Chromatography is a technique for separating chemical mixtures that relies on differences in partitioning between a moving fluid phase and a stationary phase. The chemical components interacts differently with the stationary phase, causing them to elute from the column at different rates. This allows for the separation, identification, and purification of individual chemical components from complex mixtures such as products of chemical reactions, extracts of plants, or bodily fluids like blood. It has become indispensable for many areas of science and industry such as pharmaceutical, chemical, biological, forensic and environmental research.
Types
There are several modes of it which rely on different separation principles. Some major types are:
Gas chromatography (GC)
GC involves vaporizing the sample into a gas and passing it through a long, narrow tube called a column filled with an inert material called the stationary phase. The different components of the sample interact differently with the stationary phase as they flow through under the force of a carrier gas (the mobile phase). This causes the components to elute from the column at different times, allowing their separation and detection. GC is commonly used for analyzing volatile organic components in mixtures.
Liquid chromatography (LC)
In LC, the sample is dissolved in a liquid and pumped under pressure through a column filled with tiny solid particles chromatography or porous beads called the stationary phase. The different analytes partition between the stationary and mobile liquid phases as they flow through the column. Based on their interactions, the analytes elute from the column at varying retention times. LC excels at separating both volatile and non-volatile components in mixtures and comes in different modes such as high performance liquid HPLC).
Thin layer chromatography (TLC)
TLC uses a thin, even layer of adsorbent material like silica spread on an inert backing as the stationary phase. A small spot or band of the sample mixture is applied onto the plate, which is then developed by rising movement of the mobile phase, usually a mixture of solvents. Differential migration of sample components occurs as they partition between the stationary and mobile phases, resulting in separation. TLC gives a preliminary analysis and is useful for teaching purposes.
Paper chromatography
In this, a piece of filter paper acts as the stationary phase. A small spot of the sample is applied to the paper which is then placed vertically in a glass jar containing a solvent. Capillary action causes the solvent to rise through the stationary phase carrying the sample components. They separate based on their partitioning, resulting in colored spots at different heights on the paper. It was widely used as a simple separation and analytical technique before more advanced modes developed.
Ion-exchange chromatography
Here, the stationary phase contains ionizable functional groups that can exchange or displace ions of same charge from the mixture. This separation mode relies on differential interactions between charged analytes and the immobilized exchange groups on the stationary phase. It finds applications in purifying or isolating charged compounds like proteins, peptides, amino acids, etc.
Affinity chromatography
In this mode, antibodies, antigens, hormones or other binding reagents are attached to the stationary phase. The analytes that bind strongly are retained more by the stationary phase while unbound or weakly bound components elute quickly. It enables isolation and purification of specific target compounds from complex mixtures using biological binding interactions.
Modes of Detection
Once separated components exit the column during it, they need to be detected and identified. Common detection techniques include:
- Flame ionization detector (FID) - Used for GC, detects organic compounds.
- Thermal conductivity detector (TCD) - Used for GC of inorganic gases.
- Mass spectrometer (MS) - Provides molecular weight information for compound identification in GC-MS or LC-MS.
- Ultraviolet-visible (UV-Vis) detector - Detects analytes that absorb at specific wavelengths. Used for LC.
- Evaporative light scattering detector (ELSD) - Detects non-UV absorbing compounds in LC.
- Refractive index (RI) detector - Detects changes in refractive index for quantitation in LC.
- Electrochemical detector - Sensitive to electroactive compounds separated by LC.
Applications
Given its versatility and high resolving power, it has widespread applications across many different fields:
Pharmaceutical industry - Used for analyzing drug purity, impurities profiling, stability studies and quality control during drug development, manufacturing and shelf life.
Environmental analysis - Determines concentrations of organic pollutants, pesticides and their breakdown products in air, water and soil. Helps in monitoring environmental contamination.
Food and flavor analysis - Identifies aroma compounds, separation of plant extracts, adulteration detection in edible oils, etc. Important role in ensuring food safety and quality.
Forensic science - Fingerprints drugs and poisons in body fluids, helps determine causes and times of death in toxicology investigations involving controlled substances.
Petroleum and polymer analysis - Separates, identifies and quantifies chemical components in crude oil, gasoline, plastics for refining and manufacturing applications.
Biotechnology - Purifies proteins, separates complex biomolecules like enzymes, antibodies for research and therapies. Essential tool in proteomics and metabolomics studies.
Clinical diagnostics - Measures drug, toxin and metabolite levels in serum, urine to monitor therapy and investigate diseases. Important role in personalized medicine development.
Quality control and process optimization - Helps fine tune industrial chemical production by monitoring reaction progress and isolating impurities. Ensures consistent product quality.
it has become a mainstay separation and analytical technique across science due to its high resolving power, versatility and ability to separate, identify and quantify components in even complex mixtures. Advances in instrumentation continue expanding its applications in diverse fields ranging from pharmaceuticals to forensics to environmental monitoring.
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