The techniques and methods that are developed for protein purification has played an important role for advancements made in Biotechnology. Protein purification procedure may involve one-step precipitation method to large scale validated processes. Chromatography is involved in most of protein purification procedures, because of that chromatography has become a very important part of laboratory that involves protein purification. Chromatography is the separation of a mixture in to individual components using a stationary phase and mobile phase. Chromatography was originally discovered by Russian botanist Mikhail Tswett in 1906. He separated plant leaf pigments in solution by passing it through a column of solid absorbent. The principle of separation can be either partition or adsorption. If the principle is adsorption the stationary phase should be solid and when principle is partition the stationary phase is liquid, where as in both cases moving phase the mobile phase can be a liquid or gas.
When dealing with chromatography system mainly two theories applied:
1. Plate theory which is equilibrium between the stationary phase and mobile phase. It is also used to find the volume and column efficiency.
2. Rate theory which is used to find migration of molecules in a column. This may be band shape, broadening and the diffusion of a solute.
In chromatography the basic procedure to follow is: Flow the solution containing the protein of interest through a column. Column can be packed with different materials. As different proteins has different properties they interact differently with column material and depending on the time required to pass the column, or the condition required to elute the protein from the column. As proteins elute from the column their concentration is detected by absorbance at 280nm.
In order to purify proteins scientists can use a number of different chromatographic techniques. Such as:
1. Size exclusion chromatography: separation according to size
2. Ion exchange chromatography: separation according to charged molecules
3. Hydrophobic interaction chromatography: separation according to hydrophobic interaction between the sample and the stationary phase.
When dealing with chromatography system mainly two theories applied:
1. Plate theory which is equilibrium between the stationary phase and mobile phase. It is also used to find the volume and column efficiency.
2. Rate theory which is used to find migration of molecules in a column. This may be band shape, broadening and the diffusion of a solute.
In chromatography the basic procedure to follow is: Flow the solution containing the protein of interest through a column. Column can be packed with different materials. As different proteins has different properties they interact differently with column material and depending on the time required to pass the column, or the condition required to elute the protein from the column. As proteins elute from the column their concentration is detected by absorbance at 280nm.
In order to purify proteins scientists can use a number of different chromatographic techniques. Such as:
1. Size exclusion chromatography: separation according to size
2. Ion exchange chromatography: separation according to charged molecules
3. Hydrophobic interaction chromatography: separation according to hydrophobic interaction between the sample and the stationary phase.