![]() ![]() The order in which proteins are desorbed is usually relative to the number of external hydrophobic residues each protein has (Res 3). The most common way to do this is to use a gradient that slowly increases the hydrophobicity using an organic buffer. This stage in the process involves changing the buffer conditions to elute the bound hydrophobic protein. Other proteins in the mixture will be washed out. Proteins in the mixture that have a high percentage of exposed hydrophobic amino acid residues will be adsorbed to the hydrophobic stationary phase. In this step, the sample protein is injected into the system. Because the column is hydrophobic, water molecules tend to be ordered at the junction between the column and the buffer.Īpplication of the Sample In this step the hydrophobic column is primed by applying the specific sample buffer. Once again, the process can be summarized in four steps (Res2): The stages in Reversed Phase Chromatography are very similar to the stages found in Ion exchange Chromatography. Reverse phase chromatography is commonly coupled with mass spectrometry in an effort to quantify the protein that is eluted from the column (Res1). Elution of the bound hydrophobic protein can be accomplished by increasing the concentration of organic solvent, which increases the retention time of a particular component. As mixtures of proteins are applied to the column, polar proteins will elute first while non-polar proteins will bind to the column. All peptides and proteins carry a mix of hydrophilic and hydrophobic amino acids, but those with high net hydrophobicity will be able to participate in hydrophobic interactions with the stationary phase. ![]() Utilization of these polar solvents introduces very harsh conditions for the protein, thus the method will generally work well for smaller and more stable proteins. The mobile phase on the other hand, contains relatively polar organic solvents such as methanol, butanol, isopropanol, acetonitrile (Ref1). This creates a hydrophobic stationary phase. In reverse phase, the stationary phase is packed with silica modified with silyl ethers containing non-polar alkyl groups typically C8 or C18. The term ‘reverse’ was derived from its predecessor named ‘normal’ phase chromatography, which utilized a polar stationary phase such as silica. Reverse Phase chromatography is a separation based on the solubility of the protein. Reversed Phase Chromatography - Proteins with exposed hydrophobic region (red) will be able to bind to the immobilized hydrocarbon stationary phase the rest simply wash off ![]()
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