Chromatography

1
Chromatography

• Objective
• To understand the principles of chromatography
  and know the specific types of Chromatograph used
  in the analysis of environmental samples.

2
Chromatography

• Background
• HPLC
• Gas Chromatography
• Ion Chromatography

3
Chromatography

• Definition
• Chromatography is a separation technique in which
  component molecules (solutes) are transported by
  a mobile phase over a stationary phase.
• Interaction with the stationary phase causes a
  distribution of solutes within the mobile phase.
• This interaction affects the rate at which
  solutes pass through.
• Solutes detected as they exit the stationary
  phase.

4
Development of Chromatography

• Discovered 1850
• Dyes separated on paper (water stain)
• Circular Chromatograms
• Planar Chromatography
• Paper Chromatography
• Ascending Solvent system
• Retardation Factor (Rf)
• Dyes, biochemicals, chlorophyll,
• Thin Layer Chromatography (TLC)
• Alumina with varying hydrophobicity
• 2-Dimension TLC - amino acids

5
Development of Chromatography

• Liquid Chromatography
• Mobile Phase is a liquid (water, solvent etc.)
  pumped at Low Pressure.
• Stationary Phase is a Column filled with a solid
  packing material (small beads).
• Gel Permeation Chromatography (GPC)
• Stationary Phase has pores of specific size
• Separation is on Physical Dimensions of solute
• Useful for Biopolymer separations - Enzyme
  Purification
• Solutes are Eluted in order , Largest first.
• Detected by UV Absorbance.

6
Analytical Chromatography

• Principles
• Partitioning between Phases gives Retention Time.
• Separation efficiency
• Peaks should not overlap
• Baseline Resolution
• Compromise Speed and Efficiency
• Small Stationary Phase Particles - Backpressure
• Elution rate
• Quantification
• Detector Response
• Peak shape
• Peak Area
• Standards

7
Analytical Chromatography

• External Standards
• Standard mixtures of solutes at known
  concentration.
• Injected several times
• Obtain an Average Detector Response for a given
  amount.
• Internal Standard (better)
• A known amount of a standard compound added to
  Every Sample.
• Detector response of Solute relative to Standard
  is the same in each run.
• independent of actual response of detector.

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• High Performance (Pressure) Liquid Chromatograph
  (HPLC)

• Flexible, High Resolution
• Very good for non-volatile chemicals
• sugars, labile organics, pesticides
• liquid mobile phase
• polar or non-polar
• Isocratic (same strength)
• Gradient (concentration changes)
• liquid stationary phase
• polar or non polar

9
HPLC

• Columns short, not heated, densely packed with
  small particles (5 - 10 ?m)
• Very high pressure (8000 psi)
• Detectors eg
• UV absorbance
• conductivity
• fluorescence
• Derivatisation
• pretreatment of chemical to make detection easier
• e.g. Fluorescent

10

• Gas Chromatography (GC)

• Mobile Phase - Gas
• Helium, Hydrogen - constant flow rate
• Stationary Phase Liquid (GLC)
• Gas liquid chromatography
• Liquid present as a layer on a solid particle
• Polar or Non-polar
• Stationary Phase Solid
• Gas solid chromatography
• Separates stable volatile (organics)
• e.g. THMs, Organohalogens, Solvents, PCBs,
  Organophosphates, Drugs, Fatty acids etc.

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Components of a Gas Chromatograph (GC)

• Injector
• Heated - Programmable
• Column
• Packed (2 - 10 m) diameter 5 mm
• Capillary (10 - 30 m) diameter 0.25 mm
• Oven (for Column)
• Programmable Temperature Gradients
• Very Precise Control
• Detector
• Many types, compound specific (sensitivity)
• Data Processing

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Sample Injection in GC

• Direct On-Column
• Small quantities
• Guard column protects analytical column
• Flash Vapourisation
• glass or quartz liner
• Split or Splitless for Capillary Columns
• when concentrations of compound are high
• Purge and Trap
• Good for volatiles in water (low levels)
• Sample is purged with bubbles
• Stripped Volatiles adsorb onto a trapping column
• Trapping column inserted into GC injector port

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Detectors

• Flame Ionisation Detector (FID)
• Detects most Organics
• Current across a hydrogen flame
• Sensitivity 1 ng/l, Linear Dynamic Range (LDR) is
  107
• Electron Capture Detector (ECD)
• Detects trace environmental Pollutants e.g.
  Pesticides and Herbicides that have
  Electronegative atoms (Chlorine).
• Radioactive 63Ni - electrons captured by
  compounds.
• Sensitivity 0.01 ng/l, Linear Dynamic Range (LDR)
  is 104

14
Detectors

• Thermal Conductivity Detector (TCD)
• resistance of a wire varies with temperature
• carrier gas is affected by the compounds it
  contains.
• Good for gases (methane, carbon monoxide,
  Hydrogen)
• Sensitivity 0.1 mg/l , Linear Dynamic Range
  (LDR) is 104
• Mass Selective Detector (MSD)
• Mass spectrum taken continuously
• Single ion or complete spectra
• Sensitivity 1 ng/l
• Others, Flame Photometry, Photo ionisation,
  Thermionic.

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Ion Chromatography (Dionex)

• Variation of HPLC
• Detects Anions and Cations
• ion exchange column (charged stationary phase)
• mobile phase has competing ions (exchange with
  solutes)
• Detection
• Conductivity or Absorbance of the column
  effluent.
• Sensitivity Improved by Suppression of background
  conductivity of the mobile Phase.
• Suppressor Column (ion exchange)
• Electrochemical Suppression