Chromatograph yfinal

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Chromatograph yfinal

  1. 1. CHROMATOGRAPHY
  2. 2. CHROMATOGRAPHY A laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.
  3. 3. CHROMATOGRAPHY Introduced first by the Russian botanist Mikhail Semenovich Tswett. Mixtures of solutes dissolved in a common solvent are separated from one another by a differential distribution of the solutes between two phases.
  4. 4. CHROMATOGRAPHYTwo phases in chromatography are: mobile phase is part of the chromatographic system which carries the solutes through the stationary phase. stationary phase is the part which the mobile phase flows where the distribution of the solutes between the phases occurs.
  5. 5. PRINCIPLE Fractionalism of mixtures of substances In the operation of the chromatogram, a mobile gaseous or liquid phase is use to wash the substances to be separated through a column of a porous material.
  6. 6. PRINCIPLE Capillary Action – the movement of liquid within the spaces of a material due to the forces of adhesion, cohesion, and surface tension.
  7. 7. PRINCIPLE The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.
  8. 8. PRINCIPLE The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.
  9. 9. MECHANISMS OF SEPARATION IN CHROMATOGRAPHY• Based on the interactions of solutes with mobile and stationary phases.
  10. 10. ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHY Based on the competition between the sample and the mobile phase for binding sites of the solid (stationary) phase. Molecules that are soluble in the mobile phase move fastest.
  11. 11. ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHYAdvantages An extensive separation literature is available on thin layer chromatography methods that are readily transferable to adsorption The flexibility, speed, and low cost of TLC allow its use in experimental development.
  12. 12. ADSORPTION (LIQUID-SOLID) CHROMATOGRAPHYAdvantages TLC has great value for use in the preliminary investigation of samples of unknown constituents Adsorption chromatography, particularly with silica gel, has been widely used for the separation of drugs in both the HPLC and TLC modes.
  13. 13. PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY separates molecules on the basis of sample volatility. Depends on the solubility of the solute in nonpolar (organic) or polar (aqueous) solvents
  14. 14. PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY Advantage: the stationary phase does not leave the solid support and bleed into the detector, and a uniform monomolecular layer of the stationary phase is obtained through the bonding procedure.
  15. 15. PARTITION (LIQUID-LIQUID) CHROMATOGRAPHY Since the chemical influence of the solid support may be largely ignored, the adhering film behaves essentially like a liquid stationary phase.
  16. 16. ION-EXCHANGE CHROMATOGRAPHY∞ Based on the net charge of molecules∞ It is one of the common types of separation mechanism which depends on the nature of the stationary phase.∞ It has 2 prinicipal types of ion- exchanger is cationic and anionic.
  17. 17. ION-EXCHANGE CHROMATOGRAPHY∞ Ion exchange matrices can be further categorized as either strong or weak.∞ It separates amino acid by electric charges based on their respective changes
  18. 18. ION-EXCHANGE CHROMATOGRAPHY∞ Usually performed in columns∞ Uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides and proteins.
  19. 19. ION-EXCHANGE CHROMATOGRAPHY∞ PRINCIPLE: ∞ Relies on charge to charge interactions between proteins in the sample and the charges immobilized on the resin.
  20. 20. ION-EXCHANGE CHROMATOGRAPHY∞ PRINCIPLE: ∞ Once the solutes are bound, the column is washed to equilibrate it in the starting buffer,which should be of low ionic strength ∞ Then the bound molecules are eluted off using a gradient be of low ionic strength
  21. 21. ION-EXCHANGE CHROMATOGRAPHY∞ TWO PRINCIPAL TYPES: ∞ Anion exchange ∞ Cation exchange
  22. 22. ION-EXCHANGE CHROMATOGRAPHY Factors to be considered in Ion Exchange Chromatography:⓭ Buffers – use anionic buffers for cation exchange and cationic buffers for anion exchange to avoid difficulty.⓭ pH- influence the charge on the macromoloecules in solution.
  23. 23. ION-EXCHANGE CHROMATOGRAPHY Factors to be considered in Ion Exchange Chromatography:⓭ Salts to use for elution Ions of the eluting salt must displace othermolecules from the charged groups on the stationary phase with either a gradient or step in the 0 to 1.0 range.
  24. 24. ION-EXCHANGE CHROMATOGRAPHY
  25. 25. ION-EXCHANGE CHROMATOGRAPHYMost molecules have a netcharge within a pH range of 2 to10. When the pH is altered, thenet charge on molecules canchange drastically.In this experiment, a mixture of twochemicals is absorbed onto a solidsupport ion-exchange column andseparated during elution underconditions that influence their netcharge.
