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Introduction to chromatography and its applications 2


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Introduction to chromatography and its applications 2

  2. 2. Introduction Chromatography, literally "color writing", was first employed by Russian-Italian scientist MikhailTsvet in 1900, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. The word chromatography is derived from two Greek words Chroma ….……..color Graphos ………..writing 2
  3. 3. Definition  ‘A technique by which a mixture is separated into its components on the basis of relative ability of each component to be moved along/through a stationary phase by mobile phase’  The technique of chromatography is based on the differences in the rate at which the components of a mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called moving/mobile phase).  Chromatography is a nondestructive procedure  Applied both for both qualitative and quantitative studies 3
  4. 4. Principle Like-dissolve-like or like-prefer-like. The basis-partition or distribution coefficient ‘K’ which describes the way in which a compound distributes itself between two immiscible phases. Defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase  𝐾 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 4
  5. 5. 6
  6. 6. CommonlyUsed Terms 7 Analyte The substance to be separated during chromatography Analytical chromatography Determines the existence and the concentration of analyte(s) in a sample Chromatogram Visual output of the chromatograph
  7. 7. Chromatogram 8
  8. 8. CommonlyUsed Terms 9 Chromatograph An equipment that enables the separation e.g. gas chromatographic or liquid chromatographic separation. Eluate Mobile phase Eluent The components obtained after the process. Eluotropic series List of solvents ranked according to their eluting power Elution Process of extracting a substance that is adsorbed on another surface by moving it with solvent.
  9. 9. Commonly used terms Immobilized phase Stationary phase that is immobilized on the support particles, or on the inner wall of the column Mobile phase Phase that moves over the stationary phase. It may be a liquid (LC) or a gas (GC). Retention time Time required for the mobile phase to sweep a component from the stationary phase. Sample Matter analyzed in chromatography- single component or multiple components Solute Sample components in partition chromatography.
  10. 10. Commonly UsedTerms 11 Solvent Any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography Stationary phase Substance fixed in place for the chromatography procedure- solid, gel or a liquid. e.g ; silica, alumina, cellulose Detector Instrument used for qualitative and quantitative detection of analytes after separation Rf value or Retention factor (Rf) The ratio of the distance traveled by the center of a spot (solute) to the distance traveled by the solvent front (solvent)
  11. 11. Classification 12 Analytical • To determine the chemical composition of sample Preparative • Used to purify and collect one or more components of a sample
  12. 12. Types 13 Adsorption Partition
  13. 13. 1.Adsorption Chromatography  It uses a mobile phase or gaseous phase that is adsorbed onto the surface of a stationary solid phase.The equilibration between the mobile and stationary phase accounts for the separation of different solutes. Following are the chromatographic techniques that are included in this category: 14 Techniques Gas Solid Chromatography (GSC) Gel Permeation Chromatography High Performance (pressure) Liquid Chromatography (HPLC) Ion Exchange Chromatography Column Chromatography Thin Layer Chromatography (TLC)
  14. 14. Principle 15
  15. 15. Thin Layer Chromatography  Stationary phase is a thin layer of a solid such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic.  The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobile phase) so that the liquid level is below the spot.  TLC is also called as drop, strip, spread layer, surface chromatography and open column chromatography. 16
  16. 16. TLC
  17. 17. TLC PAPER
  18. 18. Column Chromatography  A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase. The stationary phase will adsorb the components of the mixture to varying degrees. As the solution containing the mixture passes over the adsorbent, the components are distributed between the solvent and adsorbent surface.This process may be described by three-way equilibrium between the sample, the solvent and the adsorbent.The solvent and sample compete for positions on the solid adsorbent, the solvent displacing the sample reversibly and continuously in the direction of the solvent flow. Consequently, a weakly adsorbed compound will spend more time in the solvent, and will therefore be eluted first.
  19. 19. Ion Exchange Chromatography  Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type.  The column is packed with porous beads of a resin that will exchange either cations or anions.There is one type of ion on the surface of the resin and these are released when other ions are bound in their place – e.g. a basic anion exchange resin might remove nitrate ions (NO3–) from a solution and replace them with hydroxide ions (OH–).
