ChromatographyChromatography is the term used to describe a separation technique in which a mobilephase carrying a mixture is caused to move in contact with a selectively absorbent stationaryphase. There are number of different kinds of chromatography, which differ in the mobileand the stationary phase used.Chromatography is derived from a Greek word χρῶμα means chroma (colour) and γράφεινmeans graphein (to write). Chromatography is the laboratory techniques used for theseparation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through astructure holding another material called the stationary phase. The various constituents of themixture travel at different speeds, causing them to separate. The separation is based ondifferential partitioning between the mobile and stationary phases. Subtle differences in acompounds partition coefficient result in differential retention on the stationary phase andthus changing the separation.Chromatography may be preparative or analytical. The purpose of preparativechromatography is to separate the components of a mixture for more advanced use (and isthus a form of purification). Analytical chromatography is done normally with smalleramounts of material and is for measuring the relative proportions of analytes in a mixture.The two are not mutually exclusive.
History of chromatography.......Chromatography, literally "color writing", was first employed by Russian scientist MikhailTsvet in 1900. He continued to work with chromatography in the first decade of the 20thcentury, primarily for the separation of plant pigments such as chlorophyll, carotenes,and xanthophylls. Since these components have different colours (green, orange, and yellow,respectively) they gave the technique its name. New types of chromatography developedduring the 1930s and 1940s made the technique useful for many separation processes.Chromatography technique developed substantially as a result of the work of Archer JohnPorter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. Theyestablished the principles and basic techniques of partition chromatography, and their workencouraged the rapid development of several chromatographic methods: PaperChromatography, Gas Chromatography, and what would become known as highperformance liquid chromatography. Since then, the technology has advanced rapidly.Researchers found that the main principles of Tsvets chromatography could be applied inmany different ways, resulting in the different varieties of chromatography described below.Advances are continually improving the technical performance of chromatography, allowingthe separation of increasingly similar molecules.Techniques by chromatographic bed shape....... 1. paper chromatographyIn Paper Chromatography, the mobile phase is asolvent and the stationary phase is water held in thefibres of chromatography paper. A solution of themixture to be separated is spotted onto a strip ofchromatography paper (or filter paper) with a dropper.The chromatogram is developed by placing the bottomof the paper (but not the sample spot) in a tankcontaining suitable solvent. The solvent is drawn up thepaper by capillary action. The components of the mixture move up the paper with the solventat different rates due to their differing interactions with the stationary and mobile phases.Formula- Rf = Distance the solute moves Distance the solvent front move
2. Column chromatography In column chromatography, the mobile phase is again a solvent, and the stationary phase is a finely divided solid, such as silica gel or alumina. Chromatography columns vary in size and polarity. There is an element of trial and error involved in selecting a suitable solvent and column for the separation of the constituents of a particular mixture. A small volume of the sample whose constituents are to be separated is placed on top of the column. The choice of the eluting solvent should ensure that the sample is soluble. However, if the sample was too soluble the mobile phase (solvent) would move the solutes too quickly, resulting in the non-separation of the different constituents.3. Thin layer chromatographyIn thin layer chromatography, themobile phase is also a solvent, andthe stationary phase is a thin layerof finely divided solid, such assilica gel or alumina, supported onglass or aluminium. Thin layerchromatography is similar to paperchromatography in that it involvesspotting the mixture on the plateand the solvent (mobile phase) rises up the plate in the chromatographytank. It has an advantage over paper chromatography in that its
separations are very efficient because of the much smaller size of the particles in the stationary phase.Thin layer chromatography is particularly useful in forensic work, for examplein the separation of dyes from fibres. Gas chromatography and highperformance liquid chromatography are more sophisticated chromatographictechniques. 4. Gas chromatographyA gas is the mobile phase and the stationary phase can be either a solid or anon- volatile liquid.There are five basic GC components: 1) Pneumatic system – gas supply (flow control and measurement). 2) Injection system – a heated injector port, where the sample is vaporisedif necessary. 3) Column – where the separation occurs. 4) Oven –The coiled column is wholly contained in a thermostatically controlled oven. 5) Detector – integral detector or link to a mass spectrometer. How does gas chromatography work.........? 1) A carrier gas, examples of which are Helium and Neon flows through the system. A valve controls the flow rate. 2) A sample of the volatile mixture is injected into the carrier gas. The sample is vaporised in the heated injector port.
