Liquid Chromatography is a technique used in laboratories to separate dissolved molecules or ions in liquid phase. The process began in the middle of the 19th century when it was used to primarily separate chlorophyll in plant pigments. Since then this process was developed by Mikhail Semyonovich Tsvet, a Russian botanist using calcium carbonate columns in the early 20th century.
There are different kinds of chromatography such as Paper Chromatography, Column Chromatography, Gas Chromatography, Planar Chromatography, Affinity Chromatography, Thin Layer Chromatography and Liquid Chromatography. This article will delve into the details about the latter which is commonly referred to today as High Pressure Liquid Chromatography.
There are two subtypes of this process known as the Normal Phase Liquid Chromatography (NPLC) where the stationary phase is more polar than the mobile phase. The other sub-type is the Reverse Phase Liquid Chromatography (RPLC) where the mobile phase is more polar. This type is also the more commonly used separation technique nowadays.
The process of Liquid Chromatography takes the liquid subject forced in a column that is packed with particles that are spherically or irregularly shaped or porous monolithic layer using very high pressure. It uses complex instrumentations to pump out the liquid sample through a pump to provide faster analysis time and higher resolution. When doing the process, it is essential to determine which mode of separation to use. In general, water insoluble or non-polar samples use the normal phase while soluble samples take the reverse phase. Typically, laboratory experts consider certain factors when choosing the solvents to use. The strength of the solvent is based on scales of silica or alumina and the solvent’s polarity index. The most common reverse phase solvents are methanol, acetonitrile, tetrahydrofuran, and water. These solvents have low viscosity, UV transparency and are miscible with each other.
After considering which type to use – normal chromatography or reverse - the next step is to load the mixture on top of the column followed by more solvent. The different components in the sample mixture pass through this column at different rates due to differences in their partitioning behavior between the mobile liquid phase and stationary phase.
Since variations in temperature affect liquid chromatography on a minimal scale unlike other chromatography methods, Gradient Elutions have been used to reduce analysis time and improve overall resolution of the mixture. However, not all liquid chromatography methods can make use of gradient elutions as liquid-to-liquid separations tend to have unstable results while size exclusions show no good results at all. Ion exchange methods along with adsorption and bonded phase methods use gradient elutions very well.
Liquid Chromatography is most commonly used in preparatory laboratory work to purify and isolate some components of a mixture. Where there exist small disposable columns, liquid chromatography is also used in such ultra trace separations.
Today, Liquid Chromatography is most commonly used as a way to test water samples for harmful contaminants and water pollution. Scientists are still finding ways on how to better this field although current human understanding of this technology is found to be sufficient to play its part in real life application.