Chromatography
Encyclopedia
Chromatography is the collective term for a set of laboratory techniques
for the separation of mixtures.
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 changing the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.
, carotene
s, and xanthophyll
s. Since these components have different colors (green, orange, and yellow, respectively) they gave the technique its name. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes.
Chromatography technique developed substantially as a result of the work of Archer John Porter Martin
and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods: paper chromatography
, gas chromatography, and what would become known as high performance liquid chromatography
. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography described below. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules.
Chromatography is based on the concept of partition coefficient. Any solute will partition between two immissible solvents. When we make one solvent immobile (by adsorption on a solid support matrix) and another mobile it results in most common applications of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is forward phase chromatography, and if it is non polar (C-18) it is reverse phase.
In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash). The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.
In expanded bed adsorption
, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.
) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography
). Different compounds
in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel
, alumina
, or cellulose
on a flat, inert substrate
. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC
can be used.
is:
A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.
There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.
Gas chromatography (GC) is based on a partition equilibrium
of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry
; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry
, it is well suited for use in the petrochemical
, environmental monitoring
and remediation, and industrial chemical
fields. It is also used extensively in chemistry research.
(HPLC).
In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography
if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography
.
s bound to tags. These fusion protein
s are labeled with compounds such as His-tags, biotin
or antigen
s, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.
Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules.
However, HPLC techniques exist that do utilize affinity chromatogaphy properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity.
s, peptide
s, and protein
s. In conventional methods the stationary phase is an ion exchange resin
that carries charged functional group
s which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC
.
Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure
and quaternary structure
of purified proteins, especially since it can be carried out under native solution
conditions.
) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.
The sample is spotted at one corner of a square plate,developed, air-dried, then rotated by 90° and usually redeveloped in a second solvent system.
Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a vacuum. The sample is put into direct contact with a platinum wire, or placed in a quartz sample tube, and rapidly heated to 600–1000 °C. Depending on the application even higher temperatures are used. Three different heating techniques are used in actual pyrolyzers: Isothermal furnace, inductive heating (Curie Point filament), and resistive heating using platinum filaments. Large molecules cleave at their weakest points and produce smaller, more volatile fragments. These fragments can be separated by gas chromatography. Pyrolysis GC chromatograms are typically complex because a wide range of different decomposition products is formed. The data can either be used as fingerprint to prove material identity or the GC/MS data is used to identify individual fragments to obtain structural information. To increase the volatility of polar fragments, various methylating reagents can be added to a sample before pyrolysis.
Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid samples can be performed directly inside Programmable Temperature Vaporizer (PTV) injectors that provide quick heating (up to 30 °C/s) and high maximum temperatures of 600–650 °C. This is sufficient for some pyrolysis applications. The main advantage is that no dedicated instrument has to be purchased and pyrolysis can be performed as part of routine GC analysis. In this case quartz GC inlet liners have to be used. Quantitative data can be acquired, and good results of derivatization inside the PTV injector are published as well.
The operating principle of CCC equipment requires a column consisting of an open tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used. There are many types of CCC available today. These include HSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC is the latest and best performing version of the instrumentation available currently.
(with a chiral stationary phase) in both normal and reversed phase are commercially available.
Laboratory techniques
Laboratory techniques are the sum of procedures used on natural sciences such as chemistry, biology, physics in order to conduct an experiment, all of them follow scientific method; while some of them involves the use of complex laboratory equipment from laboratory glassware to electrical devices...
for the separation of mixtures.
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
Partition coefficient
In chemistry and the pharmaceutical sciences, a partition- or distribution coefficient is the ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. The terms "gas/liquid partition coefficient" and "air/water partition coefficient" are...
result in differential retention on the stationary phase and thus changing the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.
History
Chromatography, literally "color writing", was first employed by Russian scientist Michael Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyllChlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
, carotene
Carotene
The term carotene is used for several related unsaturated hydrocarbon substances having the formula C40Hx, which are synthesized by plants but cannot be made by animals. Carotene is an orange photosynthetic pigment important for photosynthesis. Carotenes are all coloured to the human eye...
s, and xanthophyll
Xanthophyll
Xanthophylls are yellow pigments that form one of two major divisions of the carotenoid group. The name is from Greek xanthos + phyllon , due to their formation of the yellow band seen in early chromatography of leaf pigments...
s. Since these components have different colors (green, orange, and yellow, respectively) they gave the technique its name. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes.
Chromatography technique developed substantially as a result of the work of Archer John Porter Martin
Archer John Porter Martin
Archer John Porter Martin, FRS was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge....
and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods: paper chromatography
Paper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...
, gas chromatography, and what would become known as high performance liquid chromatography
High performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography described below. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules.
Chromatography terms
- The analyte is the substance to be separated during chromatography.
- Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sampleSample (material)In general, a sample is a limited quantity of something which is intended to be similar to and represent a larger amount of that thing. The things could be countable objects such as individual items available as units for sale, or a material not countable as individual items. Samples of countable...
. - A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
- A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
- Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometerSpectrophotometryIn chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
, mass spectrometerMass spectrometryMass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.- A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
- Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
- The eluate is the mobile phase leaving the column.
- The eluent is the solvent that will carry the analyte.
- An eluotropic series is a list of solvents ranked according to their eluting power.
- An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
- The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLCHigh-performance liquid chromatographyHigh-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated. - Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.
- The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovats' retention index
- The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
- The solute refers to the sample components in partition chromatography.
- The solvent refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC.
- The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography
Chromatography is based on the concept of partition coefficient. Any solute will partition between two immissible solvents. When we make one solvent immobile (by adsorption on a solid support matrix) and another mobile it results in most common applications of chromatography. If matrix support is polar (e.g. paper, silica etc.) it is forward phase chromatography, and if it is non polar (C-18) it is reverse phase.
Column chromatography
Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash). The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.
In expanded bed adsorption
Expanded bed adsorption
Expanded bed adsorption is a preparative chromatographic technique which makes processing of viscous and particulate liquids possible.-Principle:...
, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.
Planar chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatographyPaper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...
) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography
Thin layer chromatography
Thin layer chromatography is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose...
). Different compounds
Chemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
Paper chromatography
Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solventSolvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
Thin layer chromatography
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatographyPaper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...
. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel
Silica gel
Silica gel is a granular, vitreous, porous form of silica made synthetically from sodium silicate. Despite its name, silica gel is a solid. It is a naturally occurring mineral that is purified and processed into either granular or beaded form...
, alumina
Aluminium oxide
Aluminium oxide is an amphoteric oxide with the chemical formula 23. It is commonly referred to as alumina, or corundum in its crystalline form, as well as many other names, reflecting its widespread occurrence in nature and industry...
, or cellulose
Cellulose
Cellulose is an organic compound with the formula , a polysaccharide consisting of a linear chain of several hundred to over ten thousand β linked D-glucose units....
on a flat, inert substrate
Substrate (chemistry)
In chemistry, a substrate is the chemical species being observed, which reacts with a reagent. This term is highly context-dependent. In particular, in biochemistry, an enzyme substrate is the material upon which an enzyme acts....
. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC
HPTLC
High performance thin layer chromatography is an enhanced form of thin layer chromatography . A number of enhancements can be made to the basic method of thin layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative...
can be used.
Displacement chromatography
The basic principle of displacement chromatographyDisplacement Chromatography
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 ...
is:
A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.
There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.
Gas chromatography
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .Gas chromatography (GC) is based on a partition equilibrium
Partition equilibrium
The most common chemical equilibrium systems involve reactants and products in the same phase - either all gases or all solutions. However, it is also possible to get equilibria between substances in different phases, such as two liquids that do not mix...
of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...
; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
, it is well suited for use in the petrochemical
Petrochemical
Petrochemicals are chemical products derived from petroleum. Some chemical compounds made from petroleum are also obtained from other fossil fuels, such as coal or natural gas, or renewable sources such as corn or sugar cane....
, environmental monitoring
Environmental monitoring
Environmental monitoring describes the processes and activities that need to take place to characterise and monitor the quality of the environment...
and remediation, and industrial chemical
Chemical industry
The chemical industry comprises the companies that produce industrial chemicals. Central to the modern world economy, it converts raw materials into more than 70,000 different products.-Products:...
fields. It is also used extensively in chemistry research.
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatographyHigh performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(HPLC).
In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography
Fast protein liquid chromatography
Fast protein liquid chromatography , is a form of liquid chromatography similar to high-performance liquid chromatography that is used to separate or purify proteins and other polymers from complex mixtures. FPLC system is a complete system for laboratory scale chromatographic separations of...
if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography
Aqueous normal phase chromatography
Aqueous normal-phase chromatography is a chromatographic technique which involves the mobile phase region between reversed-phase chromatography and organic normal-phase chromatography .-Principle:...
.
Affinity chromatography
Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s bound to tags. These fusion protein
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
s are labeled with compounds such as His-tags, biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
or antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
s, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.
Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules.
However, HPLC techniques exist that do utilize affinity chromatogaphy properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity.