3) The carrier gas carries the vaporised sample into the column. The columns are stainless steel or glass tubes. They can be up to 25 m in length and are of narrow bore (2-10 mm). Therefore the column is often wound into a coil. The packed columns contain porous support material. The sample mixture undergoes a series of interactions between the stationary and mobile phases as it is carried through the system by the carrier gas. Due to the wide choice of materials available for the stationary and mobile phases, it is possible to separate molecules that differ only slightly in their physical and chemical properties. 4) The coiled column is contained in the thermostatically controlled oven. 5) Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component comes through first. Separation is obtained when one compound is sufficiently retarded to prevent overlap with another component of the sample, as it emerges from the column. 6) Two types of detector can be used: (1) Thermal Conductivity detectors which respond to changes in the thermal conductivity of the gas leaving the column and (2) Flame Ionisation detection (FID), which is more commonly used. In thermal conductivity, as the carrier gas leaves the column, it cools the detector. When a solute emerges with the carrier gas, it does not cool the detector to the same extent. Alternatively, samples can be passed from the oven directly into a mass spectrometer, where they are analysed.Retention time is defined as the time taken for a component to go from injectionto detection. This varies depending on a) The nature of and the interactions between the solute and the stationary and mobile phases. b) The flow rate of the carrier gas, c) The temperature of the column (shorter retention times are obtained at higher temperatures), d) The length and diameter of the column,
Once GC has separated a mixture, the components can be identified usingknown retention times. For unknown compounds the solutes are collectedindividually and analysed using another method, e.g. mass spectrometry.For each compound in a mixture one peak is observed on the chromatogram. Inthe particular set of operating conditions relating to the column, the retentiontime will increase with the size and polarity of the compound. To find theconcentration of a particular compound, the peak height should be measured.GC is used to analyse blood samples for the presence of alcohol. It is also usedto analyse samples taken from athletes to check for the presence of drugs. Ineach case, it separates the components of the mixture and indicates theconcentrations of the components. Water companies test samples of water forpollutants using GC to separate the pollutants, and mass spectrometry toidentify them.GC is used to analyse blood samples for the presence of alcohol. It is also usedto analyse samples taken from athletes to check for the presence of drugs. Ineach case, it separates the components of the mixture and indicates theconcentrations of the components. Water companies test samples of water forpollutants using GC to separate the pollutants, and mass spectrometry toidentify them.
5. High performance liquid chromatographyBasic Components: 1) Solvent Reservoir. 2) The Pump System controls the flow and measures the volume of solvent (the mobile phase). The flow rates of HPLC columns are slow – often in 3 -1 the range of 0.5 - 5 cm min . 3) The Injector System: The sample to be separated is injected into the liquid phase at this point. 4) The Column is made of steel and packed usually with porous silica particles (the stationary phase). Different materials can be used depending on the nature of the liquid. A long column is not needed because separation in HPLC is very efficient. Columns are usually 10 –30 cm long, with an internal diameter of 4 mm. Different components of the sample are carried forward at different rates by the moving liquid phase, due to their differing interactions with the stationary and mobile phases. 5) The Detector: When the components reach the end of the column they are analysed by a detector. The amounts passing through the column are small, so solutes are analysed as they leave the column. Therefore HPLC is usually linked to a spectrometer (e.g. ultra violet or mass spectrometry). The length of time it takes for a compound to reach the detector allows the component to be identified. Like the GC, once the retention time of a solute has been established for a column using a particular set of operating conditions, the solute can be identified in a mixture. A chromatogram is obtained for the sample.
UsesHPLC has many uses such as drug testing, testing for vitamins in food andgrowth promoters in meat. In each case components of the mixture areseparated and detected.Comparison of HPLC over GCLess volatile and larger samples can be used with HPLC.
Chromatogram and Mass Spectrometry Data6. 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 proteins bound to tags. These fusion proteins are labelled with compounds such as his-tags, biotin or antigens, 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 bio-molecules 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 bio-molecules. However, HPLC techniques exist that do utilize affinity chromatography 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.
