Stationary phases protein type

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

There is a wide variety of commercially available chiral stationary phases and mobile phase additives.32 34 Preparative scale separations have been performed on the gram scale.32 Many stationary phases are based on chiral polymers such as cellulose or methacrylate, proteins such as human serum albumin or acid glycoprotein, Pirkle-type phases (often based on amino acids), or cyclodextrins. A typical application of a Pirkle phase column was the use of a N-(3,5-dinitrobenzyl)-a-amino phosphonate to synthesize several functionalized chiral stationary phases to separate enantiomers of... 

Weitzhandler, M., Farnan, D., Horvath, J., Rohrer, J. S., Slingsby, R. W., Avdal-ovic, N., and Pohl, C., Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases, /. Chromatogr. A, 828, 365, 1998. 

Until the introduction of capillary columns, it was not possible to separate all the amino acids found in proteins on one column. The choice of stationary phase will depend upon the types of derivatives that have been prepared and in some situations it still may be preferable to use two different columns simultaneously. 

These separations can be carried out using a silica-based bonded phase however, the important advantage of organic polymer stationary phase materials is their chemical stability. The columns can be washed by using an alkaline solution after a certain number of injections. According to the chromatograms, the proteins in serum are completely eluted and nothing remains inside the column. However, the pressure drop in this type of analysis... 

Chiral separations result from the formation of transient diastereomeric complexes between stationary phases, analytes, and mobile phases. Therefore, a column is the heart of chiral chromatography as in other forms of chromatography. Most chiral stationary phases designed for normal phase HPLC are also suitable for packed column SFC with the exception of protein-based chiral stationary phases. It was estimated that over 200 chiral stationary phases are commercially available [72]. Typical chiral stationary phases used in SFC include Pirkle-type, polysaccharide-based, inclusion-type, and cross-linked polymer-based phases. 

The use of protein immobilised to the surface of a silica gel or to another support has been a very successful approach for the chiral separation of various pharmaceuticals. The AGP stationary phase has been shown to have the broadest enantiorecognition abilities while the BSA stationary phase is especially useful for aromatic compounds. " Table 9 shows some examples of separations that were obtained on the protein-type of CSPs. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

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