Published on

Published in: Education, Technology
1 Comment
1 Like
No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide


  2. 2.  thermodynamically rigorous
  3. 3.   Distribution Ratio(D)”, Where, CA denotes concentration of „A‟ in all its form as determined analytically.
  4. 4.    “Separation factor(or coefficient)”
  5. 5. For example, suppose we haveAmount of solute in aqueous phase(xo)=300gVolume of aqueous phase = 100mlVolume of organic phase to be added = 200mlDistribution ratio of the particles = 0.5
  6. 6. NOW, The total amount of solute (x n) left non- extracted in the aqueous phase („V‟ ml) on adding the extractive organic phase („v‟ ml) can be calculated using the formulae,
  7. 7. WHERE, xo = amount of solute present before adding extractive solvent D = Distribution ratio of the solute particles n = Number of times the solvent is added
  8. 8. CASE 1: If n=1, i.e., the extractive solvent is added in one complete go, then, from the formulae, we find that, x1 = 60g, that means 240g of the solute gets extracted.CASE 2: If n=2, i.e., if we add the extractive solvent in two parts each of 100 ml, then,
  9. 9. v=100ml, and using the formulae, we findx2 to be 33g, which implies that, 267g of thesolute has been extracted.
  10. 10.
  11. 11.   By formation of a neutral metal chelate complex, or  By ion association.
  12. 12.  INNER COMPLEXES
  13. 13.   Basic strength of the chelating group  Nature of the donor atoms of chelating agent
  14. 14.  Size of the ring Resonance & steric effect
  15. 15.  
  16. 16.  The reagent and the metal complex exist as undissociated molecules in both phases. Solvation plays no significant part in the extraction process. The solutes are uncharged particle & their concentrations are so low that the solutions do not deviate much from ideality.
  17. 17. 
  18. 18.   
  19. 19. w w w
  20. 20. wo
  21. 21.  o w w
  22. 22. o w**
  23. 23. * w
  24. 24. 
  25. 25. 
  26. 26. 
  27. 27.  Those formed from the reagents yielding large organic ions, eg. Tetraphenylarsonium[(C6H5)4As+], which tend to form large ionic clusters with oppositely charged ions, like ReO4- . They do not have a hydration shell & thus, disrupt the water structure, due to which the tend to be pushed into the organic phase. Those involving a cationic or anionic
  28. 28. chelate complex of the metal ion. Thus, the chelating reagent consists of two uncharged donor atoms. Eg. 1:10 phenanthroline forms cationic complexes. Those in which solvent molecules are directly involved in the formation of ion- association complex. Eg. ethers, esters etc.
  30. 30. Partition Chromatography II Reverse Phase Chromatography – Nonpolar Stationary Phase – Polar Mobile Phase Normal Phase Chromatography – Polar Stationary Phase – Nonpolar Mobile Phase Column Selection Mobile-Phase Selection
  31. 31. Partition Chromatography III Research Applications – Parathion in Insecticides: O – CH3CH2O P O NO2 CH3CH2O – Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa
  32. 32. Adsorption Chromatography Classic Solvent Selection Non-polar Isomeric Mixtures Advantages/ Disadvantages Applications
  33. 33. What is Ion Chromatography? Modern methods of separating and determining ions based on ion-exchange resins Mid 1970s Anion or cation mixtures readily resolved on HPLC column Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations
  34. 34. The Mobile Phases are... Aqueous solutions – containing methanol, water-miscible organic solvents – also contain ionic species, in the form of a buffer – solvent strength & selectivity are determined by kind and concentration of added ingredients – ions in this phase compete with analyte ions for the active site in the packing
  35. 35. Properties of the Mobile Phase Must – dissolve the sample – have a strong solvent strength leads to reasonable retention times – interact with solutes in such a way as to lead to selectivity
  36. 36. Ion-Exchange Packings Types of packings – pellicular bead packing • large (30-40 µm) nonporous, spherical, glass, polymer bead • coated with synthetic ion-exchange resin • sample capacity of these particles is less – coating porous microparticles of silica with a thin film of the exchanger • faster diffusion leads to enhanced efficiency
  37. 37. Ion-Exchange Equilibria Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid Cation exchange resins – sulfonic acid group, carboxylic acid group Anion exchange resins – quaternary amine group, primary amine group CM Cellulose DEAE Cellulose Cation Exchanger Anion Exchanger
  38. 38. Eluent Suppressor Technique Made possible the conductometric detection of eluted ions. Introduction of a eluent suppressor column immediately following the ion-exchange column. Suppressor column – packed with a second ion-exchange resin Cation analysis Anion analysis
  39. 39. Size ExclusionChromatography(SEC) Gel permeation(GPC), gel filtration(GFC) chromatography Technique applicable to separation of high-molecular weight species Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase
  40. 40. SEC(continued) Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules – larger molecules – smaller molecules – intermediate size molecules
  41. 41. SEC Column Packing Small (~10 µm) silica or polymer particles containing a network of uniform pores Two types (diameters of 5 ~ 10 µm) – Polymer beads – silica-based particles
  42. 42. Advantages of Size ExclusionChromatography Short & well-defined separation times Narrow bands--> good sensitivity Freedom from sample loss, solutes do not interact with the stationary phase Absence of column deactivation brought about by interaction of solute with the packing