7. Super critical fluid chromatographySupercritical fluid chromatography is a separation technique in which themobile phase is a fluid above and relatively close to its critical temperature andpressure.Techniques by separation mechanism.......... 8. Ion exchange chromatographyIon exchange chromatography (usually referred to as ion chromatography) usesan ion exchange mechanism to separate analytes based on their respectivecharges. It is usually performed in columns but can also be useful in planarmode. Ion exchange chromatography uses a charged stationary phase toseparate charged compounds including anions, cations, amino acids, peptides,and proteins. In conventional methods the stationary phase is an ion exchangeresin that carries charged functional groups that interact with oppositely chargedgroups of the compound to retain. Ion exchange chromatography is commonlyused to purify proteins using FPLC. 9. Size exclusion chromatographySize-exclusion chromatography (SEC) is also known as Gel PermeationChromatography (GPC) or Gel Filtration Fhromatography and separatesmolecules according to their size (or more accurately according to theirhydrodynamic diameter or hydrodynamic volume). Smaller molecules are ableto enter the pores of the media and, therefore, molecules are trapped andremoved from the flow of the mobile phase. The average residence time in thepores depends upon the effective size of the analyte molecules. However,molecules that are larger than the average pore size of the packing are excludedand thus suffer essentially no retention; such species are the first to be eluted. Itis generally a low-resolution chromatography technique and thus it is oftenreserved for the final, "polishing" step of a purification. It is also useful fordetermining the tertiary structure and quaternary stucture of purified proteins,especially since it can be carried out under native solution conditions.
Special techniques..........10. reversed phase chromatographyReversed-phase chromatography is an elution procedure used in liquidchromatography in which the mobile phase is significantly more polar than thestationary phase.11. two-dimensional chromatographyIn some cases, the chemistry within a given column can be insufficient toseparate some analytes. It is possible to direct a series of unresolved peaks ontoa second column with different physico-chemical (Chemical classification)properties. Since the mechanism of retention on this new solid support isdifferent from the first dimensional separation, it can be possible to separatecompounds that are indistinguishable by one-dimensional chromatography. Thesample is spotted at one corner of a square plate, developed, air-dried, thenrotated by 90° and usually redeveloped in a second solvent system.12. pyrolysis gas chromatographyPyrolysis gas chromatography mass spectrometry is a method of chemicalanalysis in which the sample is heated to decomposition to produce smallermolecules that are separated by gas chromatography and detected using massspectrometry.Pyrolysis is the thermal decomposition of materials in an inert atmosphere or avacuum. The sample is put into direct contact with a platinum wire, or placed ina quartz sample tube, and rapidly heated to 600–1000 °C. Depending on theapplication even higher temperatures are used. Three different heatingtechniques are used in actual pyrolyzers: Isothermal furnace, inductive heating(Curie Point filament), and resistive heating using platinum filaments. Largemolecules cleave at their weakest points and produce smaller, more volatilefragments. These fragments can be separated by gas chromatography. PyrolysisGC chromatograms are typically complex because a wide range of differentdecomposition products is formed. The data can either be used as fingerprint to
prove material identity or the GC/MS data is used to identify individualfragments to obtain structural information. To increase the volatility of polarfragments, various methylating reagents can be added to a sample beforepyrolysis.Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquidsamples can be performed directly inside Programmable Temperature Vaporizer(PTV) injectors that provide quick heating (up to 30 °C/s) and high maximumtemperatures of 600–650 °C. This is sufficient for some pyrolysis applications.The main advantage is that no dedicated instrument has to be purchased andpyrolysis can be performed as part of routine GC analysis. In this case quartzGC inlet liners have to be used. Quantitative data can be acquired, and goodresults of derivatization inside the PTV injector are published as well.13. fast protein liquid chromatographyFast protein liquid chromatography (FPLC) is a term applied to severalchromatography techniques which are used to purify proteins. Many of thesetechniques are identical to those carried out under high performance liquidchromatography, however use of FPLC techniques are typically for preparinglarge scale batches of a purified product.14. counter-current chromatography Counter-current 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(An example of HPCCC system)the column during each rotation. This motion causes the column to see onepartitioning step per revolution and components of the sample separate in the
column due to their partitioning coefficient between the two immiscible liquidphases used. There are many types of CCC available today. These includeHSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC isthe latest and best performing version of the instrumentation available currently.15. chiral chromatographyChiral chromatography involves the separation of stereo-isomers. In the case ofen-antiomers, these have no chemical or physical differences apart from beingthree-dimensional mirror images. Conventional chromatography or otherseparation processes are incapable of separating them. To enable chiralseparations to take place, either the mobile phase or the stationary phase mustthemselves be made chiral, giving differing affinities between theanalytes. Chiral Chromatography HPLC Columns (with a chiral stationaryphase) in both normal and reversed phase are commercially available.
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