The different types of laboratory techniques used in the separation of mixtures are grouped under an umbrella term, chromatography. The process through which constituents of a mixture are separated and analyzed by physical means is referred to as chromatography. Apart from the different criteria of classification of chromatography discussed below, the basic criterion is the purpose for which this process is carried out. On the basis of this criterion, the process of chromatography is classified into analytical and preparative. The former is carried out for the purpose of measuring the amount of an analyte present in a mixture. On the other hand, preparative chromatography is used for separating the components of a mixture for their further use. Depending on the techniques used in chromatography, the process is broadly classified as adsorption and partition chromatography. An attempt to explain the different types of chromatography is made through this article. Let us find more about the different procedures.
In this form of chromatography, the chemical mixtures in question are passed over an adsorbent bed. Different compounds present in the mixture get adsorbed on the bed at different rates. This process is mostly carried out for analytical separation. Adsorption chromatography is further divided into 'affinity' and 'ion-exchange' chromatography.
The mechanism of ion-exchange which is used in this form of chromatography allows to carry out the segregation of analytes. This kind of segregation/separation can be performed in 2 different modes, i.e. planar and column. Separation of charged compounds like peptides, amino acids, proteins, etc. takes place through a charged stationary phase.
The column chromatography technique uses a set-up in which the stationary phase is placed in a column. There are two ways through which the stationary phase is placed/positioned in a column: either it entirely fills the column or lines the walls of the column.
The stationary phase is placed on a plane surface. The set-up is unlike the one used in column chromatography where stationary phase is placed in a column. Here, a plane surface is used. The plane surface could be anything from paper to glass.
The non-covalent interaction which takes place between the analyte in question and certain molecules is the basis of working of affinity chromatography. Purification of proteins bound to tags is conducted with this technique.
In this separation technique, components of the given mixture are separated through the use of partition of a solute between two solvents. In the process, one of the solvents is immobilized by means of a substance present in the filter paper or column.
Gel Filtration Chromatography
This technique is also known as gel permeation or size exclusion chromatography. Molecules of the mixture in question are separated on the basis of their size. Technically speaking, the process of separation is carried out on the basis of hydrodynamic diameter (size) of molecules. Larger molecules of the mixture are unable to enter the pores of media; therefore, molecules are washed out quickly. On the other hand, smaller molecules take more time to elute because they are able to enter the pores of media.
High Performance Liquid Chromatography
In this type of chromatography, separation of compounds is carried out on the basis of their idiosyncratic polarities. Interaction of these compounds with the stationary phase of the column too is considered. Equipment needed for carrying out high performance liquid chromatography includes a pump (used for moving the mobile phase and analyte through the column), stationary phase and a detector. Retention time for the analyte is also provided by the detector. Depending on the strength of interactions taking place between the analyte and stationary phase, retention time can vary.
This form of chromatography uses cylinders wherein gas is stored under pressure. These gases do the work of carrying the solute. The carrier gas that is commonly used in this chromatography is helium. Flame ionization detectors and thermal conductivity are used in gas chromatography. There are three sub-types of gas chromatography which include the following: gas-liquid chromatography, gas adsorption chromatography and capillary gas chromatography. In gas-liquid chromatography, an inert porous solid is used as the stationary phase. The stationary phase used in gas chromatography is a bed formed by an adsorbent. In capillary gas chromatography, the adsorbents form a layer on fused silica or glass which line the capillary walls.
Pyrolysis Gas Chromatography
This method of chromatography makes use of pyrolysis i.e. decomposition of the sample with the help of thermal power. The process of pyrolysis is followed by the regular procedure of gas chromatography. Resistive heating, inductive heating and heating in isothermal furnace are the three methods used for carrying out pyrolysis in this technique. The volatile fragments formed by heating (at a temperature of 600-1000 °C) are separated by means of gas chromatography.
This technique employs a method which is just opposite to that of normal phase chromatography. In reverse-phase chromatography, the stationary phase is made up of hydrophobic compounds; they attract the hydrophobic compounds present in the mobile phase. Here, the polarity of mobile phase is reduced in order to allow the hydrophobic molecules to elute.
The technique of chromatography which is meant for separation of compounds from mixtures thus, holds immense importance in fields like biochemistry, biotechnology and many other. An attempt to list as many types of chromatography as possible is made in this write-up.