Difference Between Reverse Phase and Hydrophobic Interaction Chromatography

The main difference between reverse phase and hydrophobic interaction chromatography is that the reverse phase chromatography (RPC) uses a more hydrophobic medium, which leads to more stronger interactions whereas the hydrophobic interaction chromatography (HIC) uses a less hydrophobic medium when compared to the medium in the reverse phase chromatography.  

Reverse phase chromatography and hydrophobic interaction chromatography are two chromatographic techniques that depend on the interactions between hydrophobic surfaces of a chromatography medium and the hydrophobic patches on the surface of biomolecules.

Key Areas Covered

1. What is Reverse Phase Chromatography
     – Definition, Steps, Applications
2. What is Hydrophobic Interaction Chromatography
     – Definition, Steps, Applications
3. What are the Similarities Between Reverse Phase Chromatography and Hydrophobic Interaction Chromatography
     – Outline of Common Features
4. What is the Difference Between Reverse Phase Chromatography and Hydrophobic Interaction Chromatography
     – Comparison of Key Differences

Key Terms

Hydrophobic Interaction Chromatography, HIC, Media, Reverse Phase Chromatography, RPC

What is Reverse Phase Chromatography

Reverse phase chromatography (RPC) is a chromatographic technique used in the purification and analysis of biomolecules such as proteins, peptides, and oligonucleotides. It gives a high-resolution separation and is ideal for the peptide mapping and purity checking. RPC is more often used in the final polishing of peptides and oligonucleotides.

Two main types of hydrophobic media can be used as the stationary phase: silica beads covered with carbon chains or, naked hydrophobic polymers. A column is packed with the hydrophobic media in a form of packed bed onto which the sample is applied. An ion-pairing agent such as trifluoroacetic acid (TFA) can be added to the mobile phase to enhance the formation of hydrophobic interactions. An organic modifier such as 5% acetonitrile is used in the beginning and elution can be initiated with the increasing the % of acetonitrile.

Figure 1: Reverse Phase Chromatography Theory

Biomolecules that are less hydrophobic/more polar will elute first while the more hydrophobic molecules will elute later. RPC is the chromatographic technique with the highest resolution.

What is Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is the other technique used in the separation of biomolecules based on the hydrophobicity under moderate conditions. It is an ideal technique for the purification of proteins when the samples have to be subjected to ammonium sulfate precipitation in the initial sample concentration and cleanup. The elevated salt level during the ammonium sulfate precipitation increases the interaction between hydrophobic components.

Figure 2: Effect of High Salt Concentration on Hydrophobic Interactions

The hydrophobic medium of HIC consists of an inert matrix of spherical particles coated with ligands containing alkyl or aryl groups. The hydrophobic interactions occur under moderately high salt concentrations (typically 1–2 M ammonium sulfate or 3 M NaCl). The start buffer ensures the binding of proteins of interest to the medium while washing the impurities away. The elution buffer contains low salt concentration, weakening the interaction between proteins and the stationary phase.

Similarities Between Reverse Phase Chromatography and Hydrophobic Interaction Chromatography

• Reverse phase and hydrophobic interaction chromatography are two techniques used in the separation of biomolecules based on their hydrophobicity.
• The mobile phase of both techniques is either water or an organic solvent.
• They use packed bed columns.
• Start buffer ensures the binding of desired molecules to the column.
• Molecules with the lowest hydrophobicity may elute first while more hydrophobic molecules may elute later.
• The final washing step elutes the most-tightly bound molecules.
• The resolution of both techniques is a combination of the selectivity (degree of separation between the peaks),  efficiency (the ability to produce narrow, symmetrical peaks) of the technique, the mass of the sample, and the retention time.

Difference Between Reverse Phase Chromatography and Hydrophobic Interaction Chromatography

Definition

Reverse phase chromatography (RPC) refers to the highest resolution separation technique based on the hydrophobicity of the molecules in a mixture while hydrophobic interaction chromatography (HIC) refers to a type of separation technique based on the hydrophobicity but, operates under relatively mild conditions.

Hydrophobicity

The RPC operates under more hydrophobic conditions while the HIC operates under relatively mild hydrophobic conditions.

Interactions

Reverse phase chromatography leads to stronger interactions between molecules and the stationary phase that has to be reversed during elution by an organic modifier while HIC leads to moderately high interactions between the stationary phase and the molecules.

Hydrophobic Media

RPC uses either silica beads covered with carbon chains or, naked hydrophobic polymers while HIC uses an inert matrix of spherical particles coated with ligands containing alkyl or aryl groups.

Start Buffer

The start buffer in RPC uses trifluoroacetic acid (TFA) while the start buffer of HIC uses moderately high salt concentrations.

Elution

The increased percentage of acetonitrile begins the elution in RPC while the decreasing salt concentration begins the elution in HIC.  

Resolution

Reverse phase chromatography is the chromatographic technique with the highest resolution while the resolution of HIC is low when compared to RPC.

Applications

RPC is used in the separation of proteins, peptides, and oligonucleotides while HIC is mainly used in the purification of proteins.

Conclusion

The Reverse phase chromatography is the chromatographic technique with the highest resolution, which operates under highly hydrophobic conditions. But, HIC operates under moderate hydrophobic conditions. RPC and HIC are the two types of separation techniques based on the hydrophobicity. The main difference between RPC and HIC is the degree of hydrophobicity used in each technique.

Reference:

1. Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods. GE Healthcare, 2006. Available Here

Image Courtesy:

1. “Reverse Phase Gradient Elution Schematic” By Nategm – Own work (CC BY-SA 4.0) via Commons Wikimedia   
2. “Hicsalt” By Daliak at English Wikibooks – Transferred from en.wikibooks to Commons. (Public Domain) via Commons Wikimedia