Proteins reversed phase HPLC

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

Capillary electrophoresis separates by differences in charge while reversed-phase HPLC separates on the basis of hydrophobicity. It turns out that these proteins have wide differences in hydrophobicity. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Reverse-phase HPLC can be used for the separation of peptides and proteins. Smaller peptides (less than 50 amino acid residues) may be satisfactorily separated on octadecylsilane (C-18) bonded phases whereas for adequate recovery of larger molecules, tetrylsilane (C-4) or octylsilane (C-8) is recommended. Porous column packing with gel permeation and reverse phase properties is usually required for proteins with relative molecular masses greater than 50 000. 

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

The injector, columns and valves reside in a low temperature chamber to minimize the loss of deuterium by back exchange (Fig. 12.2). The quenched protein solution is pumped in series through a column containing an immobilized protease and a trap column to capture the peptide fragments. The gradient pump is activated following digestion and the peptides captured on the trap column are eluted and separated over an analytical reverse-phase HPLC column directly into the mass spectrometer. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydrophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains such as butyl, octyl or... 

Figure 9. The effect of storage on protein and peptide composition in cooked ground beef stored in a refrigerator of 4 days (adapted from 7). Upper graph represents the size exclusion chromatography of acidic extracts of fresh, cooked, and cooked-stored beef. Lx)wer graph represents the reverse phase HPLC of peak II from the size exclusion chromatography.

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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