Chromatography perfusion

POROS media, made by copolymerization of styrene and divinylben-zene, have high mechanical strength and are resistant to many solvents and chemicals. The functional surface chemistry of the particles can be modified to provide supports for many types of chromatography, including ion exchange, hydrophobic interaction, immobilized metal affinity, reversed 

Transport of biomolecules through chromatographic media. A Conventional support particles. B POROS particles for perfusion chromatography. Courtesy of PerSeptive Biosystems, Cambridge, MA, 

In addition to high resolution and short analysis times, perfusion chromatography has the advantage of improved recovery of biological activity because active biomolecules spend less time on the column, where denaturing conditions may exist. 

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Adamski, RP Anderson, JL, Configurational Effects on Polystyrene Rejection from Micro-porosou Membranes, Journal of Polymer Science Part B Polymer Physics 25, 765, 1987. Adler, PM, Porous Media, Geometry and Transports Butterworth-Heinemann Boston, 1992. Afeyan, NB Fulton, SP Regnier, FE, Perfusion Chromatography Packing Materials for Proteins and Peptides, Journal of Chromatography 544, 267, 1991. 

Rodrigues, A. E., Permeable packings and perfusion chromatography in protein separation, /. Chromatogr. B, 699, 47, 1997. 

Poetsch, A., Seelert, H., Tittingdorf, J. M., and Dencher, N. A., Detergent effect on anion exchange perfusion chromatography and gel filtration of intact chloroplast H+-ATP synthase, Biochem. Biophys. Res. Commun., 265, 520, 1999. 

The effect of pore size on CEC separation was also studied in detail [70-75]. Figure 9 shows the van Deemter plots for a series of 7-pm ODS particles with pore size ranging from 10 to 400 nm. The best efficiency achieved with the large pore packing led to a conclusion that intraparticle flow contributes to the mass transfer in a way similar to that of perfusion chromatography and considerably improves column efficiency. The effect of pore size is also involved in the CEC separations of synthetic polymers in size-exclusion mode [76]. 

Fulton, S.P., Meys, M., Varady, L., Jansen, R., and Afeyan, N.B., Antibody quantitation in seconds using affinity perfusion chromatography. Biotechniques, 11, 226-231, 1991. 

Most of the applications of HPLC for protein analysis deal with the storage proteins in cereals (wheat, corn, rice, oat, barley) and beans (pea, soybeans). HPLC has proved useful for cultivar identihcation, protein separation, and characterization to detect adulterations (illegal addition of common wheat flour to durum wheat flour) [107]. Recently Losso et al. [146] have reported a rapid method for rice prolamin separation by perfusion chromatography on a RP POROS RH/2 column (UV detection at 230nm), sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and molecular size determination by MALDl-MS. DuPont et al. [147] used a combination of RP-HPLC and SDS-PAGE to determine the composition of wheat flour proteins previously fractionated by sequential extraction. 

Column packing materials such as silica gel contain a large amount of water, and separation involves partition between an immobilized aqueous phase in the gel and a mobile, often organic, solvent flowing through the column. Usually materials elute sooner when they are more soluble in the mobile phase than in the aqueous phase. These methods are closely related to perfusion chromatography, which is described in Section 2. 

M McCoy, K Kalghatgi, FE Regnier, N Afeyan. Perfusion chromatography—characterization of column packings for chromatography of proteins. J Chromatogr A 743 221-229, 1996. 

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