Chromatography is a family of physical separation techniques in which the components of a mixture distribute differently between a stationary phase and a mobile phase moving in a definite direction. These differences cause components to migrate at different rates, allowing them to be separated for analysis or collection. A central tool of analytical chemistry, chromatography encompasses methods classified by the physical state of the mobile phase, the arrangement of the separation medium, and the mechanism responsible for separation. (goldbook.iupac.org)
Historical development
Modern chromatography originated in the work of botanist Mikhail Tswett, who separated plant pigments using a column containing an adsorbent. His experiments produced distinct colored bands, including components associated with chlorophyll. The technique’s name refers to these colored separations, although chromatography is not restricted to colored substances. Tswett’s work established differential retention as a practical means of separating complex mixtures. (nobelprize.org)
A major subsequent development was partition chromatography, devised by Archer J. P. Martin and Richard L. M. Synge. Rather than relying principally on adsorption onto a solid surface, it exploited differences in distribution between phases. Martin and Synge received the 1952 Nobel Prize in Chemistry for this invention. Partition chromatography became an important technique in biochemical investigations and the separation of diverse organic compounds. (nobelprize.org)
Principles and separation mechanisms
The mobile phase transports the sample through or across the stationary phase. Components that spend more time associated with the stationary phase generally migrate more slowly. In adsorption chromatography, separation depends mainly on differences in affinity for an active solid surface. Partition chromatography instead depends chiefly on differences in solubility or distribution between phases. These mechanisms describe chemical interactions, whereas labels such as gas or liquid chromatography describe the mobile phase. (old.iupac.org)
Other mechanisms provide different kinds of selectivity. Ion exchange separates components through differences in their interactions with charged sites. Size-exclusion chromatography separates according to access to pores in the stationary phase: larger species enter fewer pores and typically emerge earlier, while smaller species explore more pore volume and emerge later. Ideally, size exclusion involves no attractive interaction between the sample and stationary phase. (old.iupac.org)
Affinity chromatography uses selective binding to capture a target from a mixture. For example, immobilized protein A can bind antibodies while other components pass through; subsequent changes in conditions release the captured material. This makes affinity methods useful for both purification and measurement of biological products. (agilent.com)
Principal formats
In column chromatography, the separation medium occupies a tube, either as packed material or as a coating on its inner surface. In planar methods, separation occurs on a flat support. Paper chromatography uses paper, while thin-layer chromatography uses a thin layer of adsorbent on a backing plate. These geometrical categories are independent of the particular retention mechanism. (publications.iupac.org)
Gas chromatography (GC) uses a carrier gas to move vaporized sample components through a column. It is suited to substances that can be vaporized without unacceptable decomposition. Many GC columns contain a thin stationary-phase film on the inner wall of a capillary. Temperature control and carrier-gas flow influence the separation, and a detector measures components as they leave the column. (agilent.com)
Liquid chromatography uses a liquid mobile phase. High-performance liquid chromatography (HPLC) employs pumps to drive liquid through a column and combines controlled sample introduction with sensitive detection. Its principal components include a pump, injector or autosampler, column, and detector. In isocratic operation, mobile-phase composition remains constant; in gradient operation, it changes during the separation to alter retention. (agilent.com)
Reversed-phase chromatography uses a relatively nonpolar stationary phase and a more polar mobile phase, often containing water mixed with an organic solvent. Hydrophobic components generally show stronger retention under otherwise comparable conditions. Normal-phase chromatography reverses this polarity arrangement, typically using a polar stationary phase and a less polar mobile phase. (agilent.com)
Chromatograms and performance
A chromatogram records the outcome of a separation, commonly as detector response plotted against time. Components reaching the detector produce peaks. Their retention positions and peak widths describe different aspects of performance: retention concerns migration through the system, while resolution measures how distinctly neighboring peaks are separated relative to their widths. Two peaks may therefore remain poorly separated despite having different retention times. (publications.iupac.org)
Column efficiency is often expressed through a theoretical plate number. The corresponding plate height is the column length divided by that number; smaller plate height indicates greater efficiency per unit length. Plates are a mathematical description of separation performance, not physical compartments within a column. Efficiency and selectivity must be considered together when evaluating a separation. (goldbook.iupac.org)
Detection, quantification, and applications
Chromatography separates components, but identification also depends on the detector and supporting evidence. Coupling chromatography to mass spectrometry adds mass-to-charge information to the separation record, helping distinguish components and investigate co-eluting substances. Quantitative measurements commonly use calibration mixtures of known concentration to establish the relationship between detector response and analyte amount. Internal standards can account for some variation in injection or detection. (agilent.com)
Applications include environmental monitoring, food analysis, petroleum characterization, and biological-product analysis. Different mechanisms answer different questions: size exclusion can reveal aggregates and fragments of proteins, ion exchange can distinguish charge variants, and reversed-phase methods can separate peptides and impurities. Preparative chromatography collects separated material for further use, whereas analytical chromatography emphasizes identification, measurement, or characterization of mixture components. (pubsapp.acs.org)