In order to study proteins individually, they need to be separated from each other. This can be done due to the fact that they have different sizes, shapes, hydrophobicity and have different affinities for other molecules.
Examples of common chromatography methods include:
- ion exchange chromatography
- Ion exchange chromatographysize exclusion chromatography
- Size exclusion chromatographyaffinity binding chromatography
- hydrophobic interaction chromatography.
Ion exchange chromatography separates molecules according to their overall charge. Positively charged molecules stick to the column whereas negatively charged molecules are eluted first with the buffer. A Salt solution can be added to the column to elute the positively charged molecules afterwards. The ions in the salt solution dissociate and break the electric interactions between the positively charged molecules and the negatively charged column. This process as a whole is called eluting and the mixture left behind is called the elute.
Affinity chromatography takes advantage of the high affinity of many proteins for specific chemical groups. Affinity chromatography is more powerful than ion exchange chromatography by means of purifying proteins. Adding free binding partner elutes the protein.
The matrix for column chromatography varies although the material is normally packed in the column in the form of small porous beads. Gel filtration columns separate proteins according to their size. Molecules small enough to enter holes in the beads are delayed and travel more slowly through the column, and those that are too large to enter the beads are washed out of the column first. These columns also allow an estimate of protein size.
Types of chromatography include: two-dimensional chromatography, thin layer chromatography (TLC) and paper chromatography.High-performance liquid chromatography (HPLC) and gas chromatography (GC) also types of chromatography. They are mainly used to separate and identify drugs in the pharmacy. It is possible to analyse the substances quantitative just like qualitative.
Without chromatography, and the work of Banting, Best and a biochemist called Collip (who perfected the purification), we would have no treatment for type I diabetes, that is, insulin would not have been discovered. Without chromatography we wouldn’t be able to sequence DNA, perform PCR, and many drugs and biological mechanisms would not have been discovered.