Transcript Measurement of Hardness

Chromatography
Objective
 To understand the principles of chromatography and
know the specific types of Chromatograph used in the
analysis of environmental samples.
Chromatography
Background
HPLC
Gas Chromatography
Ion Chromatography
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.
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
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.
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
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Detector Response
Peak shape
Peak Area
Standards
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.
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
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HPLC
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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
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.
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
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
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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
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
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.
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