Chromatography is a versatile and widely used technique in instrumental analysis for the separation, identification, and quantification of components in a mixture.
The fundamental principle behind chromatography is the differential migration of solute molecules through a medium driven by the differences in their interaction with the stationary and mobile phases.
Types of Chromatography
Based on the physical state of the mobile phase and the mechanisms of separation, chromatography can be broadly categorized
1. Gas Chromatography (GC):
The mobile phase is a gas (often helium or nitrogen), and the stationary phase can be a solid or a high boiling point liquid on a solid support.
GC is particularly suitable for volatile organic compounds.
2. High Performance Liquid Chromatography (HPLC):
Here, the mobile phase is a liquid, and the stationary phase is usually a granular solid packed into a column.
Depending on the mode of separation, HPLC can be further sub-divided into reversed-phase, normal-phase, size-exclusion, ion-exchange, and more.
3. Thin Layer Chromatography (TLC):
This uses a thin layer of stationary phase (usually a solid) on a flat plate, and the sample is allowed to move through this phase by capillary action using a liquid solvent.
4. Ion-Exchange Chromatography:
Used to separate ions based on their charge.
The stationary phase consists of charged particles that can exchange ions with the sample.
5. Size-Exclusion Chromatography (SEC):
Separates molecules based on their size.
Larger molecules are excluded and elute first, while smaller molecules enter the pores of the stationary phase and elute later.
6. Affinity Chromatography:
Separates molecules based on a specific interaction between the solute molecule and a molecule immobilized on the stationary phase.
7. Capillary Electrophoresis (CE):
Although not strictly chromatography, it's a separation technique wherein molecules are separated based on their charge and size, driven by an electric field.
Key Concepts in Chromatograph
1. Mobile Phase:
This is the phase that moves, carrying the sample with it. It can be a gas (as in GC) or a liquid (as in HPLC).
2. Stationary Phase:
This is the phase that remains stationary. It can be a solid, a solid coated with a liquid, or a gel.
3. Retention Time (tR):
The time taken for a particular compound to elute from the column. It's a characteristic property for a given compound under specific chromatographic conditions.
4. Elution:
The process by which solutes are washed out or carried out by the mobile phase as it moves through the stationary phase.
Applications of Chromatography
1. Qualitative Analysis:
Determining which components are present in a sample.
2. Quantitative Analysis:
Measuring the amount of specific compounds in a sample.
3. Purification:
Isolating a desired component from a mixture.
Detectors in Chromatography
After separation, the compounds eluting from the column are detected using various detectors like:
Flame Ionization Detector (FID): Common in GC.
Ultraviolet-Visible (UV-Vis) Detector: Common in HPLC for compounds that absorb UV or visible light.
Mass Spectrometry (MS): Can be coupled with GC or HPLC for structural identification and quantification.
Refractive Index Detector, Electrochemical Detectors, Fluorescence Detectors, and more, based on the properties of the analytes.