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Updated: Jun 19, 2026

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
Poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monolithic column for efficient hydrophobic
Yuanyuan Li1, H Dennis Tolley, Milton L Lee
1Department of Chemistry and Biochemistry and Department of Statistics, Brigham Young University, Provo, Utah 84602, USA.
Abstract:
Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monoliths were synthesized inside 75 mum i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The effect of gradient rate and initial salt concentration on the retention of proteins were investigated. Mass recovery was found to be greater than 96%, indicating the biocompatibility of this monolith. The monolith was mechanically stable and showed nearly no swelling or shrinking in different polarity solvents. The preparation of this in situ polymerized acrylate monolithic column was highly reproducible. The run-to-run and column-to-column reproducibilities were less than 2.0% relative standard deviation (RSD) on the basis of the retention times of protein standards. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles.
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