Thin-layer chromatography is a technique where the components of mixtures separate by differential migration through a planar bed of a stationary phase, the mobile phase flowing by virtue of capillary forces.
The solutes are detected in situ on the surface of the thin-layer plate by visualizing reagents after the chromatography has been completed. A variety of finely-divided particulate sorbents are used as thin-layer stationary phases. These include silica-gel, cellulose powder, ion exchange resins, restricted pore-size materials, and chiral selectors.
Single solvents or blends of two or more solvents having the appropriate overall polarity necessary to achieve the required separation are used as mobile phases. They range from non polar hydrocarbons to polar alcohols, water, and acidic or basic solvents.
Methods of visualizing solutes include spraying the surface of the thin layer plate with a chromogenic reagent, or viewing it under a UV lamp if the sorbent has been treated with a fluorescent indicator.
Alternative development procedures aimed at improving chromatographic performance have been introduced, and new stationary phases are becoming available.
Thin-layer chromatography is used primarily as a qualitative analytical technique for the identification of organic and inorganic solutes by comparisons of samples with standards chromatographed simultaneously. Quantitative analysis is possible but precision is relatively poor.
Gas chromatography
Gas chromatography is a technique for the separation of volatile components of mixtures by differential migration through a column containing a liquid or solid stationary phase. Solutes are transported through the column by a gaseous mobile phase and are detected as they are eluted.
The mobile phase is an inert gas, generally nitrogen or helium, supplied from a cylinder via pressure and flow controls, and passing through purification cartridges before entering the column. Gaseous, liquid and solid samples are introduced into the flowing mobile phase at the top of the column through an injection port using a microsyringe, valve or other device.
Columns are either long, narrow, capillary tubes with the stationary phase coated onto the inside wall, or shorter, larger diameter tubes packed with a particulate stationary phase. Stationary phases are high boiling liquids, waxes or solid sorbents.
The column is enclosed in a thermostatically-controlled oven that is maintained at a steady temperature or programmed to increase progressively during a separation.
Solutes are detected in the mobile phase as they are eluted from the end of the column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a function of time.
A dedicated microcomputer is an integral part of a modern gas chromatograph. Software packages facilitate the control and monitoring of instrumental parameters, and the display and processing of data.
High-performance liquid chromatography
High-performance liquid chromatography (HPLC) is a technique for the separation of components of mixtures by differential migration through a column containing a microparticulate solid stationary phase. Solutes are transported through the column by a pressurized flow of liquid mobile phase, and are detected as they are eluted. The mobile phase is either a single solvent or a blend of two or more having the appropriate eluting power for the sample components. It ranges from a nonpolar liquid to aqueous buffers mixed with an organic solvent.
The solvent delivery system comprises a means of degassing, filtering and blending up to four solvents which are then delivered to the top of the column under pressure by a constant flow pump.
Liquid samples or solutions are introduced into the flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector that is loaded with a syringe.
Columns are straight lengths of stainless steel tubing tightly packed with a microparticulate stationary phase. The column packings are chemically modified silicas, unmodified silica or polymeric resins or gels.
Solutes are detected in the mobile phase as they are eluted from the end of the column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a function of time.
A dedicated microcomputer is an integral part of a modern highperformance liquid chromatograph. Software packages facilitate the control and monitoring of instrumental parameters, and the display and processing of data.
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