Biotechnology Basics: Chromatography


What is chromatography?

Chromatography is the separation of molecules based on their physical properties by passing a fluid sample through a solid, stationary matrix.  The interactions between the sample and the matrix influence the way the molecules flow through the column, allowing for their physical separation.

How was chromatography discovered? 

The precursors of today’s chromatography can be traced back to the mid-19th century to analyze plant pigments.  In these experiments, scientists mixed the pigments with solvents and spotted them onto filter paper.  As the pigments traveled through the paper, they separated into distinct pools, creating bands with different colors were produced.

Screen Shot 2020-04-10 at 1.05.40 PMIn the very early 20th century, the botanist Mikhail Tsvet applied the principles of paper separation of plant pigments to develop column chromatography.  Calcium carbonate was packed into a column and used as the separate the pigments instead of paper.  Furthermore, he realized that the solvent used for chromatography was important.  Certain molecules would separate better with polar solvents than non-polar, and vice versa.

The word chromatography is from the Greek words chroma and graphein, which translates to “color writing.”  Fun fact — Tsvet may have named the technique after himself!  His last name in Russian translates to color.  So, by using the Greek chroma, which also means color, he may have been naming the technique after himself.

What properties can we use to separate molecules? 

Picture1In the mid-1900’s, researches expanded the utility of chromatography by developing chromatography matrixes that separated molecules by different physical properties.

  1. Charge: Ion Exchange separates samples based on molecular charge. The matrix holds a positive or negative charge, which uses electrostatic interactions to differentially bind molecules within the sample.  Bound molecules are eluted by flowing solutions with increasing ionic strength over the column.
  2. Size: Size exclusion, also known as gel filtration, separates molecules based on their apparent size.  The gel filtration matrix consists of microscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads.  Molecules with the same molecular weight but different secondary structure will elute differently due to their interactions with matrix.
  3. Protein binding interactions: Affinity chromatography take advantage of the biological activity of the protein to be purified.  This includes binding interactions between a protein and its substrate, or antibody-antigen interactions.
  4. Hydrophobicity: Hydrophobic Interaction Chromatography takes advantage of the surface hydrophobicity of proteins to perform a physical separation.  The column is largely hydrophobic and attracts hydrophobic regions on the molecule’s surface.  Proteins with a strongly hydrophilic surface pass through the column more quickly and will elute first.

Picture2Learn more about using chromatography in your classroom!  Last week, we live-streamed one of our most popular chromatography experiments, EdvoKit #243, Ion Exchange Chromatography.   We’ve prepared the following resources to help you teach this topic in your classroom, whether it is remote or in person!