  26. 26. ION-EXCHANGE CHROMATOGRAPHY Instruments  Eluent Generator  Water Separation Column  Sample Injector  Electrolytic Eluent Suppressor  Detector  Computer
  27. 27. ION-EXCHANGE CHROMATOGRAPHYAPPLICATIONS: SOFTENING OF WATER DEMINERALIZATION OF WATER PURIFICATION OF SOLUTIONS FREE FROM IONIC IMPURITIES SEPARATION OF INORGANIC IONS SEPARATION OF SUGARS,AMINO ACIDS
  28. 28. ION-EXCHANGE CHROMATOGRAPHYADVANTAGES: DISADVANTAGES: Long Life of Resins  Nature and properties of Cheap maintenance ion exchange resins Environmental friendly  Nature of exchanging ions because it deals only with  There are substances (such substances occurring in as organic matter or Fe3+ water. occurring in some water which can foul the resin.
  29. 29. ION-EXCHANGE CHROMATOGRAPHYFACTORS AFFECTING THEINSTRUMENTALIZATION1. Column Packing2. Detectors3. Flow Rate4. Sample Size5. Solvent and Temperature
  30. 30. Applications: Serves as liquid chromatography detectors and as quality control monitors in drug manufactures Also occurs in air and water quality, medical and clinical laboratories and industrial laboratories
  31. 31. TYPES OF CHROMATOGRAPHYA.By Chromatographic bed shapeB. By Physical State of Mobile Phase
  32. 32. By chromatographic bed shape A. Column Chromatography• A separation technique in which the stationary bed is within a tube• It works on a much larger scale by packing the same materials into a vertical glass column.
  33. 33. By chromatographic bed shapeA. Column Chromatography
  34. 34. By chromatographic bed shapeB. Plane Chromatography • A separation that takes place on a flat surface or a plane Example: • Paper Chromatography • Thin Layer Chromatography
  35. 35. Paper chromatography Based on nature of solvent, solubility of solute and rate of diffusion. Uses paper as the stationary phase and a solvent as the mobile phase.
  36. 36. Paper chromatography Solvent moves through the paper by a capillary action Separation depends on the solubility of solute and solvents, the polarity of solvent, and polarity of solutes in the sample.
  37. 37. Paper chromatography Visualization of the separated sample occurs by chemical reaction, which produces a color change.
  38. 38. Paper chromatography Considered to be the simplest and the most widely used of the chromatographic techniques because its APPLICABILITY TO THE FOLLOWING: ISOLATION IDENTIFICATION AND QUANTITATIVE DETERMINATION OF ORGANIC AND INORGANIC COMPOUNDS
  39. 39. Instrumentation of Paper chromatography1) Lid2) Paper3) SolventFront4) Solvent
  40. 40. THIN-LAYER CHROMATOGRAPHY Used as a semi-quantitative screening test screening test Uses as thin layer of silica gel, alumina gel, polyacrylamide gel, or starch gel attached to glass plate as stationary phase and the mobile phase is liquid solvent.
  41. 41. THIN-LAYER CHROMATOGRAPHY Fractions in the sample are generally quite soluble in the solvent and move with it up the stationary phase by capillary action. Separated fractions are also developed in TLC by applying a chemical reaction with the separated fractions to produce color changes
  42. 42. THIN-LAYER CHROMATOGRAPHY
  43. 43. THIN-LAYER CHROMATOGRAPHYADVANTAGE: DISADVANTAGE:  Simple and economical  Spots are often faint  Easy to perform since it  TLC is difficult to only involves spotting reproduce the stationary phase with the sample &  Not typically placing one edge of the automated stationary phase plate in the mobile phase reservoir.
  44. 44. INSTRUMENTATION
  45. 45. By Physical State of Mobile Phase Gas Chromatography • It can separate nanograms or pictograms of volatile substances. • It is principally a method for the separation and quantitative determination of gases and volatile liquids and substances.
  46. 46. Gas Chromatography  Volatile compounds can be separated in a gas chromatograph, in which the mobile phase is usually a relatively unreactive carrier gas such as helium, nitrogen or hydrogen.  Separations can be carried out in the vapor phase, most parts of a gas chromatograph are temperature controlled; selection of temperature is based on the composition of the sample.
  47. 47. Gas Chromatography It uses a special detector according to the different kinds of compound and the most widely used are: A. Mass spectrophotometer B. Thermal Conductivity C. Flame Ionization Detector D. Electron Capture Detector
  48. 48. GAS CHROMATOGRAPHYApplications: Most effectively used for analyses of organic compounds, space related, complex mixtures of volatile substances at column temperature of less than -40 °C to greater than 550° C. Geochemical research projects such as determination of various environmental pollutants at extremely low concentrations.
