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Types chrom


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Types chrom

  1. 1. Types of Chromatography
  2. 2. Classification of chromatography• According to separation mode: a) Adsorption chromatography b) Partition chromatography c) Ion-exchange chromatography d) Size exclusion chromatography e) Affinity chromatography
  3. 3. Classification of chrom. (cont.)• According to mobile phase: a) Gas Chromatography i- Gas solid chromatography ii- Gas liquid chromatography b) Liquid chromatography i- Paper chromatography ii- Thin-layer chromatography iii- Column chromatography
  4. 4. Classification of chrom. (cont.)• According to form of stationary phase a) Planar chromatography i- Paper chromatography ii- Thin-layer chromatography b) Column chromatography i- Gas chromatography ii- Liquid chromatography (LC/HPLC)
  5. 5. LIQUID-COLUMN CHROMATOGRAPHY A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample travel through the column more slowly than others resulting in the desired separation.
  6. 6. FOUR BASIC LIQUID CHROMATOGRAPHYThe basic liquid chromatography modes are named according to the mechanism involved:1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC
  7. 7. L C used for samples: containing large molecules/ionic containing substances with low vapor pressure (non-volatile substances) Substances thermally unstable Substances can’t be vaporized without decomposing
  8. 8. LIQUID SOLID CHROMATOGRAPHY Normal phase LS Reverse phase LS δ− δ+ Si - O - H 30 µ Silica Gel The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
  10. 10. Adsorption chromatography Stationery phase is solid and mobile phase is liquid Distribution between two phases (adsorption and desorption) Attractive forces (ionic, dipole-dipole, dipole induced dipole) Good adsorbent has large surface area and more active sites Equilibration occurs at: surface-solute, surface- solvent, solvent-solute
  11. 11. Adsorption isotherms An adsorption isotherm is a plot of the concentration or amount of analyte on a surface as a function of its concentration in the bulk phase. In liquid chromatography, the bulk phase is, of course, the mobile phase.
  12. 12. Adsorption isotherms Linear (k = Cs/Cm) = 1 Convex (k = Cs/Cm1/n) where n >1 Concave
  13. 13. Adsorbents Adsorbing power depends on 1- chemical nature of the surface 2- area available 3- pretreatment
  14. 14. Commonly used adsorbents Alumina, Al2O3 ,(Aluminum oxide). It may be acidic, basic and neutral in nature. Available in various grades Silica gel (silicon dioxide). It is acidic in nature. Available in various grades. CaCO3 Sucrose Starch cellulose
  15. 15.  Adsorbent should have uniform size and large surface area Weight of the adsorbent should be 20-50 times more than the sample
  16. 16. Solvents & adsorbents Since adsorbents are polar, non-polar elute first. Usually, the elusion order is as: alkyl halids < saturated hydrocarbons< unsaturated hydrocarbons <ethers < esters < ketones < amines < alcohols < phenols < acids and bases. Polymeric compounds and salts often don’t elute The solvents in the order of polarity Hexane/Pet ether < CCl4 < toluene < dichloromethane < chloroform < diethyl ether < acetone < ethyl acetate < propanol < ethanol < methanol < acetic acid < water
  17. 17. Columns and packing Various sizes are available Wet method Dry method Sample loading and running the column
  18. 18. WATER-SOLUBLE VITAMINS1. Niacinamide 2. Pyridoxine H 3C N N HO CH 2OH CONH 2 CH 2OH3. Riboflavin CH 2OH HOCH HOCH 4. Thiamin HOCH CH 2H 3C N N O H 3C N NH 2 S CH 2CH 2OH NH ClH 3C N N N CH 2 CH 3 O
  19. 19. WATER-SOLUBLE VITAMINS 2 3Inject 4 1 Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins 0 5 10 15 20
  20. 20. LIQUID-LIQUID CHROMATOGRAPHY ODPN(oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH3 CH2 OCH2 CH2 CN(Normal) CH3 (CH2 ) 16 CH3 (Reverse)The stationary solid surface is coated with a 2nd liquid (theStationary Phase) which is immiscible in the solvent (Mobile) phase.Partitioning of the sample between 2 phases delays or retains somecomponents more than others to effect separation.
