1. Chromatography vObjective  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 vDefinition  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 vDiscovered 1850  Dyes separated on paper (water stain)  Circular Chromatograms vPlanar Chromatography  Paper Chromatography  Ascending Solvent system  Retardation Factor (R f ) –Dyes, biochemicals, chlorophyll,  Thin Layer Chromatography (TLC)  Alumina with varying hydrophobicity  2-Dimension TLC - amino acids

5. Development of Chromatography vLiquid 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 vPrinciples  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 vExternal Standards  Standard mixtures of solutes at known concentration.  Injected several times  Obtain an Average Detector Response for a given amount. vInternal 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.

8. High Performance (Pressure) Liquid Chromatograph (HPLC) n Flexible, High Resolution n Very good for non-volatile chemicals n sugars, labile organics, pesticides n liquid mobile phase – polar or non-polar – Isocratic (same strength) – Gradient (concentration changes) n liquid stationary phase – polar or non polar

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

10. Gas Chromatography (GC) vMobile Phase - Gas vHelium, Hydrogen - constant flow rate vStationary Phase Liquid (GLC)  Gas liquid chromatography  Liquid present as a layer on a solid particle  Polar or Non-polar vStationary Phase Solid  Gas solid chromatography vSeparates stable volatile (organics)  e.g. THMs, Organohalogens, Solvents, PCBs, Organophosphates, Drugs, Fatty acids etc.

11. Components of a Gas Chromatograph (GC) vInjector  Heated - Programmable vColumn  Packed (2 - 10 m) diameter 5 mm  Capillary (10 - 30 m) diameter 0.25 mm vOven (for Column)  Programmable Temperature Gradients  Very Precise Control vDetector  Many types, compound specific (sensitivity) vData Processing

12. Sample Injection in GC vDirect On-Column  Small quantities  Guard column protects analytical column vFlash Vapourisation  glass or quartz liner vSplit or Splitless for Capillary Columns  when concentrations of compound are high vPurge 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

13. Detectors vFlame Ionisation Detector (FID)  Detects most Organics  Current across a hydrogen flame  Sensitivity 1 ng/l, Linear Dynamic Range (LDR) is 10 7 vElectron Capture Detector (ECD)  Detects trace environmental Pollutants e.g. Pesticides and Herbicides that have Electronegative atoms (Chlorine).  Radioactive 63 Ni - electrons captured by compounds.  Sensitivity 0.01 ng/l, Linear Dynamic Range (LDR) is 10 4

14. Detectors vThermal 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 10 4 vMass Selective Detector (MSD)  Mass spectrum taken continuously  Single ion or complete spectra  Sensitivity 1 ng/l vOthers, Flame Photometry, Photo ionisation, Thermionic.

15. Ion Chromatography (Dionex) vVariation of HPLC vDetects Anions and Cations  ion exchange column (charged stationary phase)  mobile phase has competing ions (exchange with solutes) vDetection  Conductivity or Absorbance of the column effluent.  Sensitivity Improved by Suppression of background conductivity of the mobile Phase.  Suppressor Column (ion exchange)  Electrochemical Suppression