Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
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 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.
Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates analytes on the basis of size. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964 and the proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
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 some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
Archer John Porter Martin was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge.
Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography. A paper chromatography variant, two-dimensional chromatography involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids. The setup has three components. The mobile phase is a solution that travels up the stationary phase, due to capillary action. The mobile phase is generally mixture of non-polar organic solvent, while the stationary phase is polar organic solvent in water. Paper is used to support stationary phase. Difference between TLC and paper chromatography is that stationary phase in TLC is a layer of adsorbent, and stationary phase in paper chromatography is water.
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.
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.
In chemical processing, a packed bed is a hollow tube, pipe, or other vessel that is filled with a packing material. The packing can be randomly filled with small objects like Raschig rings or else it can be a specifically designed structured packing. Packed beds may also contain catalyst particles or adsorbents such as zeolite pellets, granular activated carbon, etc.
A theoretical plate in many separation processes is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other. Such equilibrium stages may also be referred to as an equilibrium stage, ideal stage, or a theoretical tray. The performance of many separation processes depends on having series of equilibrium stages and is enhanced by providing more such stages. In other words, having more theoretical plates increases the efficiency of the separation process be it either a distillation, absorption, chromatographic, adsorption or similar process.
Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column and is then displaced by a solute that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive “rectangular” zones of highly concentrated pure substances rather than solvent-separated “peaks”. It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.
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.
The history of chromatography spans from the mid-19th century to the 21st. Chromatography, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll and carotenoids. New forms of chromatography developed in the 1930s and 1940s made the technique useful for a wide range of separation processes and chemical analysis tasks, especially in biochemistry.
Partition chromatography theory and practice was introduced through the work and publications of Archer Martin and Richard Laurence Millington Synge during the 1940s. The process of separating mixtures of chemical compounds by passing them through a column that contains a solid stationary phase that was eluted with a mobile phase was well known at that time. Chromatographic separation was considered to occur by an adsorption process whereby compounds adhered to a solid media and were washed off the column with a solvent, mixture of solvents, or solvent gradient. In contrast, Martin and Synge developed and described a chromatographic separation process whereby compounds were partitioned between two liquid phases similar to the separatory funnel liquid-liquid separation dynamic. This was an important departure, both in theory and in practice, from adsorption chromatography.
Erika Cremer was a German physical chemist and Professor Emeritus at the University of Innsbruck who is regarded as one of the most important pioneer in gas chromatography, as she first conceived the technique in 1944.
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.
Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by centrifugal 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.
