Difference Between RP HPLC and HIC

Thekey differencebetween RP HPLC and HIC is thatRP HPLC uses a more polar移动阶段and a less polarstationary phase. WHEES，HIC使用hydrophobicstationary phase that allows the hydrophobic molecules to attach to it.

Chromatographyis an analytical technique that helps to separate a mixture into its components. First, we have to dissolve the sample to be separated into a solution. This solution-substance mixture is called the mobile phase. Then the mobile phase is passed through another material called the stationary phase. The stationary phase determines the separation of the components. The separation occurs due to the partitioning of material between the mobile phase and the stationary phase.

内容

1.Overview and Key Difference
2.What is RP HPLC
3.什么是HIC
4.Side by Side Comparison – RP HPLC vs. HIC in Tabular Form
5.概括

What is RP HPLC?

The term RP HPLC stands forReverse Phase High-Performance Liquid Chromatography。根据疏水性，它涉及混合物中的组件分离。流动相的疏水成分与固定相的固定疏水配体连接，而亲水成分在固定相中洗脱而没有附着在固定相的表面上。

Moreover, this method has more reproducibility compared to other chromatographic techniques, and it shows broad applicability as well. Therefore, we use this method in laboratories for over 75% of all HPLC methods. Most of the time, we use an aqueous blend of water with a miscible polar organic solvent as the mobile phase. Thus, it will ensure the attachment of the hydrophobic components in the solution to the surface of the stationary phase.

什么是HIC？

HIC一词代表疏水相互作用色谱。It is a type of reverse phase HPLC, and this method is used mainly for the separation of large biomolecules such as proteins. In this method, we need to use an aqueous medium to make the sample of the biomolecule. It is because organic solvents can cause denaturing of the proteins. Moreover, this technique uses the interaction between large molecules with a slightly hydrophobic surface of the stationary phase. Furthermore, we have to use a high salt concentration in the sample because it encourages the protein to be retained on the packing; this process is called salting out. By decreasing the salt concentration gradually, we can make the biomolecules elute separately according to their hydrophobicity.

Usually, this method uses the conditions opposite to that of ion-exchange chromatography. In this technique, first, we need to pass a buffer solution through the column in order to reduce the solvation of solute in the sample. It makes the hydrophobic regions of the protein to expose. However, when the molecule is more hydrophobic, less salt is required to promote the binding. Here, the less hydrophobic solutes are eluted first, and according to the change in salt concentration, more hydrophobic solutes are eluted last.

What is the Difference Between RP HPLC and HIC?

Chromatography is an analytical technique for the separation of components in a mixture. RP HPLC and HIC are two specialized chromatographic techniques. The key difference between RP HPLC and HIC is that RP HPLC uses a more polar mobile phase and less polar stationary phase, whereas HIC uses a hydrophobic stationary phase, which allows the hydrophobic molecules to attach on to it.

The below infographic summarizes the difference between RP HPLC and HIC.

概括– RP HPLC vs. HIC

Chromatography is an analytical technique for the separation of components in a mixture. RP HPLC and HIC are two specialized chromatographic techniques. The key difference between RP HPLC and HIC is that RP HPLC uses a more polar mobile phase and a less polar stationary phase, whereas HIC uses a hydrophobic stationary phase, which allows the hydrophobic molecules to attach to it.

Reference:

1. “HPLC Separation Modes.” Waters,Available here。

Image Courtesy:

1. “Preparative HPLC” By GYassineMrabetTalk✉This W3C-unspecified vector image was created with Inkscape. – Own work(CC BY 3.0)viaCommons Wikimedia