Chromatography: A Powerful Analytical Technique
What is Chromatography?
Chromatography refers to a set of laboratory techniques that are used to separate the components or solutes of a mixture for qualitative or quantitative analysis. The basic principle of chromatography is that the components of a mixture travel through a stationary phase (solid or liquid) at different rates depending on their chemical properties and affinity. A mobile phase (liquid or gas) carries the sample molecules and interacts differently with the stationary phase, causing the separation of components.
The separated components are then detected and identified, usually by their retention times and peak characteristics. Chromatography effectively acts like a sieve that filters and separates mixtures based on molecular properties. This allows the identification, purification, and quantification of each component in a sample.
Types of Chromatography
There are several major types of chromatography that are commonly used based on the stationary and mobile phases:
Gas Chromatography (GC): Used to separate and analyze volatile mixtures that can be vaporized without decomposition. The mobile phase is an inert gas (helium, nitrogen etc.) and the stationary phase is coated on an internal surface of a glass or metal tube. GC is best used for thermally stable compounds with lower molecular weights.
Liquid Chromatography (LC): The mobile phase is a liquid like water or organic solvents while the stationary phase is usually small solid particles packed into a column. LC methods include normal phase, reverse phase, ion exchange and size exclusion chromatography based on the interactions between components and stationary phase. It is used for thermally unstable, polar, high molecular weight compounds dissolved in liquid samples.
Ion-Exchange Chromatography: Used to separate ionic compounds based on their charge. The stationary phase contains ion exchangers that bind ions of opposite charge from the mobile phase solution. It is commonly used in water purification and analysis of amino acids, peptides and proteins.
Affinity Chromatography: Employs biological molecules like antigens, enzymes or ligands immobilized onto the stationary phase. It specifically isolates molecules that have high binding affinity for the ligand, used for enzyme purification and studied biomolecular interactions.
Thin Layer Chromatography (TLC): A simple planar separation technique where the stationary phase is a thin layer of adsorbent like silica gel coated onto a flat surface. Used for checking purity and determining Rf values as a fast analytical method.
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