Chromatography involves the physical separation of a mixture of compounds. Chromatography can be used as a purification method but also sees wide use for the identification of compounds based on their chromatographic behavior.

Theory

There are many variations of chromatography, but all involve the dissolution of an analyte into a fluid known as the mobile phase and the passage of this fluid solution across a stationary phase, often a solid or liquid-coated solid.

As the mobile phase comes into contact with the stationary phase, some of the analyte molecules dissolve or adsorb onto the mobile phase. The more the molecules of that substance are retained, the slower their progress through the chromatographic apparatus. Different substances will then move through at different rates, ideally resulting in distinctly identifiable retention times for each substance.

Commonly used chromatographic techniques are identified through the nature of the stationary and mobile phases used, the method for passing the mobile phase through the apparatus, and how separated components are identified.

Paper chromatography

In paper chromatography the stationary phase is a specialized paper made to absorb water to a high level. The mobile phase is usually water or a concentrated salt solution. Paper chromatography has many uses in forensic chemistry due to it's simplicity and availability. However, paper chromatography is limited by the characteristics that only water soluble components can be separated and inaccuracy in RF values. This makes paper chromatography mostly useful to distinguish the differences between two residues rather than their similarities.

Thin layer chromatography

In thin layer chromatography (TLC) a plastic or glass plate is coated with the stationary phase, often alumina, silica, or alkylated silica. The analyte is dissolved in a quick-drying solvent and spotted near the bottom of the plate. The edge of the plate beneath the spot or spots is then dipped and left in a solution of the mobile phase, either an organic solvent or aqueous solution (depending on the nature of the analyte and stationary phase). Capillary action is then allowed to draw the solvent front through the spotted analyte, carrying with it and in the process separating out the analyte's components.

Gas chromatography

In gas chromatography (GC) the analyte and mobile phase must both be gases or be readily introduced into the gas phase by heating. The mobile phase gas must be inert and not reacting with the sample to be analysed. Examples of inert gases are helium and nitrogen gas; while not as inert as helium and nitrogen, hydrogen may also be used.

The gases are passed through a long, narrow (and most often, coiled) tube either packed with a porous stationary phase or whose inner walls are coated with a stationary phase, and the analyte components are detected as they emerge from the far end of the tube. The tube is commonly known as GC column.

Often a time-varying temperature gradient, from lower temperature to higher temperature, is applied to the tube. This first allows the analyte components to partition into the stationary phase and then, as the temperature rises, to differentially force them back into the mobile phase.

Common detectors for gas chromatography are flame ionization detector (FID), electron capture detector (ECD) and mass spectrometry (MS). Different types of sample analysis would require the use of a different type of detectors.

Column chromatography

Column chromatography, like gas chromatography, uses a tube packed with a stationary phase, but the mobile phase is a liquid instead of a gas (It is sometimes known as liquid chromatography or LC). Instead of temperature gradients, a gradient in the composition of the liquid phase can be used to separate components.

Column chromatography can be performed on larger molecules which may not be readily introduced into the gas phase. On the other hand, because of the increased viscosity of liquids compared with gases, liquid chromatography can be a more ponderous process. HPLC (variously high-pressure liquid chromatography or high-performance liquid chromatography) speeds the process and improves its selectivity and sensitivity to a significant degree by forcing the mobile phase through the chromatographic column with high-pressure pumps.

Detection methods

The root of the word chromatography, chroma (Greek khrōma, color) and grafein is "to write", indicates that the separated components in some forms of the technique can be identified by their color alone. But chromatography has now long been performed on colorless compounds that can be identified in other ways.

Analyte components on thin-layer chromatography plates are often identified under ultraviolet light, or by chemical staining in, for example, an iodine chamber or potassium permanganate. Gas chromatographic analytes are detected by changes in the ionization levels of a flame at the output end of the column or by changes in the electrical conductivity of the gas mixture at the end of the column. Liquid chromatography fractions are often analyzed through spectrophotometric techniques, notably UV-visible spectroscopy. When separation with GC or LC is performed in tandem with mass spectrometry (the "hyphenated" techniques of GC-MS and LC-MS), masses of individual fractions are rapidly determined. These methods are frequently employed in analytical and forensic science.