Ion-exchange separation of proteins

The Swedish firm Pharmacia contributed to the development of CF by producing special amphoteric buffers (cf., 4.5.3.3) and ion exchangers (cf., 4.5.2.4). These products are described in detail in a brochure [51] available on request, which serves as a laboratory manual for CF. There is no doubt that chromatofocusing is an effective method for ion exchange separation of proteins. However, because it is a new method, we cannot yet evaluate its contribution to the progress in the field of biochemistry. The author hopes that this method will be found as useful as the electrofocusing method. 

Unsal E, Irmak T, Durusoy E, Tuncel M, Tuncel A (2006) Monodisperse Porous Pol3uner Particles with Polyionic Ligands for Ion Exchange Separation of Proteins. Anal. Chim. Acta 570 240-248. 

Nash DC. Comparison of diffusion and diffusion-convection matrices for use in ion-exchange separations of proteins. J Chromatogr 1998 807 185-207. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

FIGURE 11.5 (Panel a) Reversed phase separation of rHuBDNF feedstock showing the positions of variants and host cell proteins. (Panel b) Ion exchange separation of the same feedstock on a 50 pm cation exchange resin. Loading was 20 pg of rHuBDNF. (Reprinted with permission from Elsevier from Zhao, G.F. and Sun, Y., J. Chromatogr., 1165, 109, 2007. Copyright.)... 

The hydrophilic silica-based diol packings have been modified by derivation through some of the diol groups with carboxymethyl and diethy-laminoethyl functions to make weak anionic and cationic protein size-separation columns. These provide the HPLC equivalent of the CM- and DEAE-cellulose columns used in protein purification on open columns and are used with the same type of buffers to provide ion exchange purifications of proteins. 

Gallant, S., Vunnum, S., and Cramer, S. M. (1996). Optimization of preparative ion-exchange chromatography of proteins-linear gradient separations. /. Chromatogr. 725, 295-314. 

Unfortunately, there have apparently been no descriptions in the literature of similar peak tracking efforts applied to protein separations. In our laboratory we have applied this to an ion-exchange separation of a protein mixture and followed the change in elution via the spectral contrast technique (B. Warren, private eommunication). Although protein spectra are visually quite similar in appearance, the technique was able to distinguish all the proteins in a mixture of... 

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