A Chromatography column is a device used in chromatography for the separation of chemical compounds. A chromatography column contains the stationary phase, allowing the mobile phase to pass through it. Chromatography columns of different types are used in both gas and liquid chromatography.
While small-scale columns range from inner diameters of 0.5 cm and withstand pressures of up to 130 MPa, [2] industrial large scale columns reach diameters of up to 2 m and operate at considerable lower pressures (below 1 MPa). While it is favorable to view the packed bed of a column large scale columns are manufactured from steel due to its superior resilience.
Chromatography columns can be used as stand-alone devices or in combination with manual or automated chromatography systems. Medium to large columns are almost exclusively operated together with automated systems to decrease the risk of process failure and loss of product.
Transitions between scales are always fluent. There is no sharp cut that defines the end of small- and the beginning of medium/pilot scale. However, chromatography columns with an inner diameter (ID) of up to 5 cm are generally considered small scale or laboratory scale columns.
Small scale chromatography columns are mostly intended for design of experiments (DoE); proof of concept; validation (drug manufacture) or research and development experiments. Columns of this scale category are distinguished by their small dimensions in comparison to chromatography columns intended for larger scales as well as relatively high pressure tolerance and selection of materials in contact with the liquid phase. This is especially important for applications in the biopharmaceutical industry which underlie close scrutiny by regulatory agencies (U.S. Food and Drug Administration; European Medicines Agency).
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Distillation, or classical distillation, is the process of separating the components or substances from a liquid mixture by using selective boiling and condensation. Dry distillation is the heating of solid materials to produce gaseous products. Dry distillation may involve chemical changes such as destructive distillation or cracking and is not discussed under this article. Distillation may result in essentially complete separation, or it may be a partial separation that increases the concentration of selected components in the mixture. In either case, the process exploits differences in the relative volatility of the mixture's components. In industrial applications, distillation is a unit operation of practically universal importance, but it is a physical separation process, not a chemical reaction.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.
Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC-MS is also appropriate for Metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC-MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries.
The van Deemter equation in chromatography, named for Jan van Deemter, relates the variance per unit length of a separation column to the linear mobile phase velocity by considering physical, kinetic, and thermodynamic properties of a separation. These properties include pathways within the column, diffusion, and mass transfer kinetics between stationary and mobile phases. In liquid chromatography, the mobile phase velocity is taken as the exit velocity, that is, the ratio of the flow rate in ml/second to the cross-sectional area of the ‘column-exit flow path.’ For a packed column, the cross-sectional area of the column exit flow path is usually taken as 0.6 times the cross-sectional area of the column. Alternatively, the linear velocity can be taken as the ratio of the column length to the dead time. If the mobile phase is a gas, then the pressure correction must be applied. The variance per unit length of the column is taken as the ratio of the column length to the column efficiency in theoretical plates. The van Deemter equation is a hyperbolic function that predicts that there is an optimum velocity at which there will be the minimum variance per unit column length and, thence, a maximum efficiency. The van Deemter equation was the result of the first application of rate theory to the chromatography elution process.
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.
Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography.
Electrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. It is a combination of size exclusion chromatography and gel electrophoresis. These separation mechanisms operate essentially in superposition along the length of a gel filtration column to which an axial electric field gradient has been added. The molecules are separated by size due to the gel filtration mechanism and by electrophoretic mobility due to the gel electrophoresis mechanism. Additionally there are secondary chromatographic solute retention mechanisms.
In gas chromatography, the Kovats retention index is used to convert retention times into system-independent constants. The index is named after the Hungarian-born Swiss chemist Ervin Kováts, who outlined the concept in the 1950s while performing research into the composition of the essential oils.
Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.
A monolithic HPLC column, or monolithic column, is a column used in high-performance liquid chromatography (HPLC). The internal structure of the monolithic column is created in such a way that many channels form inside the column. The material inside the column which separates the channels can be porous and functionalized. In contrast, most HPLC configurations use particulate packed columns; in these configurations, tiny beads of an inert substance, typically a modified silica, are used inside the column. Monolithic columns can be broken down into two categories, silica-based and polymer-based monoliths. Silica-based monoliths are known for their efficiency in separating smaller molecules while, polymer-based are known for separating large protein molecules.
Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.
A separation process is a method that converts a mixture or solution of chemical substances into two or more distinct product mixtures. In other words, it's a scientific process of distinguishing to two or more substance in order to obtain purity. At least one product mixture of the separation is enriched in one or more of the source mixture's constituents. In some cases, a separation may fully divide the mixture into pure constituents. Separations exploit differences in chemical properties or physical properties between the constituents of a mixture.
Capillary electrochromatography (CEC) is a chromatographic technique in which the mobile phase is driven through the chromatographic bed by electroosmosis. Capillary electrochromatography is a combination of two analytical techniques, high-performance liquid chromatography and capillary electrophoresis. Capillary electrophoresis aims to separate analytes on the basis of their mass-to-charge ratio by passing a high voltage across ends of a capillary tube, which is filled with the analyte. High-performance liquid chromatography separates analytes by passing them, under high pressure, through a column filled with stationary phase. The interactions between the analytes and the stationary phase and mobile phase lead to the separation of the analytes. In capillary electrochromatography capillaries, packed with HPLC stationary phase, are subjected to a high voltage. Separation is achieved by electrophoretic migration of solutes and differential partitioning.
Centrifugal partition chromatography is a special chromatographic technique where both stationary and mobile phase are liquid, and the stationary phase is immobilized by a strong centrifugal force. Centrifugal partition chromatography consists of a series-connected network of extraction cells, which operates as elemental extractors, and the efficiency is guaranteed by the cascade.
Emanuel Gil-Av (Zimkin) was an Israeli chemist. The main emphasis of his work constituted chiral chromatography for the analytical separation of enantiomers.
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