Title: Learn: Column Chromatography
Course: BTEC-2020-301-302-303-F21 > Section: Chromatography

Module3 Column Chromatography

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HIC purification of GFP. From left to right, the image shows the HIC column after application of binding buffer, then wash buffer, and finally elution buffer. The photo was taken under UV light to cause the GFP to glow. (Image from BioRad.)

In the simplest forms of liquid column chromatography, a glass or plastic tube with a narrow outlet at the bottom is filled with a bed of microscopic beads. Those beads are the stationary phase of column chromatography. They don’t move, and they have some chemical or physical property that helps separate the mixture of proteins you apply to the column. Flowing through the column, and surrounding the stationary beads, is some sort of buffer. The buffer is the mobile phase that is pulled by gravity, or sometimes pushed with pressure, through the stationary bed. When a cell lysate is applied to the column, the various components will have different affinities for the stationary and mobile phases; some components will “stick” to the stationary phase, while others will “prefer” to be in the mobile phase. By altering the buffer conditions, we can control when the POI (protein of interest) comes off the column (elutes), and hopefully capture it in a tube that contains no other proteins.

In BTEC 1000, you performed hydrophobic interaction chromatography to purify GFP from lysed bacterial cells. Read the following excerpt from the BioRad GFP Purification Kit Instruction Manual to refresh your memory of the process.

Protein Chromatography

In this final step of purifying the Green Fluorescent Protein, the bacterial lysate you prepared will be loaded onto a hydrophobic interaction column (HIC). Remember that GFP contains an abundance of hydrophobic amino acids making this protein much more hydrophobic than most other bacterial proteins. In the first step, you will pass the supernatant containing the bacterial proteins and GFP over an HIC column in a highly salty buffer. The salt causes the three-dimensional structure of proteins to actually change [denature] so that the hydrophobic regions of the protein move to the exterior of the protein and the hydrophilic ("water-loving") regions move to the interior of the protein.

The chromatography column at your workstation contains a matrix of microscopic hydrophobic beads. When your sample is loaded onto this matrix in very salty buffer, the hydrophobic proteins should stick to the beads. The more hydrophobic the proteins, the tighter they will stick. The more hydrophilic the proteins, the less they will stick. As the salt concentration is decreased, the three-dimensional structure of proteins change again [renature] so that the hydrophobic regions of the proteins move back into the interior and the hydrophilic ("water-loving") regions move to the exterior.

You will use these four solutions to complete the chromatography:

Equilibration buffer—A high salt buffer (2 M (NH_4)2SO_4)

Binding buffer—A very high salt buffer (4 M (NH_4)2SO_4)

Wash buffer—A medium salt buffer (1.3 M (NH_4)2SO_4)

Elution buffer—A very low salt buffer (10 mM Tris/EDTA)

Hydrophobic interaction chromatography works well for proteins that have lots of hydrophobic amino acids, like GFP. However, most proteins don’t have lots of hydrophobic amino acids. To purify most proteins, we have to rely on other properties that make them more or less attracted to a stationary phase.

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