Supercritical fluid chromatography
Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.Ion exchange chromatography
Ion exchange chromatography (usually referred to as ion chromatography) uses an ion exchange mechanism to separate analytes based on their respective charges. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acidAmino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s, peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
s, and protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s. In conventional methods the stationary phase is an ion exchange resin
Ion exchange resin
An ion-exchange resin or ion-exchange polymer is an insoluble matrix normally in the form of small beads, usually white or yellowish, fabricated from an organic polymer substrate. The material has highly developed structure of pores on the surface of which are sites with easily trapped and...
that carries charged functional group
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
s which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC
Fast protein liquid chromatography
Fast protein liquid chromatography , is a form of liquid chromatography similar to high-performance liquid chromatography that is used to separate or purify proteins and other polymers from complex mixtures. FPLC system is a complete system for laboratory scale chromatographic separations of...
.
Size-exclusion chromatography
Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume).Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
and quaternary structure
Quaternary structure
In biochemistry, quaternary structure is the arrangement of multiple folded protein or coiling protein molecules in a multi-subunit complex.-Description and examples:...
of purified proteins, especially since it can be carried out under native solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
conditions.
Reversed-phase chromatography
Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.Two-dimensional chromatography
In some cases, the chemistry within a given column can be insufficient to separate some analytes. It is possible to direct a series of unresolved peaks onto a second column with different physico-chemical (Chemical classificationChemical classification
Chemical classification systems attempt to classify as elements or compounds according to certain chemical functional or structural properties. Whereas the structural properties are largely intrinsic, functional properties and the derived classifications depend to a certain degree on the type of...
) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.
The sample is spotted at one corner of a square plate,developed, air-dried, then rotated by 90° and usually redeveloped in a second solvent system.
Simulated moving-bed chromatography
Pyrolysis gas chromatography
Pyrolysis gas chromatography mass spectrometry is a method of chemical analysis in which the sample is heated to decomposition to produce smaller molecules that are separated by gas chromatography and detected using mass spectrometry.Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a vacuum. The sample is put into direct contact with a platinum wire, or placed in a quartz sample tube, and rapidly heated to 600–1000 °C. Depending on the application even higher temperatures are used. Three different heating techniques are used in actual pyrolyzers: Isothermal furnace, inductive heating (Curie Point filament), and resistive heating using platinum filaments. Large molecules cleave at their weakest points and produce smaller, more volatile fragments. These fragments can be separated by gas chromatography. Pyrolysis GC chromatograms are typically complex because a wide range of different decomposition products is formed. The data can either be used as fingerprint to prove material identity or the GC/MS data is used to identify individual fragments to obtain structural information. To increase the volatility of polar fragments, various methylating reagents can be added to a sample before pyrolysis.
Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid samples can be performed directly inside Programmable Temperature Vaporizer (PTV) injectors that provide quick heating (up to 30 °C/s) and high maximum temperatures of 600–650 °C. This is sufficient for some pyrolysis applications. The main advantage is that no dedicated instrument has to be purchased and pyrolysis can be performed as part of routine GC analysis. In this case quartz GC inlet liners have to be used. Quantitative data can be acquired, and good results of derivatization inside the PTV injector are published as well.
Fast protein liquid chromatography
Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography, however use of FPLC techniques are typically for preparing large scale batches of a purified product.Countercurrent chromatography
Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids.The operating principle of CCC equipment requires a column consisting of an open tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used. There are many types of CCC available today. These include HSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC is the latest and best performing version of the instrumentation available currently.
Chiral chromatography
Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography HPLC columnsChiral column chromatography
Chiral column chromatography is a variant of column chromatography in which the stationary phase contains a single enantiomer of a chiral compound rather than being achiral...
(with a chiral stationary phase) in both normal and reversed phase are commercially available.
See also
- Aqueous normal-phase chromatography
- Multicolumn countercurrent solvent gradient purificationMulticolumn countercurrent solvent gradient purificationMulticolumn Countercurrent Solvent Gradient Purification is a form of chromatography that is used to separate or purify biomolecules from complex mixtures. It was developed at the Swiss Federal Institute of Technology Zürich by Aumann and Morbidelli...
(MCSGP) - Purnell equation
- Chromatography in blood processingChromatography in blood processingChromatographic techniques have been used in blood processing and purification since the 1980s. It has emerged as an effective method of purifying blood components for therapeutic use.-Human blood plasma:...
- Chromatography softwareChromatography softwareA chromatography software, also known as a Chromatography data system , collects and analyzes chromatographic results delivered by chromatography detectors....
- Van Deemter equation
- Binding selectivityBinding selectivityBinding selectivity refers to the differing affinities with which different ligands bind to a substrate forming a complex. A selectivity coefficient is the equilibrium constant for the reaction of displacement by one ligand of another ligand in a complex with the substrate...
External links
- IUPAC Nomenclature for Chromatography
- Chromedia On line database and community for chromatography practitioners (paid subscription required)
- Library 4 Science: Chrom-Ed Series
- Overlapping Peaks Program – Learning by Simulations
- Chromatography Videos – MIT OCW – Digital Lab Techniques Manual
- Chromatography Equations Calculators – MicroSolv Technology Corporation