Hydrophobic interaction chromatography

Table of Contents

Hydrophobic interaction chromatography is an extremely valuable chromatographic analysis technique in which macromolecules are separated on the basis of their hydrophobicity. So far, we have learnt about a diverse variety of compounds that can analyzed by many different types of chromatography. How then is hydrophobic interaction chromatography different? What is its working principle, the special kind of stationary phases used in it and how can its mobile phase compositions vary? All this and much more in this particular article on hydrophobic interaction chromatography.

What is hydrophobic interaction chromatography

Hydrophobic interaction chromatography often abbreviated as HIC is a type of liquid column chromatography in which a non-polar, hydrophobic stationary phase is used. High concentration salt solutions are commonly employed as mobile phase solvents against the hydrophobic stationary phase in HIC. This type of chromatography is specifically important for separation and purification of biological macromolecules such as proteins.

A protein consists of both hydrophobic as well as hydrophilic functional group regions. Proteins bury a part of their hydrophobic core inside their extensively folded tertiary structures while leaving the polar, hydrophilic regions exposed for interaction with aqueous solvents.  But still about 50% of the total surface area of a protein have hydrophobic areas. These hydrophobic areas can be effectively utilized for protein adsorption onto an HIC stationary phase. Larger the protein molecules, greater their exposed hydrophobic regions.

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A large number of hydrophobic amino acids attached in a polypeptide chain will make the end protein structure highly hydrophobic.

Examples of amino acids with hydrophobic side chains

All this information indicates that hydrophobic interaction chromatography is a type of reversed-phase chromatography that employs an adsorption mode for analyte retention. In this case, the stationary phase is less-polar than the mobile phase while the analytes get retained onto the stationary phase by adsorption.

Read more on normal-phase and reverse-phase chromatography and about the different types of chromatography here: what is chromatography.

Historical background of hydrophobic interaction chromatography

Hydrophobic interaction chromatography was developed and used for the first time by Tiselius in 1948. It was initially called ‘salting-out’ chromatography. The term hydrophobic interaction chromatography was introduced in 1973 by Hjerten. The concept of ‘salting out’ will be explained to you later in this article.

Working principle of hydrophobic interaction chromatography

Hydrophobic interaction chromatography is performed on the principle of hydrophobic solute retention from the sample mixture onto the stationary phase. Meanwhile the more polar, less hydrophobic analyte components pass out of the column with the mobile phase.

A high salt concentration is initially applied inside the column to induce protein binding with the stationary phase. The targeted proteins are then eluted out by decreasing the concentration of salt solution inside the column. Proteins become more soluble in water and easily elute out of the column with the salt solution as a consequence.

Stationary phase in hydrophobic interaction chromatography

As is obvious from what we have already learnt, a hydrophobic stationary phase is packed into an HIC column. The stationary phase in hydrophobic interaction chromatography is essentially based on the following two components:

  • Support Matrix
  • Ligand

In hydrophobic interaction chromatography, the matrix itself is hydrophilic while hydrophobic ligands are immobilized on it. Inert, porous gels such as an agarose gel is used as the backbone matrix in HIC while hydrophobic phenyl or alkyl (butyl, octyl or isopropyl) functional groups are attached to it.

Greater the ligand immobilization onto the stationary phase matrix, higher its hydrophobicity. Generally speaking, straight chain alkyl groups function as stronger hydrophobic ligands than aryl ligands.

Mobile phase in hydrophobic interaction chromatography

Buffer/ salt solutions are used as mobile phases in hydrophobic interaction chromatography. Salts such as the solutions of ammonium sulfate (NH4)2SO4, sodium chloride (NaCl) and sodium sulfate (Na2SO4) are some of the most commonly used mobile phases in HIC. These salts can be used in pure form and/or as their binary or ternary mixtures for mobile phase preparation in hydrophobic interaction chromatography.

Increasing salt concentration increases protein binding to the stationary phase while decreasing salt concentration liberates these proteins out of the column. Read more on this concept in the next section where we have discussed in detail how to perform hydrophobic interaction chromatography.

How to perform hydrophobic interaction chromatography

The following step-by-step guide explains how a hydrophobic interaction chromatography is performed in the laboratory:

Step I: Column packing

The column is packed with the stationary phase material as discussed above.

Step II: Preliminary concentration and clean up

The protein samples are treated with kosmotropic, highly concentrated salt solutions such as a 1M solution of ammonium sulfate. The salt reduces any risk of sample solvation in aqueous media and facilitates sample binding with the hydrophobic stationary phase. The more hydrophobic a targeted analyte is, less salt concentration required to promote its binding.

