What is Chromatography? - Definition, Types & Uses

Thin Layer Chromatography, or TLC, uses a flat plate coated with a stationary phase instead of a packed column. One edge of the plate is dipped into the mobile phase, which moves up through the solid material, carrying and separating the analytes. Colorless analytes can be sprayed with a chemical that colors them, so the chemist can see the result. TLC is usually preliminary to more in-depth analysis; it is, however, fast and requires very little equipment.

Gas Chromatographyor GC in which the mobile phase is an inert gas, usually helium, which simply carries along any molecules in the gaseous state. A GC system has an oven for heating the column, and so works best for samples that evaporate readily and do not decompose at high temperature. The stationary phase is a finely divided solid (often coated) packed into a coiled column. The analytes interact either directly with the solid surface (adsorption chromatography) or with the surface coating (partition chromatography). A popular variant on the packed column is the capillary column, a very long, coiled, open, narrow-bore tube with a coating on the inside walls. Capillary columns are particularly efficient at separating analytes with similar chemical structures.

Liquid Chromatography which has various abbreviations, the most common being HPLC, for high-performance liquid chromatography, the mobile phase is a liquid rather than a gas, often water or a simple organic compound, pumped through the stationary phase in a column under pressure. Better for temperature-sensitive compounds and those that do not evaporate easily, the analytes can undergo either adsorption or partition-type interactions as in gas chromatography. HPLC is used widely in the biotechnology industry for separating biological materials such as DNA segments.

Size Exclusion Chromatography or SEC is a little different from either GC or HPLC. Instead of analytes interacting directly with surfaces or coatings, the analytes slow down through the column by entrapment in stationary phase pores. The smallest particles are more likely to get trapped, so the largest particles separate and elute first (thus the name size exclusion) while the smaller ones lag behind. SEC is often used for the separation of very large molecules like proteins and the polymers used to make plastics.

Affinity Chromatography is used extensively in the biotechnology industry, affinity chromatography can purify and concentrate specific species, such as enzymes, proteins, or nucleic acids. The chemist applies the sample to the column, and the desired analyte sticks to the stationary phase. After washing impurities through, a different mobile phase detaches and elutes the analyte.

Lesson Summary

Chromatography is a versatile separation technique widely used to obtain pure compounds from mixtures. All chromatographic techniques depend on a stationary phase, usually a finely divided solid or coated solid, that a mobile phase, usually a gas or liquid, moves through. Each analyte on a system will have a particular distribution ratio between the two phases, and as they progress through, they will separate. A detector can measure both the concentration and retention time of the analytes as they elute from or leave the system. The five main types of chromatography include thin layer chromatography, gas chromatography, high-performance liquid chromatography, size exclusion chromatography, and affinity chromatography.

Things to Remember

• Chromatography: Chromatography is a separation technique used to get pure compounds from different mixtures.
• Stationary Phase: A sample needing separation will move along a stationary phase which is usually a solid, thick liquid or bonded coating that stays contained in the same place.
• Mobile Phase/Eleunt: With a mobile phase, a gas or liquid passes through the sample.
• Analyte: A sample compound is an analyte.
• Distribution Ratio: When an analyte passes through the two phases the separation will have a certain distribution ratio
• Detector: A detector is necessary to measure the the concentration and retention time of analytes leaving the system.

Learning Outcomes

This lesson on chromatography should prepare you to confidently do the following:

• Recount the history of chromatography
• Write the definition of chromatography and list the main phases used for separation testing
• Specify the five main types of chromatography