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 that allows the hydrophobic molecules to attach to it.
Chromatography is 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.
CONTENTS
1. Overview and Key Difference
2. What is RP HPLC
3. What is HIC
4. Side by Side Comparison – RP HPLC vs. HIC in Tabular Form
5. Summary
What is RP HPLC?
The term RP HPLC stands for Reverse Phase High-Performance Liquid Chromatography. It involves the separation of components in a mixture, according to hydrophobicity. The hydrophobic components in the mobile phase link with the immobilized hydrophobic ligands on the stationary phase, while hydrophilic components elute through the stationary phase without getting attached to the surface of the stationary phase.
Figure 01: HPLC Apparatus
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.
What is HIC?
The term HIC stands for Hydrophobic Interaction Chromatography. 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.
Summary – 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) via Commons Wikimedia