HIC Chromatography (Hydrophobic Interaction Chromatography)

Short answer

HIC chromatography is hydrophobic interaction chromatography, a liquid chromatography mode that separates proteins by how strongly their hydrophobic surface regions interact with the stationary phase. Proteins bind in a high-salt buffer and are eluted by lowering the salt concentration, so the more hydrophobic a protein is, the later it comes off. It runs under non-denaturing conditions with no organic solvent.

Definition and scope

An LCGC North America review on biopharmaceutical analysis describes HIC as a milder form of reversed-phase liquid chromatography. Elution order reflects the relative hydrophobicity of the molecules, and the separation takes place at physiological pH without organic solvents or high temperatures.¹ Because the protein is not exposed to those conditions, HIC is widely used where the folded form needs to be preserved.

A Methods in Enzymology chapter on the theory and use of HIC calls it a valuable tool in protein purification applications, usable at analytical and preparative scales.² HIC is therefore both a purification step, covered in the protein purification guide, and an analytical HPLC method, covered more broadly in the HPLC guide.

How the salt gradient works

HIC uses a reversed salt gradient: it starts with a high salt concentration and moves to a low one.¹ Higher salt promotes binding, and more hydrophobic molecules need less salt to bind.¹ A vendor explanation adds the mechanism: salt reduces solvation of the protein, which exposes its hydrophobic regions so they can attach to the column, and lowering the salt lets the protein release, with proteins released in order of increasing hydrophobicity.³

In practice this means the sample is loaded in the high-salt starting buffer, and the gradient to low salt is what produces the chromatogram. For how to read the resulting peaks, see the HPLC chromatogram guide.

Ligands, salts and mobile phases

The LCGC review reports the following typical materials and conditions.¹

• Stationary phase: silica bonded with ligands of limited hydrophobicity, such as butyl, phenyl, ether, amide and propyl. Alkyl ligands are hydrophobic, while aryl ligands give mixed-mode behavior. Particles are usually nonporous; some porous polymethacrylate phases exist.
• Salts: ammonium sulfate at 1-2 M or sodium chloride at 3 M.
• Buffer: phosphate buffer at about pH 6-7.
• Column hardware: columns operated at 100-400 bar, with 5, 3 or 2.5 µm particles.

The same review warns that high salt may precipitate proteins, so protein solubility should be checked under the starting conditions.¹

Where HIC is used

Removing aggregates

The Methods in Enzymology abstract notes that HIC is often used to remove product aggregates, which differ in hydrophobicity from the target monomer.²

Monoclonal antibodies and antibody-drug conjugates

For monoclonal antibodies (mAbs), the LCGC review points to heterogeneity from post-translational modifications as a target for HIC.¹ For antibody-drug conjugates (ADCs), species with different drug-to-antibody ratios (DAR) separate because the attached cytotoxins are hydrophobic. Antibody that carries no drug elutes first, and a weighted DAR is determined from the percentage peak areas and the drug-load numbers.¹

This is a worked reading of elution order: the first peak is antibody with no drug, and later peaks are species with more attached hydrophobic drug, not larger or smaller molecules. Size-based separation is a different mode, covered in the size-exclusion chromatography guide.

Checklist for method and supply decisions

Each row turns a reported point into something a lab can check against its own instrument and consumables list.

Check	What is reported	Source
Salt and buffer on hand	Ammonium sulfate 1-2 M or sodium chloride 3 M; phosphate buffer near pH 6-7	1
Protein tolerates the starting salt	High salt may precipitate proteins, so solubility is checked under the initial binding conditions	1
Ligand family	Alkyl ligands are hydrophobic; aryl ligands give mixed-mode behavior	1
Column pressure rating and particle size	Columns run at 100-400 bar with 5, 3 or 2.5 µm particles	1
Detector plan	HIC is generally incompatible with mass spectrometry because of the high salt; some studies used ammonium tartrate, and multidimensional setups with reversed-phase desalting allow online ADC characterization	1
Gradient direction	High salt to low salt	1, 3

The mass spectrometry row matters most for detector planning. The ion exchange HPLC guide covers the same issue for salt gradients in another separation mode: nonvolatile salts are typically incompatible with mass spectrometry, so volatile salts or pH gradients are used.

Common misunderstandings

• “The gradient goes up in salt.” In HIC it goes down: binding happens in high salt and elution in low salt.^1,3
• “HIC is the same as reversed-phase.” It is described as a milder form of reversed-phase LC, run at physiological pH without organic solvents or high temperatures.¹ For the reversed-phase concept itself, see the reversed-phase vs normal-phase HPLC comparison.
• “HIC separates by size.” Elution order reflects relative hydrophobicity.¹ Size-based separation is a different mode.
• “HIC pairs easily with mass spectrometry.” It is generally incompatible with MS because of the high salt.¹

FAQ

What does HIC stand for in chromatography?

HIC stands for hydrophobic interaction chromatography, a protein separation mode based on hydrophobic interaction between the protein and the stationary phase.^1,2

Does HIC bind at high or low salt?

High salt. Proteins bind in a high-salt buffer and are eluted as the salt concentration falls.^1,3

Can HIC be used for preparative work?

Yes. The Methods in Enzymology chapter describes use at analytical and preparative scales.²

Why is HIC used for antibody-drug conjugates?

Species with different drug-to-antibody ratios differ in hydrophobicity because the attached cytotoxins are hydrophobic, so they separate by drug load.¹

Sources

1. LCGC North America, Hydrophobic Interaction Chromatography for Biopharmaceutical Analysis. Checked 2026-10-29.
2. McCue JT. Theory and use of hydrophobic interaction chromatography in protein purification applications. Methods Enzymol 2009;463:405-14. PubMed 19892185, doi 10.1016/S0076-6879(09)63025-1. Abstract checked 2026-10-29.
3. AAT Bioquest, How does hydrophobic interaction chromatography (HIC) work? Vendor FAQ, checked 2026-10-29.