SEPARATION BY SIZE-EXCLUSION FILTRATION

Ronde, N.J. and Vogt, D. (2006) Separation by size-exclusion filtration - homogeneous catalysts applied in membrane reactors, in Catalyst Separation, Recovery and Recycling -Chemistry and Process Design (eds D.-J. Cole-Hamilton and R.P. Tooze), Springer, Dordrecht, pp. 73-104. 

Biocatalysts are not always immobilized on membranes in bioreactors, though. As enzymes are macromolecules and often differ greatly in size from reactants they can be separated by size exclusion membrane filtration with ultra- or nano-filtration. This is used on an industrial scale in one type of enzyme membrane reactor for the production of enantiopure amino acids by kinetic racemic resolution of chemically derived racemic amino acids. The most prominent example is the production of L-methionine on a scale of 400 t/y (Liese et al, 2006). The advantage of this method over immobilization of the catalyst is that the enzymes are not altered in activity or selectivity as they remain solubilized. This principle can be applied to all macromolecular catalysts which can be separated from the other reactants by means of filtration. So far, only enzymes have been used to a significant extent. 

A number of potential methods for homogeneous catalyst separation and recovery have been discussed in the preceding chapters. This chapter addresses the separation of homogeneous catalysts by means of advanced filtration techniques. Separation of homogeneous catalysts by size exclusion (ultra- or nanofiltration, defined in detail in Section 4.3.1) offers several advantages ... 

Separation of molecules by size exclusion chromatography (SEC) also known as either gel filtration or gel permeation chromatography, is accomplished by exploiting the differences in molecular dimensions. Soft gels that are hydrophilic in nature, and made... 

The native molecular weight was estimated by size exclusion chromatography. On this basis, the native enzyme apparently existed as a monomer at pH 7.5 but dimerized (Afr70,000) at pH 8.8. Lucas et al. (1983) found that the enzyme from cowpea nodules also separated into monomers at pH 7.5 (M 50,000) and the monomer could then be separated from catalase activity by gel filtration. 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

In addition to the insoluble polymers described above, soluble polymers, such as non-cross-linked PS and PEG have proven useful for synthetic applications. However, since synthesis on soluble supports is more difficult to automate, these polymers are not used as extensively as insoluble beads. Soluble polymers offer most of the advantages of both homogeneous-phase chemistry (lack of diffusion phenomena and easy monitoring) and solid-phase techniques (use of excess reagents and ease of isolation and purification of products). Separation of the functionalized matrix is achieved by either precipitation (solvent or heat), membrane filtration, or size-exclusion chromatography [98,99]. 

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