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

Module3 Size Exclusion Chromatography

The elution profile of a size-exclusion chromatography experiment.

Size exclusion chromatography is also known as gel filtration, gel permeation, and molecular sieve chromatography. As you saw in the introductory video, it uses microscopic beads that have pores of various sizes. Some proteins are small enough to fit inside the pores, which makes them travel through the column more slowly. Large proteins that don’t fit into the pores will travel through quickly. (Note that this is the opposite of what happens in gel electrophoresis.)

The video below shows you what is happening inside the column. The opening shot shows you what the column looks like; this column is designed for use with an FPLC (Fast Protein Liquid Chromatography) instrument. The FPLC instrument has an awesome feature: as liquid comes off the column, a built-in spectrophotometer monitors the absorbance at 280 nm. 280 nm is the wavelength that is most strongly absorbed by proteins. This means that, as the liquid comes off the column, you have a real-time view of how much protein is coming off the column. You will use the FPLC in your final project for this course.

In the video, you will see a graph on the right side (see screenshot below). This is like the graph that the FPLC instrument will generate for you. The x-axis is time/volume; it reflects how much time has passed in the experiment, which correlates which how much volume has passed through the column. The y-axis is absorption: the absorbance at 280 nm, which reflects how much protein is coming off the column. The peaks on the graph represent proteins of distinct sizes, and they have color coded the peaks to match the color used in the video.

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