Displacement chromatography peptide

Displacement Chromatography in the Separation and Characterization of Proteins and Peptides... 

Displacement chromatography was named and introduced by Tiselius [1] who demonstrated its utility in both preparative and analytical applications. Tiselius explored separations of many different biological substances including amino acids and peptides [2,3]. The technique was also used... 

Chromatographic separation of these mixtures in the elution mode is incapable of resolving many thousands of peptides present in these mixtures, even when orthogonal, two-dimensional separations are performed. The investigator is left with little option for low-abundance peptide iden-tihcation other than affinity approaches that target certain subclasses (e.g., phosphopeptides). While effective for certain applications, the latter allow for enrichment of only a small subset of low-abundance peptides. Because of its potential for broad applicability to the problem of low-abundance peptide enrichment, displacement chromatography remains a technique that offers great possibilities in this area. 

New concepts presented in this edition include monolithic columns, bonded stationary phases, micro-HPLC, two-dimensional comprehensive liquid chromatography, gradient elution mode, and capillary electromigration techniques. The book also discusses LC-MS interfaces, nonlinear chromatography, displacement chromatography of peptides and proteins, field-flow fractionation, retention models for ions, and polymer HPLC. 

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

II. PURIFICATION OF AMINO ACIDS AND PEPTIDES BY DISPLACEMENT CHROMATOGRAPHY... 

Anita, F. D. and Cs. Horvath, Displacement chromatography of peptides and proteins, in HPLC of Peptides and Proteins Separation, Analysis, and Conformation (C. Mant and R. Hodges, eds.), CRC Press, Boca Raton, FL, 1990, pp. 809-821. 

Frenz ef al. [16,17] used displacement chromatography for the analysis of the peptides obtained by tryptic digestion of recombinant proteins. This analysis was done by LC/MS, using microcoliunns because of the low flow rate acceptable by the MS ion source. Displacement chromatography was the procedure adopted to... 

Figure 21.5 Separation of peptides by displacement chromatography [reproduced with permission from G. Subramanian, M.W. Phillips and S.M. Cramer, J. Chromatogr., 439, 431 (1988)]. Conditions column, 25cm x 4.6mm i.d. stationary phase, Zorbax ODS 5 [xm mobile phase, 1 ml min phosphate buffer 50mM, pH 2.2-methanol (60 40) temperature, 45 C. Peaks 1=9.6mg /V-benzoyl-L-arginine 2 = 14mg /V-carboxybenzoxy-i-alanyl-L-glycyl-L-glycine 3 =15 mg /V-carboxybenzoxy-i-alanyl-L-alanine. Displacer (horizontally shaded) 30 mg ml of 2-(2-butoxy-ethoxy)ethanol in the mobile phase. The composition of the 150 [xl fractions was analysed by reversed-phase HPLC.

One of the three basic modes of chromatographic operation, displacement chromatography is useful for preparative separations and trace enrichment. Most liquid chromatographic methods have been performed in displacement mode. Solutes purified by displacement chromatography include metal cations, small organic molecules, antibiotics, sugars, peptides, proteins, and nucleic acids. 

Frenz J, Bourell J, Hancock WS. High-performance displacement chromatography-mass spectrometry of tryptic peptides of recombinant human growth hormone. J Chromatogr 1990 512 299-314. 

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