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

Module3 Introduction to Chromatography

Introduction

Operators preparing a programmable chromatography skid with packed column for Sterilization-In-Place prior to virus purification run. Photographer : Randy Monceaux - 12/2008 (c) Sanofi Pasteur - Full rights issue and unlimited usage

As you’ve already learned, we can engineer cells to make many things: therapeutic proteins like insulin, enzymes that help clothes get cleaner in cold water, antivenom antibodies, and more. Once the engineered cells make the protein of interest (POI), though, we still have to purify it away from all the other cellular components - and that usually involves chromatography.

Chromatography is a technique that separates molecules from a mixture on the basis of how the molecules interact with a stationary phase (which does not move) and a mobile phase (which does move). A very simple chromatography experiment can be performed using coffee filter paper, a glass of water, and a black magic marker. If you draw a line on the filter paper with the black magic marker, and place one end of the filter paper in the water, water will soak up the filter. When it hits the black ink, you will see the colors that make up the black separate out. This is because the various pigments that together make black have different affinities for the water (the mobile phase) and the filter paper (the stationary phase). In the experiment pictured below, the green pigment is more mobile than the reddish one.

Paper chromatography of black marker ink.

The tools you will use to purify a POI in this course are more sophisticated than filter paper and water, but the general principle of chromatography is the same: separation of molecules using a stationary and mobile phase.  In this module, you’ll learn about three commonly used variants of column chromatography, and perform a separation using size exclusion. In the final project for the course, you’ll use affinity chromatography to purify the BglA enzyme from the cells you transformed in BTEC 1100.

Objectives

- Describe chromatography.
- Explain how affinity, size exclusion, and ion exchange chromatography methods differ from each other.
- Predict when the desired molecule will elute.
- Analyze elution profiles to identify fractions of interest.
- Purify small molecules via chromatography.