Chromatography

By Levi Clancy for Student Reader on
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In chromatrography, molecules are dissolved in a solution (phenol) and migrate by simple diffusion into a matrix (usually a special type of paper).

The osmotic pressure and mass of the molecule determines the migration speed/location. Chromatography has come a long way. We used liquid chromatography, but now use high performance liquid chromatography (HPLC) which utilized pressures and a complex matrix of beads and electrical charges to separate molecules very thoroughly. HPLC also gives a readout of properties of the molecules (molecular weight, charge, size).

There are many types of chromatography: affinity chromatography (by ligand-binding), column chromatography (chemical nature), gel filtration chromatography (size) and ion exchange chromatography (charge).

Affinity chromatography

Affinity chromatography separates molecules according to their affinity to specific ligand, such as antibody. Proteins are separated according to ability to bind to ligands in cylindrical columns filled with water permeable solid matrix (also called bead, resin or gel) of different properties. Bead can be covalently linked to the ligand that the target protein will bind to. This method allows highly specific purification of a single protein. Affinity chromatography does not detect the target protein according to special properties such as enzymatic activity, ligand-binding activity, special calorimetric reaction, etc.

Column chromatography

Column chromatography is a huge procedure used to purify pharmaceutical proteins. The column chromatography apparatus is usually composed of: (1) A cylindrical column filled with resins (matrix, beads, etc). The type of chromatography is determined by the chemical nature of the resin. (2) A pump to push solution flow through the column. (3) A fraction collector to collect solutions flew out from the column. (4) A detector to detect proteins in different fractions.

Gel filtration chromatography

Gel filtration chromatography (or sizing column chromatography) involves gel filtration to separate molecules according to their size. The larger molecules migrate faster in the gel filtration column. The smaller molecules migrate slower because they travel through pores of the porous beads. This separates according to size.

Ion exchange chromatography

Ion exchange chromatography separates according to charge. Anion exchange resins bind to negatively-charged anions.Cation exchange resins bind to positively-charged cations.