  49. 49. GAS CHROMATOGRAPHYADVANTAGES: DISADVANTAGES: Ability to provide  LIMITED to volatile qualitative information and samples quantitative information  Not suitable for thermally labile samples FAST ANALYSIS  Fairly difficult for large Efficient, providing high preparative samples resolution  Requires spectroscopy Sensitive usually mass Nondestructive spectroscopy for confirmation of peak Requires small samples identity Inexpensive
  50. 50. COMPONENTS Autosampler- provides the means to introduce a sample automatically into the inlets. Automatic insertion provides better reproducibility and time- optimization. Column inlet (or injector)- provides the means to introduce a sample into a continuous flow of carrier gas. The inlet is a piece of hardware attached to the column head.
  51. 51. COMPONENTS Carrier Gas (mobile phase) - must be chemically inert, include helium, hydrogen and nitrogen. It should be of high purity, and the flow must be tightly controlled to ensure optimum column efficiency and reproducibility of test results. Detector
  52. 52. GAS CHROMATOGRAPHY GAS-LIQUID GAS-SOLID CHROMATOGRAPHY CHROMATOGRAPHY Separates molecules on  Uses a solid material as an the basis of sample absorbent volatility  Based upon a solid Mobile phase is a gas such stationary phase on which retention of analytes is the as helium and the consequence of physical stationary phase is a high adsorption boiling point liquid  Relies upon a large absorbed onto a solid. granular surface to aid in the separation of the substances.
  53. 53. GAS CHROMATOGRAPHYInterferences Volatility of compound Polarity of compounds Column temperature Column packing polarity Flow rate of the gas Length of the column
  54. 54. Application Use in biomedical research, routine clinical determination and drug researching programs
  55. 55. LIQUID CHROMATOGRAPHY The mobile phase is percolated through the column by means of either gravity , under pressure generated by a suitable pump or centrifugal force.
  56. 56. SIZE EXCLUSION CHROMATOGRAPHY Particles of different size will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided, that all the particles are loaded simultaneously or near – simultaneously of the same size should elute together.
  57. 57. SIZE EXCLUSION CHROMATOGRAPHY The support material has certain range of pore sizes. As solutes travel through, the small molecules can enter the pores, whereas the larger ones cannot and will elute first from column. The determination of molecular weight, e.g. , of enzymes, and estimation of equilibrium constants can be achieved with relative ease
  58. 58. SIZE EXCLUSION CHROMATOGRAPHYADVANTAGES DISADVANTAGES RAPID ROUTINE ANALYSIS  FILTRATIONS MUST BE PERFORMED BEFORE USING IDENTIFYING HIGH MASS THE INSTRUMENT BAD COMPONENTS EVEN IN RESPONSE FOR VERY LOW CONCENTRATION SMALL MOLECULAR WEIGHTS CAN ANALYZE  STANDARDS ARE NEEDED POLYDISPERSED  SENSITIVE FOR FLOW RATE SAMPLES,BRANCHING VARIATION. INTERNAL STUDIES CAN BE DONE, STANDARD SHOULD BE ABSOLUTE MOLECULAR USED WHENEVER POSSIBLE WEIGHTS CAN BE  HIGH INVESTMENT COST OBTAINED.
  59. 59. SIZE EXCLUSION CHROMATOGRAPHY Instruments Eluent Degasser Pump Auto Injector Size Exclusion Column Computer
  60. 60. Instrumentation
  61. 61. LIQUID CHROMATOGRAPHYHigh-performance liquidchromatography (HPLC) Uses a pressure for the pumping of aqueous or organic solution through a column. The mobile phase is forced under pressure through a long, narrow column, yielding an excellent separation in a relatively short time. Highly sensitive and specific.
  62. 62. LIQUID CHROMATOGRAPHYHigh-performance liquidchromatography (HPLC) Become the primary means of monitoring the use of drugs and of detecting drug abuse. Also used to separate the compounds contributing to the fragrance of the flowers.
  63. 63. LIQUID CHROMATOGRAPHY High-Performance Liquid ChromatographyADVANTAGES: DISADVANTAGES:An automated process that Difficult to detect coelution, takes only a few minutes to which may lead to inacurrate produce results. compound categorization.Uses gravity instead of high High cost for equipment speed pump to force compounds through the needed to conduct HPLC. densely packed tubing. Operation is complex, requiringResults are of high resolution a trained technician to operate. and are easy to read. Equipment has low sensitivityCan be reproduce easily via to some compounds because automated process. of the speed of the process.
  64. 64. Application Use in biomedical research, routine clinical determination and drug researching programs
  65. 65. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Instruments  Fraction Collector  Auto Sampler  Pumping systems  Columns & Packing  Detectors  Control Data & Processing
  66. 66. Instrumentation
  67. 67. Application Use in monitoring the use of therapeutic drugs and detecting drug abuse. also use to separate compounds contributing to the fragrance of flowers

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