Hydrophobic interaction chromatography (HIC) uses a filler with moderate hydrophobicity as the stationary phase and a salt-containing aqueous solution as the mobile phase, and uses the difference in the hydrophobic properties of the solute molecules to achieve separation by the difference in the strength of the hydrophobic interaction with the stationary phase, chromatographic method. Because the separation principle of hydrophobic interaction chromatography is completely different from chromatographic techniques such as ion exchange chromatography or gel filtration chromatography, this technique is often used in combination with the latter two to separate complex biological samples. At present, the main application field of this technology is in the purification of proteins, and it has become an effective means for the separation of serum proteins, membrane-bound proteins, nuclear proteins, receptors, recombinant proteins, etc., as well as some drug molecules, and even cells.

For small molecules, they can be divided into hydrophilic molecules and hydrophobic molecules according to their polarity. Generally speaking, hydrophilic small molecules are difficult to interact with HIC media. But for biological macromolecules such as proteins, which are the main targets of HIC, their hydrophilicity or hydrophobicity are relative. Even hydrophilic molecules will have local hydrophobic regions, which may cause hydrophobic interactions with HIC media. Therefore, it can be separated according to the relative strength of its hydrophobicity.

HIC media are composed of hydrophobic ligands such as alkyl or aromatic groups attached to a specific matrix such as agarose. The interaction between HIC media and hydrophobic biomolecules is believed to be driven by entropy increase and change in free energy, just like the spontaneous aggregation of hydrophobic molecules in aqueous systems. Salts play a very important role in the hydrophobic interaction. The presence of high concentrations of salts can strongly interact with water molecules, resulting in the reduction of water molecules that can form holes around the hydrophobic molecules, which promotes the hydrophobicity between the hydrophobic molecules and the chromatographic medium. binding between ligands.

Therefore, in the HIC process, a solution with high salt concentration is used in the sample adsorption stage, so that the target molecules are bound in the chromatographic column, and in the elution stage, the hydrophobicity between the solute and the chromatographic medium is made by reducing the salt concentration in the eluent. The effect is weakened, so it is desorbed from the column and eluted. For chromatographic media with aromatic groups as hydrophobic ligands, there is also a potential possibility of π-π interaction. When the surface of the substance to be separated has aromatic groups, it will show a mixed separation of hydrophobic interaction and π-π interaction. model.

In theory, HIC and RPC are two closely related liquid chromatography techniques, both of which are based on the flow-water interaction between the hydrophobic regions on the surface of biomolecules and the hydrophobic ligands (alkyl or aromatic) on the chromatographic medium , however, the chromatographic mechanisms at the molecular level as well as the practical level differ between the two techniques.

The degree of substitution of hydrophobic ligands on RPC media is much higher than that on HIC media. The RPC medium can be considered as a continuous hydrophobic phase, and the degree of substitution of ligands such as C4~C18 alkyl groups is usually in the hundreds of micromoles/mL gel: while the HIC medium has ligands such as C2~C alkyl groups or simple aromatic groups. The degree of substitution is usually in the range of 10–50 mmol/mL gel, which can be regarded as a discontinuous hydrophobic phase, which is participated by one or several ligands when binding to biomolecules.

Obviously, the interaction between the hydrophobic solute and the RPC medium is much stronger than that of the HIC medium, and severe elution conditions such as organic solvent gradients are required to elute the solute from the column. Denaturation tends to occur under the elution conditions of RPC, so RPC is suitable for the separation and purification of peptides and small protein molecules with good stability in water-organic solvent systems, while the elution conditions of the HIC process are much milder and usually reduce the elution Therefore, HIC not only takes advantage of the hydrophobic properties of proteins, but also can be carried out in a more polar and less denaturing environment, so it has a wider application in protein purification.

The influence of mobile phase conditions on HIC is mainly manifested in the type and concentration of salts used, the pH of mobile phase, and the influence of other additives. The HIC process achieves the adsorption of the sample at high salt concentration, and then completes the elution process at low salt concentration. Clearly, the type and concentration of salts in the mobile phase are crucial parameters in HIC. Different ions, especially anions, have different roles in HIC. Some ions in solution promote the precipitation of proteins, and they can increase the hydrophobic interaction; while the presence of other ions promote the dissolution of proteins, called solubilizing salts, their presence destroys the hydrophobic interaction. The following table indicates the effect of different ions on the hydrophobic interaction. The ions on the left in the table can promote the hydrophobic interaction and are therefore often used in HIC, while the ions on the right belong to the solubilizing ions, which can destroy the hydrophobic interaction and are sometimes used in chromatographic media. It can be used to elute some particularly strongly bound impurities during cleaning.

Under the condition that the type of salt used has been determined, the level of salt concentration will affect the binding strength of solute molecules and the chromatographic medium and the binding capacity of the chromatographic medium. Elevated salt concentration can promote hydrophobic interaction, so HIC is usually loaded and adsorbed at high salt concentration, and eluted by reducing the salt concentration in the eluent. In addition, the initial salt concentration of the chromatography process will also affect the binding capacity of the chromatography medium for proteins.

The pH value of the solution mainly considers the pH environment that can maintain the biological activity of biological macromolecules. In general, when the pH of the solution is close to the isoelectric point of the protein, its hydrophobicity increases, which is conducive to the interaction with the immobilized ligand; far from its isoelectric point, its hydrophobicity decreases, which is not conducive to binding with the immobilized ligand, but is beneficial to the protein elute. Therefore, the hydrophobicity of the protein can also be changed by changing the pH of the solution.

Different salts have an impact on the strength of hydrophobic interaction, and the selectivity in chromatography is also different; the salt concentration also varies with the hydrophobicity of the target molecule, (NH4)2SO4 is usually 0.75~2mol/L, and NaCl is 1~4mol /L; the most common elution method is to reduce the salt concentration in the mobile phase. Mobile phase B is a buffer with lower salt content. The buffer type is the same as that of mobile phase A, and the B% is from 0 to 100. Add organic solvents to the mobile phase, such as ethylene glycol, propanol, isopropanol, etc., to reduce the polarity of the mobile phase to elute; add detergents to the mobile phase, and the detergent itself can interact with the medium It adsorbs strongly, thereby displacing the target components bound to it.

Categories: News


Leave a Reply

Avatar placeholder

Your email address will not be published. Required fields are marked *