However, it is important to keep in mind that treating a sample with a high salt concentration promotes binding but at the same time it increases the risk of protein precipitation inside the column. Decreasing solubility and precipitating out a sample in this way is called ‘salting-out’’. You can read more about this concept here: The mechanism behind salting out.

Strictly monitoring the concentration, temperature and pH conditions is thus very important for a successful hydrophobic interaction chromatography as we discussed in our article: affinity chromatography.

Step III: Sample loading

The analyte mixture is applied onto the hydrophobic stationary phase in the presence of aqueous buffers/salt solutions. The hydrophobic molecules bind to the alkyl chains present on the stationary phase via reversible London dispersion forces. On the other hand, π-π interactions are involved for sample binding to the aromatic ends of the hydrophobic stationary phase. 

Step IV: Washing

All the non-bound sample components are washed out of the column by using the same high concentration salt solution as used in Step II.

Step V: Elution

The elevated salt concentration present inside the column is suppressed by passing the mobile phase in a decreasing concentration gradient. Diluting the salt solution, reverses protein-ligand binding. Consequently, solubilizing the protein into the mobile phase thus a ‘salting-in’ effect as opposed to ‘salting-out’ discussed in step II.

Usually, slightly acidic or neutral buffer solutions are preferred in order to maintain a low pH environment inside the column. Depending upon mobile phase pH, the net charge present on a protein and/or its conformation can change. As a general matter-of-fact, it is said that a mobile phase pH close to the isoelectric point of the protein, induces higher hydrophobicity and vice versa. Learn more on isoelectric point of a protein and its importance in chromatography here: gel electrophoresis.

Organic modifiers/detergents are also sometimes added into the HIC mobile phase at the elution stage. Inclusion of such solvents reduce the hydrophobic interaction between the proteins and the stationary phase. The proteins in turn interact strongly with the mobile phase and get eluted out of the column.

Step VI: Detection

Least hydrophobic protein is eluted the first followed by other proteins in an increasing order of hydrophobicity. Eluate reaches the detector and a detector response is recorded. A UV-Visible detector and/or a mass spectrometry (MS) detector are the most popular choices to be coupled with HIC. Chromatogram is then obtained at the final step as a plot of detector response versus retention time.

For more on different types of detectors and the chromatographic terms we recommend our articles: High-performance liquid chromatography (HPLC) and detectors in chromatography.

Why do we need hydrophobic interaction chromatography

  • Protein purification: The chemical conditions and the stationary phase matrices used in hydrophobic interaction chromatography are a special feature for protein purification. The sensitive protein structures stay intact under less denaturing conditions of HIC. Hydrophobic interaction chromatography serves both as an analytical as well as a preparative chromatographic tool. Different types of bio-samples can be separated and detected such as therapeutic proteins, hydrophobically tagged proteins, antibodies and DNA vaccines etc.
  • Capturing impurities: Treating analytical samples with high salt concentrations (step II) also helps as a source of clean up. The protein molecules are precipitated while the impurities are captured and thus removed.


HIC is important because it can separate proteins with as little a difference as a single amino acid. It can also distinguish between correctly and incorrectly folded protein components so it is very valuable for bio-medicinal purposes.

For a more detailed discussion on the application of hydrophobic interaction chromatography for studying therapeutic proteins, you may read this: HIC for the characterization of therapeutic monoclonal antibodies.

In addition to the salting out theory, there are other theories involved in protein purification by HIC which you can check out from the sources given below.

  1. Thermodynamic theory of protein purification
  2. Vander Walls theory

Watch this video and revise all the concepts learnt here. 

If you found this article useful, you may also like to discover the other special type of chromatography in our article: hydrophilic interaction chromatography (HILIC).


1. Bertin, L., D. Frascari, H. Domínguez, E. Falqué, F. A. Riera Rodriguez and S. A. Blanco (2015). Chapter 7 – Conventional purification and isolation. Food Waste Recovery. C. M. Galanakis. San Diego, Academic Press: 149-172.

2. McCue, J. T. (2009). Chapter 25. Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications. Methods in Enzymology. R. R. Burgess and M. P. Deutscher, Academic Press. 463: 405-414.

3. Sun, Y., Q. H. Shi, L. Zhang, G. F. Zhao and F. F. Liu (2011). 2.47 – Adsorption and Chromatography. Comprehensive Biotechnology (Second Edition). M. Moo-Young. Burlington, Academic Press: 665-679.

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