Protein separation systems

FIG. 22-85 Phase diagram for a PEG/Dextran, hiphasic, aqueous-polymer system used in liquid-liquid extraction operations for protein separations. Alheiisson, Partition of Cell Particles and Macromolecules, 3d ed., Copyright 1986. Reprintedhy petTTUssion of John Wiley Sons, Inc.)... 

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

Selection of an Ion-Exchange-Reversed-Phase Separation System for Protein-Level Separations... 

In general, a comprehensive separation strategy implies the desire to resolve/analyze all components within a sample. In the specific context of a multidimensional chromatographic method, the term is more narrowly applied to indicate that all analytes introduced to the first-dimension separation are also subjected to a second-dimension separation. There are two basic configurations used by our laboratory to carry out comprehensive multidimensional (IEX/RP) protein separations—IEX— Dual Column RP system and IEX—Dual Trap RP system (Figs. 13.1 and 13.2), respectively. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

Proteins have, to date, only rarely been purified by SMB. The first attempt was made by Huang et al. in 1986 [42]. They isolated trypsin from porcine pancreas extracts using an SMB made of only six columns. In addition, this example also demonstrates that SMB systems with a very limited number of columns can be efficient. Another example for a successful protein-separation by SMB is the purification of human serum albumin (HSA) using two SMB-systems connected in series [43]. The first SMB was used for removing the less strongly retained components and the second one for removing the more strongly retained components of the sample matrix. 

According to these equations, for a given separation system, the main parameters involved in the separation of SDS-protein complexes are the electric force, the frictional force, and the retardation coefficient. These parameters are in turn affected by the strength of the electric field, molecular charge, analyte shape and size, polymer concentration, and temperature. 

The former polymer system represents a reversed-phase material, providing C4-alkylchains, which has most frequently been employed for protein separation [53], whereas the latter carries reactive moieties that can easily be converted in order to yield the desired surface functionalities. 

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