  20. 20. Principle
  21. 21. High Performance (pressure) Liquid Chromatograp hy (HPLC)  HPLC involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent under pressure. Normal Phase HPLC • A non-polar, non-aqueous mobile phase (e.g. Chloroform), the analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. Reversed Phase HPLC • A non-polar stationary phase and an aqueous, moderately polar mobile phase.With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis).
  22. 22. HPLCWorking
  23. 23. Gel Filtration or Gel Permeation Chromatography  The separation of large molecules, often in biochemical situations, can be achieved in a column which works on the basis of molecular exclusion. The mixture of solutes is carried through the column by a solvent. The stationary phase (the gel) typically consists of particles of a cross-linked polyamide which contains pores. Separation occurs according to molecular size – the larger molecules passing through the column fastest. Different gels are available that allow the separation of proteins with relative masses. The greatest resolution is achieved by using very small gel particles, but the flow rate through the column then becomes much slower.
  24. 24. 2. Partition Chromatography  This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. Paper Chromatography Gas Liquid Chromatography (GLC)
  25. 25. Principle
  26. 26. Gas Chromatography  Gas-liquid chromatography (GLC):  In GLC the mobile phase is a gas and stationary phase is a thin layer of a non-volatile liquid bound to a solid support thus a partition process occurs. In such case small inert particles such as  Diatomaceous earth is coated with the liquid so that a large surface area exists for the solute to equilibrate with.  Gas-solid chromatography (GSC):  GSC utilizes a solid adsorbent as the stationary phase while gas as a mobile phase and an adsorption process takes place.  The separation method can be affected by the polarity of stationary phase, temperature, carrier gas flow, length of column, material amount etc.
  27. 27. Principle
  28. 28. Working
  29. 29. Paper Chromatography  Cellulose filter paper is often used as stationary phase in paper chromatography. Since it is hydrophilic, it is usually covered with a thin film of water.That’s why it is often called as liquid- liquid chromatography.  Principle: The substances are distributed between liquid phases. One phase is the water which present in pores of filter paper and other phase is mobile phase which moves on the paper.The separation of mixture is due to different attraction force towards stationary phase (water) and mobile phase (solvents).
  30. 30. Principle
  31. 31. Affinity chromatography
  32. 32. Detectors  The components elute from the column can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on- line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC- MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV- VIS absorption spectroscopy (HPLC-UV-VIS)
  33. 33. Visualization of Chromatogram It can be done by following method, Use of fluorescent material like manganese with activated zinc silicate Spray of Iodine vapors UV exposure Ninhydrin for identification of amino acids
  34. 34. Applications  It is commonly used technique for the separation of molecule. For example, it is used to remove pesticides and insecticides like DDT in the water and poly chlorinated biphenyls.  It is widely used to determine the purity of a substance.  In pharmaceutical companies, it is used for producing pure materials for medicines and also for checking the contamination presence in medicines.  It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical isomers).  In the food industry, this technique is very useful for analyzing and the separation of additives, proteins and amino acids etc.  It used in forensic science for detecting the presence of drugs.  Chromatography has been extensively used in the isolation, characterization and determination of the 60 or so carotenoids at present known.
  35. 35. References  M.Younus, Ilmi Kitab Khana, 2011. Organic Spectroscopy And Chromatography, Chromatography, pg. 269, ed. 3rd.  Scott, R. P.W. Introduction to Analytical Gas Chromatography; 2nd ed.; Marcel Dekker, 1998.  Jennings,W. G.; Mittlefehldt, E.; Stremple, P.Analytical Gas Chromatography; 2nd ed.; Academic Press, 1997.  McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997.  Grant, D.W. Capillary Gas Chromatography; Wiley, 1996.  Fowlis, I. Gas Chromatography; 2nd ed.;Wiley, 1995.  Scott, R. P.W.Techniques and Practices of Chromatography; 2nd ed.; Marcel Dekker, 1995.  Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.;Wiley, 1995.  Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994.  Hinshaw, J.V.; Ettre, L. S. Introduction to OpenTubularColumnGas Chromatography; Advanstar, 1994.  Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd ed.; Hüthig, 1993.  Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley, 1992.  Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.; Hüthig, 1991.  Poole, C. F.; Poole, S. K. ChromatographyToday; Elsevier, 1991.  Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994.  Kolb, B. Gaschromatographie in Bildern;Wiley-VCH, NewYork, 1999.  Kenndler, E.; Huber, J. F. K. In AnalytikerTaschenbuch; Springer, 1989