Wim Th. Kok

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.

A comprehensive 2‐D separation method was developed for the characterization of methacrylate copolymers. In both dimensions conditions were employed that give a critical separation for the homopolymer of one of the monomers in the copolymer, and exclusion behaviour for the other. The 2‐D separation was realized by using a normal‐phase column in one dimension and a reversed phase column in the other, and by precisely tuning the compositions of the two mobile phases employed. In the normal‐phase dimension mixtures of THF and n‐hexane or n‐heptane were used as mobile phase, and in the reversed‐phase dimension mixtures of ACN and THF. Moreover, stationary phase particles had to be selected for both columns that gave an exclusion window appropriate for the molecular size of the sample polymers to be characterized. The 2‐D critical chromatography principle was tested with a polystyrene (PS)‐polymethylmethacrylate (PMMA) block copolymer and with block and random polybutylmethacrylate (PBMA)‐PMMA copolymers. Ideally, the retention time for a copolymer in both dimensions of this system would depend on the size of only one of the blocks, or on the contribution of only one of the monomers to the size of a random copolymer. However, it was found that the elution of the PS‐PMMA block copolymer depended on the size of both blocks, even when the corresponding homopolymer of one of the monomers showed critical elution behaviour. Therefore, the method could not be calibrated for block sizes by using homopolymer standards alone. Still, it was shown that the method can be used to determine differences between samples (PS‐PMMA and PBMA‐PMMA) with respect to total molecular size or block sizes separately, or to average size and chemical composition for random copolymers. Block and random PBMA‐PMMA copolymers showed a distinctly different pattern in the 2‐D plots obtained with 2‐D critical chromatography. This difference was shown to be related to the different procedures followed in the polymerization process, and the different molecular distributions resulting from these.