hydrophobic interaction chromatography molecules

When New Purification Challenges Require a Precise Approach

Hydrophobic Interaction Chromatography (HIC)

Today’s bioprocessing requires more precision and more efficient operations. Hydrophobic interaction chromatography (HIC) separates molecules based on differences in their surface hydrophobicity. This technology is widely used in chromatographic purification, especially as a more precise purification step from other methods like ion exchange (IEX) or affinity. HIC typically requires high salt concentration for efficient binding of the target protein. Therefore, it is ideally used after chromatographic steps where elution is accomplished with high salt concentrations, i.e. ion exchange chromatography.

Sartorius has a range of HIC modalities (resins, membranes, and monoliths) to match your application.


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Aggregate removal

Powerful method for removing aggregates


Complementary purification

Ideal after initial sample cleanup and IEX chromatography


Efficient biomolecule polishing

Ideal for final polishing steps or intermediate large-molecule purification


Find the Right Solution for Your Hydrophobic Interaction Chromatography Process

HIC is primarily used for aggregate removal due to its superior performance in that function. 

Resins exhibit good binding capacities for small and medium-size proteins, while mixed-mode resins often combine HIC and IEX mechanisms.

Membranes have a higher binding capacity to viruses, virus-like particles, and other large complexes compared to resins, and also offer the advantage of higher flow rates resulting in shorter process time and higher productivity.

HIC monoliths are key in separating plasmid DNA isoforms.


Membranes

Monoliths

Application
 

Capture of large biomolecules, polishing of all biomolecules

Capture and polishing of large biomolecules

Implementation 

Intrabatch multi-use, single-use between batches

Intrabatch multi-use, single-use between batches

Ease of use 

Ready-to-use

Ready-to-use

Scalability 

Capsules and cassettes for linear scale-up

Wide range of device sizes

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Sartobind® Membranes in HIC

Sartobind® Phenyl – Easy Aggregate Removal

Sartobind® Phenyl is a hydrophobic interaction membrane with low ligand substitution. This allows for mild elution conditions for the purification of all biomolecules.

Sartobind® Phenyl membranes can be considered as a replacement to columns for polishing (flow-through) operations and a number of bind-and-elute applications, as they work at much higher flow rates, reduced complexity and without size exclusion effects when purifying large biomolecules.

The typical dynamic binding capacity (DBC) at 10 % breakthrough is:
•    9-13 mg/mL mAb
•    10 mg/mL BSA
•    23 mg/mL Lysozyme


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Monoliths for HIC

The Choice for Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is a must for nucleic acid separations. When combined with the advantages of monolithic chromatography, HIC meets this need while also providing an excellent solution for the purification of large biomolecules including adenoassociated viruses (AAV).


CIMmultus® OH

CIMmultus® C4-HLD

Channel sizes 

1.3, 2, 6 µm

2, 6 µm

Column volume 

1 mL – 8 L

1 mL – 8 L

Working range, pH 

2 – 10

2 – 10

Functionality 

Weak hydrophobic

Strong hydrophobic

Typical application 

Viruses, (i.e. AAV), and VLPs 

Nucleic acids (pDNA, mRNA) 

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CIMmultus® OH – Hydrophobic Interaction Chromatography

  • Uncharged (neutral) ligand

  • Working range, pH 2–10

  • Retains very large solutes (virus particles, VLP, vesicles) in the presence of precipitating salts or polyethylene glycol

  • Elutes large solutes with high resolution in order of increasing size

  • Elution with descending salt or polyethylene glycol

  • Sanitizable with 1 M NaOH

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CIMmultus® C4-HLD - Hydrophobic Interaction Chromatography

  • Strongly hydrophobic, (uncharged) ligand

  • Working range, pH 2–10

  • Highly effective for removing proteins from nucleic acids (pDNA, RNA)

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