  43. 43. Disadvantages Only limited number of bands can be accommodated because the time scale of the chromatogram is short Inapplicability to samples of similar size, such as isomers. – At least 10% difference in molecular weight is required for reasonable resolution
  44. 44. Instrumentation Instruments required: – Mobile phase reservoir – Pump – Injector – Column – Detector – Data system
  45. 45. Schematic of liquidchromatograph
  46. 46. Mobile phase reservoir Glass/stainless steel reservoir Removal of dissolved gases by degassers – vacuum pumping system – heating/stirring of solvents – sparging – vacuum filtration
  47. 47. Elution methods Isocratic elution – single solvent of constant composition Gradient elution – 2 or more solvents of differing polarity used
  48. 48. Pumping System I Provide a continuous constant flow of the solvent through the injector Requirements – pressure outputs up to 6000 psi – pulse-free output – flow rates ranging from .1-10 mL/min – flow control and flow reproducibility of .5% or better – corrosion-resistant components
  49. 49. Pumping System II Two types: – constant-pressure – constant-flow Reciprocating pumps – motor-driven piston – disadvantage: pulsed flow creates noise – advantages: small internal volume (35-400 L), high output pressures (up to 10,000 psi), ready adaptability to gradient elution, constant flow rates
  50. 50. Pumping System III Displacement pumps – syringe-like chambers activated by screw-driven mechanism powered by a stepper motor – advantages: output is pulse free – disadvantage: limited solvent capacity (~20 mL) and inconvenience when solvents need to be changed Flow control and programming system – computer-controlled devices – measure flow rate – increase/decrease speed of pump motor
  51. 51. Sample Injection Systems For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 L Sampling loops – interchangeable loops (5-500 L at pressures up to 7000 psi)
  52. 52. LC column LC injector
  53. 53. Liquid Chromatographic Column  Smooth-bore stainless steel or heavy-walled glass tubing  Hundreds of packed columns differing in size and packing are available from manufacturers ($200- $500)  Add columns together to increase length
  54. 54. Liquid ChromatographicColumns II Column thermostats – maintaining column temperatures constant to a few tenths degree centigrade – column heaters control column temperatures (from ambient to 150oC) – columns fitted with water jackets fed from a constant temperature bath
  55. 55. Detector Mostly optical Equipped with a flow cell Focus light beam at the center for maximum energy transmission Cell ensures that the separated bands do not widen
  56. 56. Some Properties of Detector Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening
  57. 57. More Properties of Detector High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of analytes Non-destructive
  58. 58. Types of Detector Refractive index UV/Visible Fluorescence Conductivity Evaporative light scattering Electrochemical
  59. 59. Refractive Index I Measure displacement of beam with respect to photosensitive surface of dectector
  60. 60. Refractive Index II Advantages – universal respond to nearly all solutes – reliable – unaffected by flow rate – low sensitive to dirt and air bubbles in the flow cell
  61. 61. Refractive Index III Disadvantages – expensive – highly temperature sensitive – moderate sensitivity – cannot be used with gradient elution
  62. 62. UV/Visible I Mercury lamp = 254nm = 250, 313, 334 and 365nm with filters Photocell measures absorbance Modern UV detector has filter wheels for rapidly switching filters; used for repetitive and quantitative analysis
  63. 63. UV/Visible II
  64. 64. UV/Visible III Advantages – high sensitivity – small sample volume required – linearity over wide concentration ranges – can be used with gradient elution
  65. 65. UV/Visible IV Disadvantage – does not work with compounds that do not absorb light at this wavelength region
  66. 66. Fluorescence I For compounds having natural fluorescing capability Fluorescence observed by photoelectric detector Mercury or Xenon source with grating monochromator to isolate fluorescent radiation
  67. 67. Fluorescence II Advantages – extremely high sensitivity – high selectivity Disadvantage – may not yield linear response over wide range of concentrations
  68. 68. Conductivity Measure conductivity of column effluent Sample indicated by change in conductivity Best in ion-exchange chromatography Cell instability
  69. 69. Evaporative Light Scattering I Nebulizer converts eluent into mist Evaporation of mobile phase leads to formation of fine analyte particles Particles passed through laser beam; scattered radiation detected at right angles by silicon photodiode Similar response for all nonvolatile solutes Good sensitivity
  70. 70. Evaporative Light Scattering II
  71. 71. Electrochemical I Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current
  72. 72. Electrochemical II Advantages – high sensitivity – ease of use Disadvantages – mobile phase must be made conductive – mobile phase must be purified from oxygen, metal contamination, halides
  73. 73. Data System For better accuracy and precision Routine analysis – pre-programmed computing integrator Data station/computer needed for higher control levels – add automation options – complex data becomes more feasible – software safeguard prevents misuse of data system
  74. 74. Electrophoresis…charged speciesmigrate in electric fieldSeparation based on charge ormobility
  75. 75. Capillary electrophoresishigher voltages can be used asthe heat can be dissipated
  76. 76. Capillary electrophoresis