Chromatography 120726045329-phpapp02


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Chromatography 120726045329-phpapp02

  1. 1. Chromatography Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. There are number of different kinds of chromatography, which differ in the mobile and the stationary phase used. Chromatography is derived from a Greek word χρῶμα means chroma (colour) and γράφειν means graphein (to write). Chromatography is the laboratory techniques used 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 more advanced 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.
  2. 2. History of chromatography....... Chromatography, literally "color writing", was first employed by Russian scientist Mikhail 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 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 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. Techniques by chromatographic bed shape....... 1. paper chromatography In Paper Chromatography, the mobile phase is a solvent and the stationary phase is water held in the fibres of chromatography paper. A solution of the mixture to be separated is spotted onto a strip of chromatography paper (or filter paper) with a dropper. The chromatogram is developed by placing the bottom of the paper (but not the sample spot) in a tank containing suitable solvent. The solvent is drawn up the paper by capillary action. The components of the mixture move up the paper with the solvent at different rates due to their differing interactions with the stationary and mobile phases. FormulaRf = Distance the solute moves Distance the solvent front move
  3. 3. 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 chromatography In thin layer chromatography, the mobile phase is also a solvent, and the stationary phase is a thin layer of finely divided solid, such as silica gel or alumina, supported on glass or aluminium. Thin layer chromatography is similar to paper chromatography in that it involves spotting the mixture on the plate and the solvent (mobile phase) rises up the plate in the chromatography tank. It has an advantage over paper chromatography in that its
  4. 4. 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 example in the separation of dyes from fibres. Gas chromatography and high performance liquid chromatography are more sophisticated chromatographic techniques. 4. Gas chromatography A gas is the mobile phase and the stationary phase can be either a solid or a non- 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 vaporised if 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.
  5. 5. 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 injection to 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,
  6. 6. Once GC has separated a mixture, the components can be identified using known retention times. For unknown compounds the solutes are collected individually and analysed using another method, e.g. mass spectrometry. For each compound in a mixture one peak is observed on the chromatogram. In the particular set of operating conditions relating to the column, the retention time will increase with the size and polarity of the compound. To find the concentration 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 used to analyse samples taken from athletes to check for the presence of drugs. In each case, it separates the components of the mixture and indicates the concentrations of the components. Water companies test samples of water for pollutants using GC to separate the pollutants, and mass spectrometry to identify them. GC is used to analyse blood samples for the presence of alcohol. It is also used to analyse samples taken from athletes to check for the presence of drugs. In each case, it separates the components of the mixture and indicates the concentrations of the components. Water companies test samples of water for pollutants using GC to separate the pollutants, and mass spectrometry to identify them.
  7. 7. 5. High performance liquid chromatography Basic 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.
  8. 8. Uses HPLC has many uses such as drug testing, testing for vitamins in food and growth promoters in meat. In each case components of the mixture are separated and detected. Comparison of HPLC over GC Less volatile and larger samples can be used with HPLC.
  9. 9. Chromatogram and Mass Spectrometry Data 6. 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-molecule'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 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.
  10. 10. 7. Super critical 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. Techniques by separation mechanism.......... 8. 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 acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain. Ion exchange chromatography is commonly used to purify proteins using FPLC. 9. Size exclusion chromatography Size-exclusion chromatography (SEC) is also known as Gel Permeation Chromatography (GPC) or Gel Filtration Fhromatography 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 and quaternary stucture of purified proteins, especially since it can be carried out under native solution conditions.
  11. 11. Special techniques.......... 10. 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. 11. 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 classification) 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. 12. 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
  12. 12. 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. 13. 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. 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 one partitioning step per revolution and components of the sample separate in the
  13. 13. 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. 15. chiral chromatography Chiral chromatography involves the separation of stereo-isomers. In the case of en-antiomers, 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 Columns (with a chiral stationary phase) in both normal and reversed phase are commercially available.
  14. 14. Source page 1. 2. 3. Physics and Chemistry Support Science (PCSS)