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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Published on: September 21, 2011

Managing sample introduction problems in hydrophilic interaction liquid chromatography.

Mark R Taylor¹, Jane Kawakami², David V McCalley³

¹Pfizer Global Research and Development, Discovery Park, Ramsgate Road, Sandwich, CT13 9NJ, UK.

Journal of Chromatography. A

|May 11, 2023

Summary

Sample injection issues in hydrophilic interaction liquid chromatography (HILIC) are addressed by using pre-column dilution with weak solvents like acetonitrile. This method improves peak shape and efficiency when injecting samples dissolved in stronger solvents than the mobile phase.

Keywords:

HILIC HPLC Sample injection

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Area of Science:

Analytical Chemistry
Chromatography

Background:

Sample injection can cause peak asymmetry and poor efficiency in hydrophilic interaction liquid chromatography (HILIC).
This occurs when the injection solvent has higher elution strength than the mobile phase (MP), often due to high water content needed for dissolving polar samples.
Acetonitrile-rich MPs exacerbate this mismatch.

Purpose of the Study:

To investigate strategies for overcoming injection solvent mismatch in HILIC.
To improve peak shape, efficiency, and overall chromatographic performance.

Main Methods:

Investigated pre-column dilution using weak solvents (e.g., acetonitrile) delivered with the sample.
Explored alternative organic solvents (isopropanol, acetone, tetrahydrofuran) for injection.
Evaluated the impact of injection solvent volume and mass overload.

Main Results:

Pre-column dilution effectively mitigates injection solvent mismatch issues.
Alternative organic solvents may enhance sample solubility.
Minimizing injection solvent volume is crucial for reducing mismatch effects.

Conclusions:

Pre-column dilution is a simple and effective strategy to resolve injection solvent mismatch in HILIC.
Careful selection of injection solvent and volume can significantly improve chromatographic results.
Small sample volumes are recommended to minimize adverse effects.