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Analytical Chem JoVE (Open Access) Citable · DOI

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

DOI: 10.3791/3060-v
What you'll learn
  • ✓ Set up and configure automated medium-pressure liquid chromatography system for HIC
  • ✓ Execute column scouting protocol to identify optimal hydrophobic interaction chromatography media
  • ✓ Interpret chromatogram data and fluorescent fraction analysis for protein purification optimization
Protocol

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Difficulty
advanced
Total time
~4–6 hours per column screening run (including equilibration, sample loading, and data collection)

Steps

1
Prepare buffers and protein sample

Prepare and characterize buffers and protein sample required for HIC screening. Ensure sample is properly diluted and ready for injection into the automated chromatography system.

▶ 01:30
2
Assemble DuoFlow chromatography system plumbing

Connect and configure the medium-pressure liquid chromatography system including pump, detector, automated buffer blending module, sample loop, and column selection valve according to manufacturer specifications.

▶ 02:48
3
Prime system lines and equilibrate HIC columns

Prime all fluidic lines with appropriate solvents, program the chromatography run method, and equilibrate each HIC column media to baseline conditions before sample injection.

▶ 04:11
4
Execute automated column scouting protocol

Load sample into the dynamic injection loop, configure software parameters for sequential column selection and multi-wavelength detection, and run the automated screening protocol to test multiple HIC media.

▶ 05:43
5
Analyze chromatograms and fluorescent fraction data

Review representative chromatograms and fluorescent fraction collection data to identify the optimal HIC column media for your protein purification workflow.

▶ 07:36
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