The interaction between hydrophobic proteins and a HIC resin is greatly influenced by the running buffer. HIC and RPC are closely related techniques since both are based upon interactions between hydrophobic patches on the surface of biomolecules and the hydrophobic surfaces of a chromatography medium. Theory of reversed phase chromatography The separation mechanism in reversed phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the immobilised hydrophobic ligand, i.e.

From these observations two modes of protein chromatography have been developed, hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC). In hydrophobic interaction chromatography the matrix material is lightly substituted with hydrophobic groups.
Hydrophobic interaction chromatography (HIC) separates proteins according to differences in their surface hydrophobicity. Ion exchange chromatography iv. The use of a hydrophobic stationary phase can be considered the opposite, or "reverse", of normal phase chromatography - hence the term "reversed-phase chromatography". Frequently Asked Questions What is the difference between Reversed Phase (RP) HPLC and hydrophobic interaction chromatography (HIC)? First, we have to dissolve the sample to be separated into a … In other words, it may be difficult for a reversed-phase method or bonded chemistry to differentiate between molecules that are structurally similar. The actual Hydrophobic interaction chromatography. Normal phase chromatography uses activated silica or alumina as the stationary phase. The variable hydrophobic nature of proteins allows their separation through differential hydrophobic surface interactions. the development of reversed-phase separation materials, polar–ionic compounds were found to be eluted at or near the dead volume so that effective analysis of these types of compounds was difficult. Whereas, HIC uses a hydrophobic stationary phase that allows the hydrophobic molecules to attach to it.. Chromatography is an analytical technique that helps to separate a mixture into its components.

The base matrix for the commercially available reversed phase media is generally composed of silica or a synthetic organic polymer such as polystyrene.

These groups can range from methyl, ethyl, propyl, octyl, or phenyl groups. Mode of Separation Reverse Phase Chromatography A reversed phase chromatography medium consists of hydrophobic ligands chemically grafted to a porous, insoluble beaded matrix. The term HIC stands for Hydrophobic Interaction Chromatography. Reversed-phase chromatography is a technique using alkyl chains covalently bonded to the stationary phase particles in order to create a hydrophobic stationary phase, which has a stronger affinity for hydrophobic or less polar compounds.
For historical reasons, it has been reported that HILIC is a variant of normal phase liquid chromatography, but the separation mechanism used in HILIC is more complicated than that in NP-LC. Reverse phase chromatography i. Affinity Chromatography It is a selective (specific to study function and interaction of protein) technique in which binding affinity is exploited. The key difference between RP HPLC and HIC is that RP HPLC uses a more polar mobile phase and a less polar stationary phase. Ion exchange chromatography iv. The key difference between affinity and ion exchange chromatography is that we can use affinity chromatography to separate charged or uncharged components in a mixture whereas we can use ion exchange chromatography to separate charged components in a mixture.. Chromatography is a technique that we can use to separate the desired components in a mixture. We attach any component with the beads, those components which … Reverse phase chromatography i. Affinity Chromatography It is a selective (specific to study function and interaction of protein) technique in which binding affinity is exploited. We attach any component with the beads, those components which … It is because organic solvents can cause denaturing of the proteins. Typical reversed-phase materials filled an important need in chromatography when they were developed; retention of hydrophobic compounds and the Also, the mobile phase of the reverse phase chromatography is polar, washing out polar analytes in the sample mixture. The mobile phase of both techniques is either water or an organic solvent. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or butyl, are attached to the stationary column.

It is a type of reverse phase HPLC, and this method is used mainly for the separation of large biomolecules such as proteins. In RPC, hydrophobic substances dissolved in a polar solvent are separated due to differences in their preferential interaction with nonpolar ligands (the so-called “reversed phase”) that are either attached to a chromatography matrix, or are a part of a chromatography matrix structure itself, i.e., polymeric stationary phases. Hydrophilic interaction liquid chromatography (HILIC) is an alternative high-performance liquid chromatography (HPLC) mode for separating polar compounds. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin. In this method, we need to use an aqueous medium to make the sample of the biomolecule. the stationary phase.