HPLC
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HPLC

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HPLC HPLC Presentation Transcript

  • High Performance Liquid Chromatography FEROSEKHAN S FNB-41 CIFE MUMBAI
  • Liquid Chromatography
  • Elution chromatography
    • Increasing polarity of pure solvents
    • hexane
    • ether
    • acetone
    • methanol
    • water
    • acetic acid
    • Solvents mixed
    • %hexane and % methanol
    • miscible
    • can be mixed continuously (solvent programming)
  • Outline
    • What is HPLC?
      • An Overview
    • Types of HPLC
      • Partition Chromatography
      • Adsorption Chromatography
      • Ion Chromatography
      • Size-Exclusion Chromatography
  • What is HPLC?
    • The most widely used analytical separations technique
    • Utilizes a liquid mobile phase to separate components of mixture
    • uses high pressure to push solvent through the column
    • Popularity :
      • sensitivity
      • ready adaptability to accurate quantitative determination
      • suitability for separating nonvolatile species or thermally fragile ones
  • HPLC is….
    • Popularity:
      • widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public
    • Ideally suited for separation and identification of,
      • proteins,
      • amino acids
      • nucleic acids,
      • hydrocarbons,
      • carbohydrates,
      • pharmaceuticals,
      • pesticides,
      • pigments,
      • antibiotics,
      • steroids, and a variety of other inorganic substances
  • History
    • Early LC carried out in glass columns
      • diameters: 1-5 cm
      • lengths: 50-500 cm
    • Size of solid stationary phase
      • diameters: 150-200  m
    • Flow rates still low! Separation times long!
    • Decrease particle size of packing causes increase in column efficiency!
      • diameters 3-10  m
    • This technology required sophisticated instruments
      • new method called HPLC
  • Advantages to HPLC
    • Higher resolution and speed of analysis
    • HPLC columns can be reused without repacking or regeneration
    • Greater reproducibility due to close control of the parameters affecting the efficiency of separation
    • Easy automation of instrument operation and data analysis
    • Adaptability to large-scale, preparative procedures
  • Advantages to HPLC
    • Advantages of HPLC are result of 2 major advances:
      • stationary supports with very small particle sizes and large surface areas
      • appliance of high pressure to solvent flow
  • Instrumentation
    • Instruments required:
      • Mobile phase reservoir
      • Pump
      • Injector
      • Column
      • Detector
      • Data system
  • Schematic of liquid chromatograph
  • LC column LC injector
  • Partition Chromatography
    • Most widely used
    • Bonded-phase Chromatography
    • Silica Stationary Phase:
    • OH OH OH OH
    • O O O
    • Si Si Si Si
    • Siloxanes: O CH 3
    • Si O Si R R= C 8 , C 18
    • O CH 3
  • Partition Chromatography
    • Reverse Phase Chromatography
      • Nonpolar Stationary Phase
      • Polar Mobile Phase
    • Normal Phase Chromatography
      • Polar Stationary Phase
      • Nonpolar Mobile Phase
    • Column Selection
    • Mobile-Phase Selection
  • Partition Chromatography
    • Applications
      • Parathion in Insecticides:
      • O
      • CH 3 CH 2 O P O NO 2
      • CH 3 CH 2 O
  •  
  • Adsorption Chromatography
    • Classic
    • Solvent Selection
    • Non-polar Isomeric Mixtures
    • Advantages/ Disadvantages
    • Applications
  • 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
  •  
  • Size Exclusion Chromatography(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
    • 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
    SEC(continued)
  • 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
  •  
  • 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
  • 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
  • Mobile phase reservoir
    • Glass/stainless steel reservoir
    • Removal of dissolved gases by degassers
      • vacuum pumping system
      • heating/stirring of solvents
  • Elution methods
    • Isocratic elution
      • single solvent of constant composition
    • Gradient elution
      • 2 or more solvents of differing polarity used
  •  
  • Sample Injection Systems
    • For injecting the solvent through the column
    • Minimize possible flow disturbances
    • Volumes must be small
    • .1-500  L
    • Sampling loops
      • interchangeable loops (5-500  L at pressures up to 7000 psi)
  • Types of Detector
    • Refractive index
    • UV/Visible
    • Fluorescence
    • Conductivity
    • Evaporative light scattering
    • Electrochemical
  • Detectors
    • Refractive Index detector
      • All solutes
      • Measures change in RI of the mobile phase due to solutes
      • Carbohydrates and lipids
  • Refractive Index
    • Measure displacement of beam with respect to photosensitive surface of dectector
  • Refractive Index
    • Advantages
      • universal respond to nearly all solutes
      • reliable
      • unaffected by flow rate
      • low sensitive to dirt and air bubbles in the flow cell
  • Refractive Index
    • Disadvantages
      • expensive
      • highly temperature sensitive
      • moderate sensitivity
      • cannot be used with gradient elution
  • Detectors
    • UV-VIS absorption detector (190-800 nm)
      • Aromatics
      • Conjugated molecules
  • UV/Visible
    • Mercury lamp
    • Photocell measures absorbance
  • UV/Visible
    • Advantages
      • high sensitivity
      • small sample volume required
      • linearity over wide concentration ranges
      • can be used with gradient elution
    • Disadvantage
      • does not work with compounds that do not absorb light at this wavelength region
  • Fluorescence
    • For compounds having natural fluorescing capability
    • Fluorescence observed by photoelectric detector
    • Mercury or Xenon source with grating monochromator to isolate fluorescent radiation
  • Fluorescence
    • Advantages
      • extremely high sensitivity
      • high selectivity
    • Disadvantage
      • may not yield linear response over wide range of concentrations
  • Conductivity
    • Measure conductivity of column effluent
    • Sample indicated by change in conductivity
    • Best in ion-exchange chromatography
    • Cell instability
  • 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