analytes on the basis of their size in solution, not necessarily on the basis of analyte molar mass. A consequence of this is the phenomenon of local polydispersity, in which molecules which differ from each other in architecture, chemical composition, or both may co-elute from the SEC column because of the similar hydrodynamic volumes occupied by these molecules. Although it may be possible to address this problem by use of multiple physical detectors, this requires highly accurate band-broadening corrections and interdetector delay calculations and, quite often, for the co-eluting species to have specific refractive index increments that are extremely different from each other. Another approach to the problem of local polydispersity has been to circumvent it altogether. This is seen in the review by Gilbert who, rather than determining the MMD of heterogeneously branched polysaccharides for which local architectural polydispersity might be observed in size-based separations, instead examines the size distribution of the analytes to derive conclusions therefrom. Another limitation of SEC stems from the large shear rates to which macromolecules are exposed during their passage through the packed, porous medium of the chromatographic column. The ease with which large polymers may degrade under even the mildest of SEC conditions (employing ultra-low flow rates and columns packed with large-diameter particles) has encouraged the development and use of gentler, alternative size-based techniques. Chief among the latter is the family of methods known as field-flow fractionation (FFF) and, in particular, flow FFF, also known as A4F. As demonstrated by a multiplicity of papers in this special issue, A4F has found favor in the characterization of biopolymers, especially Published in the special issue on Separation Science of Macromolecules with Guest Editor Andre Striegel.