HIC columns – what they do and how to find the right one

HIC columns – hydrophobic interaction chromatography for (U)HPLC analysis

Hydrophobic interaction chromatography (HIC) is a technique that separates molecule mixtures according to their hydrophobicity. It is popular for analyzing and purifying proteins because HIC works with aqueous salt buffers which preserves their native state. In addition to proteins, it is used to analyze nucleotide-based molecules such as plasmids. This page overviews how HIC works, how to select a suitable HIC column, and their applications.

How Hydrophobic Interaction Chromatography works

The goal of HIC is separating a molecule mixture from low hydrophobicity to high hydrophobicity. To that end, the sample is injected onto a HIC column at high ionic strength, meaning in a buffer with high salt concentration. The column contains a non-polar stationary phase. The high salt concentration results in low surface tension and low solvation of both stationary phase and sample molecules. Accordingly, hydrophobic patches on the sample molecules are accessible and open to interact with the stationary phase finally leading to retention. Inhibition of the non-polar interaction along with elution takes place when the salt concentration in the mobile phase is reduced. Molecules with low hydrophobicity elute early at comparatively high salt concentrations while elution of very hydrophobic molecules elute late in low/no salt conditions.

HIC separation mechanism

HIC columns and how to select the right one

HPLC or UHPLC HIC columns share they contain particles that with a hydrophobic surface. The particles can be of different size (e.g. 2-13 µm in diameter), porous or non-porous and of different base materials such as silica or polymethacrylate. In addition, the surface hydrophobicity is achieved by hydrophobic ligands. Popular functional groups that are coupled to the base matrix of the particles are phenyl, butyl, ether, or ethyl. The table below gives insight into the advantages and disadvantages of the different materials available.


Advantage Disadvantage Suited for
Base Matrix Polymethacrylate/
High pH stability Tendency to swell in organic solvents (not typical for HIC) Any separation independent from pH, standard analyses
Silica High mechanical stability Limited pH stability (2.5-8) Separations at low pH and if organic solvents of > 20 % are required
Porosity Porous Large surface area and high binding capacity High mass transfer resistance, slower separations Best for preparative applications due to high binding capacity
Non-porous Reduced mass transfer resistance (high separation performance and fast separations) Low binding capacity Standard for analytical applications
Particle Size Up to 5 µm Best separation performance, mainly compatible with UHPLC Higher back-pressure as compared to larger particles UHPLC and optimized HPLC analyses
5-10 µm Medium separation performance and back-pressure Optimal for HPLC analyses
> 10 µm Low back-pressure Low separation performance For preparative applications

The ligand dictates the hydrophobicity of the stationary phase, though the base matrix and ligand density also impact on the overall hydrophobicity of the stationary phase. The most common ligand used for protein analysis is butyl, while phenyl is used for less hydrophobic molecules such as nucleic acids.

As a rule of thumb, less hydrophobic molecules require stationary phases with high hydrophobicity while highly hydrophobic molecules are better separated on stationary phases with lower hydrophobicity.

Applications of HIC columns

Drug-Antibody-Ratio (DAR) analysis of Antibody-Drug-Conjugates (ADCs)

ADCs are therapeutics that combine the specific binding capability of antibodies with chemotherapeutics to achieve site-specific cytotoxicity. The average number of cytotoxins (drugs) an antibody carries determines the efficacy of the therapeutic. As each drug modification increases the overall hydrophobicity of the ADC, the drug-antibody-ratio is typically determined by HIC. Tosoh offers columns designed for ADC analysis: TSKgel HIC-ADC columns

Protein modifications analysis in proteins

HIC is sensitive to small modifications in proteins as they alter the overall hydrophobicity of the protein. These include oxidations and reductions as well as deamidations that can be detected by HIC.

Hydrophobicity screening in protein drug candidates

Highly hydrophobic molecules are more prone to aggregate and faster clearance. Analyzing promising protein-based drug candidates by HIC elucidates their overall hydrophobicity and helps excluding molecules early in the development that would likely generate hydrophobicity-related issues in later development stages.

TSKgel hydrophobic interaction chromatography columns

Application proteins, mAbs, fast analyses
Hydrophobicity low-medium hydrophobic
Particle size (mean): 2.5 µm
Pore size (mean): non-porous
pH range: 2.0 - 12.0
Functional group butyl

More Information

Application medium-large proteins, semi-preparative applications
Hydrophobicity medium hydrophobic
Particle size (mean): 10 µm
Pore size (mean): 100 nm
pH range: 2.0 - 12.0
Functional group ether

More Information

Application nucleic acids, smaller proteins, semi-preparative applications
Hydrophobicity highly hydrophobic
Particle size (mean): 10, 13, and 20 µm
Pore size (mean): 100 nm
pH range: 2.0 - 12.0
Functional group phenyl

More Information

Application analysis of antibody-drug-donjugates (ADCs), mAb analysis
Hydrophobicity low-medium hydrophobic
Particle size (mean): 5 µm
Pore size (mean): non-porous
pH range: 2.0 - 12.0
Functional group butyl

More Information