Electrokinetic Separations Synthetic Polymers for
Wim Th. Kok Remco Stol Robert Tijssen University of Amsterdam (The Netherlands)
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In terms of speed and sample throughput, CE-like methods have an enormous advantage over size-exclusion chromatography for large numbers of samples.
E has matured over the past decade into a separation technique that is now routinely used in pharmaceutical, clinical, and biochemical analyses. The inherent high efficiency of the technique is easily exploited in full, with plate numbers in practice at or close to fundamental limits set by the diffusive dispersion of the analyte zones during separation. It’s easy to predict that CE performance will be especially high for separating large molecules, since the diffusion rate is related to molecular size. In their 1981 paper marking the development of modern CE, Jorgenson and Lukacs already recognized that “the possibilities for high-resolution separations of macromolecules will be quite promising” (1). These promises have been amply fulfilled. CE is important in protein analysis and, with the introduction of high-speed and full automation, is now a serious alternative to slab gel electrophoresis in DNA sequencing. Several separation techniques originally developed in slab gel format have been transferred to capillary systems, such as sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. So far, little attention has been paid to applying these techniques for synthetic polymer separations. There are several reasons why polymer chemists have failed to exploit the techniques. First, solvent compatibility is a problem. CE remains mostly an aqueous technique, thereby limiting its application to water-soluble polymers. An early application of CE to synthetic polymers involved separating water-soluble poly(amino acids) with a gel-filled capillary (2); use of CE separation was feasible because of similarities between these compounds and natural poly(amino acids)—peptides and pro-
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teins. Experience with organic solvents in CE so far has been limited. In micellar electrokinetic capillary chromatography (MEKC), mixtures of water and an organic modifier are sometimes used as the separation medium, thereby extending the analyte hydrophobicity range that can be separated. Occasionally, nonaqueous solvents are used to obtain a different selectivity than occurs in water, via solvent effects on the acid–base properties of the analytes (3) or by special interaction effects with constituents of the separation medium (4). New CE techniques may be required for separating waterinsoluble polymers, which constitute a significant portion of the synthetic polymer market. Interest in CE for polymer separation also suffers because polymer chemists may have gotten used to the low separation efficiency of traditional size-exclusion chromatography (SEC). A complete separation of polymer molecules differing by one or a few monomer units, as is required in DNA analysis, is usually not sought. Absent an alternative, scientists are often satisfied when the analysis method yields a few characteristic data on a sample, such as average molecular mass and polydispersity. Investment would be required in method development to obtain the superior data that can result from an electrokinetic separation technique. This endeavor is certainly not straightforward for industrial polymer chemists. A third explanation may be that, in many cases, the polymer analysis customer is not interested in the accuracy of
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temperature-rise elution fractionation (7 ) or critical chromatography (8). CE also provides interesting possibilities for polymer separation. Electrokinetic methods have been developed based on differences in ionization, degree of interaction with solvent constituents, and molecular size and conformation.
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Diamine oligomers
Differential migration
Monoamine oligomers
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10 12 Time (min)
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FIGURE 1. CE separation of monoamine oligomer byproducts of PEO diamine polymer (average molecular mass 1500). (Adapted with permission from Ref. 9.)
analysis results, but rather in their precision and reproducibility. A molecular weight (or better mass) distribution of a synthetic polymer may be determined as a control on the production process or to explain differences in physical properties between lots of different origin. For example, small differences in the average molecular mass have to be detected from changes in elution or migration times. Especially during the early stages of CE, migration time repeatability and reproducibility were a weakness. Reproducibility has improved over the years with the practical experience obtained using CE and the development of sophisticated instrumentation. Still, CE cannot compete with SEC performance in this respect. Despite arguments against the use of electrokinetic separation techniques for polymer analysis, there are reasons to favor it. In terms of speed and sample throughput, CElike methods have an enormous advantage over SEC for large numbers of samples. CE’s low solvent consumption is desirable especially if very toxic or expensive solvents are used. Miniaturization of standard SEC is possible but requires adaptation of instrumentation to avoid extra-column peak broadening effects (5, 6)—wide-bore columns are still very much en vogue in SEC. In some cases, such as in forensic analysis, small samples may necessitate the use of a capillary technique. In our opinion, one of the main arguments for using electrokinetic techniques is that they facilitate some chemical analysis of synthetic polymers as part of the separation process. SEC separates on molecular size only and does not provide much information for end-group determination needed for chemical characterization of copolymers or polymer blends, except when combined with sophisticated and costly detectors. In chromatography, this void is filled using techniques such as
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For separating polymers with ionizable end groups, ordinary capillary zone electrophoresis (CZE) in free solution can be applied. Polymer samples with a fixed end group composition will separate according to size, with the largest molecules migrating the slowest. CZE was applied by Bullock (9) to analyze Jeffamine samples—technical mixtures of poly(oxypropylene)–poly(oxyethylene) copolymers with amino end groups and of poly(ethylene oxide) (PEO) diamines. In aqueous solutions with pH
