Chromatography is a technique used to isolate the various components of a mixture and this makes its application in analysis of biomolecules very important. It is used to separate and analyse the complex DNA sequences and other compounds, and also the concentration of the samples. There are many types of chromatography used in the study of biomolecules which range from DNA/RNA to recombinant proteins and antibodies. Here are some types of chromatography that you should know about.
High Performance Liquid Chromatography
Small particles and High pressure is required to carry out this type of liquid chromatography. HPLC has many forms and its application revolves around drug analysis and other forensic applications. There are forms of HPLC which specifically deal with enzymology and purification of other biomolecules.
The reversed phase chromatography has a larger application in industry. In this the stationary phase is non-polar, while the solvent or mobile phase used is polar which is opposite to normal chromatography where stationary phase is polar and the mobile phase is non-polar. The advantage of reverse phase liquid chromatography is that it allows the separation of a large variety of samples, with a wide range of molecular weights and polarities involved. It is easy to use and results are attained rapidly.
Fast Protein Liquid Chromatography
FPLC is also a form of liquid chromatography and it specializes in separating proteins from complexes, as the name suggests. FPLC is popularly used in enzymology, with a complete setup designed especially for separation of proteins and other biomolecules. Cross linked agarose beads are used.
Aqueous- Normal Phase Chromatography
This type of chromatography has a special feature, it has a mobile phase which is somewhere between polar and non-polar. The mobile phase is based on an organic solvent and a small amount of water which results in it being semi polar.
This type is again used in the purification of proteins which are bound to tags. The proteins being analysed are marked or labelled with compounds like antigens or biotins. To get pure proteins in the end, the labels are removed; the labels are just there to provide accurate separation of proteins. The mechanism uses a property of biomolecules i.e. affinity for metals, hence various metals are used in the chromatography columns. Immobilized Metal Affinity Chromatography is an advanced and much refined version of affinity chromatography used in identification of biomolecules these days.