What is Chromatography?
One of the most helpful technique till date in the field of biochemistry is the Chromatography. This technique analyses the seperation of the closely related compound from any given mixture. These compounds can be amino acids, proteins, lipids, peptides, carbohydrates, vitmains, and minerals.
The term “Chromatography” was first coined in the late 19th century, where the technique was used to separate the molecules or pigments of the complex mixture.
Principle and Classification
Chromatography is the Greek word, where ‘Chroma‘ means ‘color‘ and ‘graphein ‘- ‘to write‘. This process has two phases – the stationary phase and the mobile phase.
- Mobile Phase – The liquid or gas containing the dissolved mixture of substances that is to be separated is refers as the mobile phase. This is the moving part of the process.
- Stationary Phase – The porous solid mixture, through which the sample contained in the mobile phase has to be run or percolates is the stationary phase. This is the static part of the process. The stationary phase can be either liquid or solid.
The interaction of the mobile phase and the stationary phases out-turn to the separation of the compounds from the mixture (mobile phase). For this process the Physico-chemical properties like ion-exchange, molecular sieving, adsorption, partition, and affinity are involved.
Such interactions between the mobile and the stationary phase, divides the method under different categories like adsorption, ion-exchange, partition, etc. Though the clear differentiation of the types of chromatography is on the ground of either on the stationary phase (thin layer chromatography, paper chromatography) while it can be also on the basis of the both stationary and mobile phases (gas-liquid chromatography).
Various components travel through the stationary phase at different speeds, that results them to separate molecules from one another. The time travelled by these molecules either slowly or quickly and the way they got dispersed on the stationary phase reflects the nature of the components. This travelling time is known as retention time.
Making alterations in the stationary and the mobile phase additionally the conditions deciding the travelling speed, decides the chromatographic methods which have been made, purposefully serving ideal for various mixtures.
1. Partition Chromatography
Depending on the relative affinity to each one of the phase the molecules of the mixture get separated between the mobile and the stationary phase.
2. Adsorption Column Chromatography
Adsorbents such as charcoal powder, silica gel, alumina or calcium hydroxyapatite are been put into the chamber of a glass tube. This play the role of the stationary phase. The mixture is loaded on the column, gradually the absorption of the components took place in a different way onto the absorbent. The separated compounds reach to the exit point at different rates, where they are collected in different tubes and identified later on.
3. High-Performance Liquid Chromatography (HPLC)
Generally, the process of the chromatography is proof-full for the separation of compounds, but on the other hand it is time taking process, therefore to enhance the speed of the process high pressure is applied on the components to be separated which varies from the 5000 – 10, 000 psi (pounds per square inch).
So, this is all about chromatography.