What is chromatography?
- Chromatography is a vital biophysical technique that enables the separation, differentiation, and purification of mixture constituents for quantitative and qualitative analysis.
- Chromatography was first used by Russian botanist Mikhail Tswett in 1906.
- In 1952, James and Martin first explored the utilisation of chromatography for analysis, while researchers examined fatty acid combinations using gas chromatography.
- Various chromatographic techniques isolate substances by utilising differences in size, binding affinities, charge, and other characteristics.
- It is a very effective isolation technique used in a variety of scientific domains and is frequently the only means to isolate ingredients from complex mixtures.
Principle of Chromatography (how does chromatography work)
Chromatography is based on the principle that mixtures of molecules placed on surfaces or solids and fluid stationary phases (stable phases) separate when migrating with the aid of a mobile phase.
Influencing this separation process are the molecular properties associated with adsorption (liquid-solid), partition (liquid-solid), and attraction or changes in their molecular weights.
Based on the differences, some mixture components move rapidly into the mobile phase and depart the chromatographic system more rapidly, while others move gradually into the stationary phase and transit slowly through the system.
Thus, the chromatography process is based on three components.
- Solid phase:A “solid” phase or “a layer of a liquid adsorbed on the surface of a solid support” consistently constitutes the stationary phase.
- Mobile phase:A “liquid” or “gaseous component” always makes up this phase.
- Separated molecules
The kind of interaction between the stationary phase, mobile phase, and chemicals present in the mixture is the determining factor in the separation of molecules.
Types of Chromatography
- To isolate compounds, several strategies and characteristics can be used, like size and shape, total charge, hydrophobic groups on the surface, and binding capacity to the stationary phase.
- As a consequence, there exist a variety of chromatography techniques utilising distinct equipment and operating principles.
- For example, ion exchange, surface adsorption, partitioning, and size exclusion are four processes employed in four isolation methods depending on molecular characteristics and interaction type.
- Several chromatography techniques, including column, thin-layer, and paper chromatography, are founded on the stationary bed.
Commonly employed chromatography techniques include:
- Column chromatography
- Ion-exchange chromatography
- Gel-permeation (molecular sieve) chromatography
- Affinity chromatography
- Paper chromatography
- Thin-layer chromatography
- Gas chromatography (GS)
- Dye-ligand chromatography
- Hydrophobic interaction chromatography
- Pseudoaffinity chromatography
- High-pressure liquid chromatography (HPLC)
Applications of Chromatography
- identifying and analysing samples for the existence of chemicals or trace components.
- Separating substances according to their elemental composition and molecular weight.
- It detects the purity of the mixture and unidentified chemicals.
- In drug development
- evaluating samples of water and examining the state of the air.
- Polychlorinated biphenyl (PCB) in oils and pesticides is one of several pollutants that may be found using HPLC and GC.
- In several applications of the biological sciences,
- In the detection of additives and food rotting,
- evaluating the nutrient content of food.
Blood and hair samples from crime scenes are analysed in forensic pathology and crime scene testing.
Studies in Molecular Biology
Along with nucleic acid research, metabolomics and proteomics are studied using a variety of hyphenated chromatography methods, such as EC-LC-MS.
In addition to the fuel industry, biotechnology, and biochemical processes, HPLC is utilised in protein separation operations such as insulin purification, plasma fractionation, as well as enzyme purification.