  21. 21. Advantages of Partition chromatography – Advantage over adsorption chromatography More reproducible and predictable Distribution coefficient is constant over a much greater range of concentration yielding sharper and symmetrical peaks It is also of two types 1-Normal phase 2-Reversed phase
  22. 22. Supports for stationary phase Silica gel Kieselguhr/Celite Cellulose
  23. 23. steps Sample preparation Sample loading Elution Detection: chemical methods; diverse types of detectors can be used
  24. 24. ION-EXCHANGE CHROMATOGRAPHY - + SO3 NaSeparation is based on the competition of different ioniccompounds of the sample for the active sites on the ion-exchange resin (column-packing).
  25. 25. Ion exchange chromatography A process where ions held by solid matrix are exchanged for counter ions in the solution Synthetic ion exchange resins are used for water purification and separation of ions
  26. 26. MECHANISM OF ION-EXCHANGECHROMATOGRAPHY OF AMINO ACIDS pH2 - + + SO3 Na H3N COOH Ion-exchange Resin - + SO3 H3N - COO pH4.5 + Na
  27. 27. Chromatography of Amino Acids Stationary Phase Mobile Phase + H3N - SO3 Na+ COOH + Na OH - + SO3 H3 N COOH Exchange Resin - SO3 H3N+ COOH pH3.5 OH - SO3 + H3 N + - + - Na COO H OH = H 2 O + Na - SO3 H3 N + - + - COO H OH = H 2 O - SO3Na+ pH4.5
  28. 28. Ions exchange resins Consist of three dimensional polymeric chains, cross linked by short chains, which carry ionisable functional groups. Based on ions these are of two types – Cation exchangers (weak or strong) – Anion exchangers (weak or strong)
  29. 29. Formation of resin Styrene and divinylbenzene The number of cross linkers determine by the ratio of Styrene : divinylbenzene Increasing cross linkers increases the rigidity and reduces swelling
  30. 30. Ion-exchange chromatography can be used toperform preparative separation of amino acidsNegatively charged resin binds selectively topositively charged amino acids
  31. 31. Behavior of resin Important properties which determine behavior of resin are:1- size of particles2- degree of cross linking3- nature of functional groups4- number of functional groups
  32. 32. Theoretical principles Ion exchange equilibrium distribution coefficient (KD) indicates affinity of the resin for ions relative to hydrogen Generally, if KD is large, resin will incline to attract the ion Polyvalent ions are more attracted to the resin compared to mono-valent
  33. 33.  In groups where charges are same, the difference between KD is related to the size of the ion
  34. 34. Uses of ion exchange chromatography Ion separation Concentration of trace matters Separation of alkali and alkali earth metals Separation of amino acids, proteins, peptides, nucleic acid and nucleotides immunoglobulin
  35. 35. Steps Same to that of the others Detection: conduction measuring
  36. 36. Size exclusion chromatography Molecular gel chromatography, gel permeation, molecular sieving or molecular exclusion Stationary phase serves as molecular sieve Separate molecules based on size via sieving or filtration Adsorption and electrical charge play role in separation
  37. 37. Gels Open three dimensional network formed by cross linking large ploymeric chains Polar groups absorb water and swell Have an exclusion limit i.e critical size of a molecule that can just penetrate the interior
  38. 38. Theoretical principles There are 2 kinds of solvent in the gel Vi = Volume within gel Vo = volume outside the beads of the gelLarge molecules will not be able to enter or penetrate the pores of the gel, hence their elution volume (Ve ) will be Ve = VoWhereas, smaller molecules must be swept through Vo plus some additional volume which is a fraction of Vi
  39. 39. Ve = Vo + KDVi, where KD = distribution coefficientKD = Average concentration of solute in gel/ Average concentration of solute outside gelKD value should be between 0 and 1 If sieving action is the only mechanism of separation (KD=1) then Ve = Vo + ViIf K<1, It indicates solvent interacts with the gel (adsorption, hydrogen bonding)
