Chromatography is a chemical technique that physically separates a mixture into its component parts. It is a highly effective technique that is used in multiple industries, from food to pharmaceutical and across all branches of science. Often, it is the only way to separate components, down to the molecular level, from complex mixtures.
Liquid chromatography and gas chromatography are the dominant forms of chromatography. These broadly used techniques include multiple types of chromatography. For instance, high-performance liquid chromatography (HPLC) is a fast, effective process used to maintain product purity and to separate different molecules- like drug APIs, proteins and nucleic acids. Ultra high-performance liquid chromatography (UHPLC) takes this to another extreme, using very small particle size columns and instrumentation capable of dealing with pressures in excess of 600 bar.
Chromatography usually begins with a sample that has individual components that require separation and identification. Components in the sample can be of either solid or liquid origins. A sample mixture can be liquid (for liquid chromatography) or a gas state (for gas chromatography).
There are two phases in chromatography, the mobile phase and the stationary phase. The mobile phase either carries the sample mixture to the stationary phase or disrupts the sample mixture from stationary phase during elution. In liquid chromatography the sample should be compatible with the mobile phase. For gas chromatography the sample, when injected into the process, will convert into a gas—if the sample began in a liquid state.
With regards to the stationary phase, the sample mixture moves through a second material, often a silica based phase, which interacts with the sample analytes differently depending on their chemical and physical properties. This leads to a differential migration through the column which then separates the molecules that are subsequently detected by the system and show as peaks in a chromatogram.
Reversed phase HPLC separates analytes based on their hydrophobicity, while ion exchange is utilized for charged analytes. Other techniques include size exclusion chromatography SEC (GPC/GFC) which can separate molecules by size/shape and hydrophobic interaction chromatography (HIC) which, like reversed-phase, separates based on hydrophobicity but is more commonly employed for biomolecules since it is a more gentle technique keeping protein structure intact.
Normal phase chromatography is used for more polar analytes as is hydrophilic liquid chromatography (HILIC). While more specialized types of chromatography can also be used for chiral molecules and very specific types of molecules can be retained and captured using affinity chromatography.
