Module3 Introduction to Chromatography

Introduction

Two people wearing cleanroom suits work on a stainless steel piece of equipment that is about 6 feet wide, tall, and deep.

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.

The first photo shows the experimental setup: a beaker with about half an inch of water, a black marker, and a piece of filter paper with a black line made by the marker. The second photo shows the start of the experiment: the marker laid across the top of the beaker, with the filter paper attached to the marker and the end of the filter paper hanging into the water in the beaker. The water is starting to wick up the filter paper toward the black line. The third photo shows that, once the water has wicked up another inch or so, the black ink has separated into green pigment, which has moved up the paper quickly, and a reddish pigment, which has traveled more slowly.
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