  40. 40. Types of the gels Sephadex, dextran gel (classified by the amount of water regain) Biogel, polyacrylamide gel, inert series of gels, insoluble in water and common organic solvents Styragel rigid cross linked polystyrene gel useful at temperature > 150oC with organic solvents, it can be used under high pressure
  41. 41. Gel Filtration
  42. 42. Applications Desalting (removal of salts and small molecules from macromolecules) Concentrating (concentration of dilute solutions of macromolecules with MW> exclusion limit Fractionation (separation of mixture of closely related molecules having small difference in KD values namely proteins, peptides, nucleic acids, polysaccharides, enzymes and hormones)
  43. 43. Types of ChromatographyMOBILE PHASE LIQUID Liquid-Liquid Liquid-SolidFORMAT Chromatography Chromatography (Partition) (Adsorption)STATIONARY Solid LiquidPHASE Normal Phase Normal Phase Reverse Phase Reverse Phase Mobile Phase - Mobile Phase - Nonpolar Polar Stationary phase - Stationary phase - Polar Nonpolar
  44. 44. Detectors1. Ultraviolet Detector 200-400nm 254 nm2. Reflective Index Detector Universal Detector
  45. 45. Affinity chromatography Separation where surface of inert phase has been modified to selectively bind compounds having specific functional group Binding force should be strong enough to effect separation but weak enough to get the compound when desired
  46. 46. Properties of Inert matrix Mechanically and chemically stable Large surface area Easily derivatized Good flow characteristics Examples (agarose, controlled pore glass, cellulose)
  47. 47. Spacer An arm to move active group away from the bead so that steric hindrances are at minimum Effectiveness depend on their – length – stability of the attachment to the bead – hydrophobic nature – presence of fixed charges and their concentration Affi-gel has spacer arm –O- (CH2)3 NH2 [Oxypropylamine]
  48. 48. Affinity Chromatography
  49. 49. Affinity Chromatography
  50. 50. Selected ligands and their affinity compoundsLigands Affinity compoundsDiazo-NAD- dehydrogenasesAMP analogues NADP- binding proteinsBlue dextran Yeast phosphofructokinase2000Methotrexate Dihydrofolate reductaseB12 Transcobalamin I and II
  51. 51. Chromatographic techniques Classical LC TLC/ paper chromatography Modern LC
  52. 52. Classical chromatography technoques Glass or plastic columns Need skill Solvent flow (gravity, suction) and individual samples collected manually Detected using different detectors Detection and quantification achieved by manual analysis of fractions Results are recorded in the form of chromatogram (sample concentration vs fraction number)
  53. 53. Disadvantages Column packing procedure tedious Low column efficiency, long analysis time Technique depends on user Detection of solutes is labor intensive and takes a lot of time
  54. 54. Plane chromatography Plane surface rather than column 2 dimensional Selective properties (use of two solvents) Include – Paper chromatography and – thin layer chromatography
  55. 55. Principles Principles are similar to column Successive equilibrations of the analyte between two phases Non ideal processes may cause zone spreading Degree of retention is Rf – Ratio between distance traveled by solute/distance traveled by solvent
  56. 56. Relation between Rf and K Rf = number of moles of solute in mobile phase/total moles in both phases = Cm Am/CmAm+ CsAsAm and As are the cross sectional areas of two phases. By dividing CmRf = Am/Am+AsCs/Cm = Am/Am+ KAs
  57. 57. Paper chromatography Mainly qualitative and semi quantiative Easy to perform Mechanisms1- liquid liquid2- adsorption3- hydrogen bonding4- ion exchange
  58. 58. Nature of the paper Highly purified cellulose Great affinity for water and polar solvents Paper may be impregnated with alumina, silica or ion exchange resin
  59. 59. Procedure Sample application Development1- ascending Simple and popular Solvent flow through capillary action Slow development Slow rate enhances partition, separation
  60. 60. 2- descending Flow is downward Paper folded U shape Solvent flow capillary and gravity Much faster
  61. 61. detection Visible Application of Reagents UV absorbance Florescence IR Radioactivity Chemical tests Bioautography
  62. 62. qualitative Based on Rf valuesSemi-quantitative Extraction and spectroscopy densitometry