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MiBTP Masterclass Chromatography

MiBTP Masterclass Chromatography. 5 /12/2012 Hendrik Schäfer and Yin Chen. Overview. Introductions What is chromatography Principles, glossary Applications of chromatography Different kinds of chromatography. Chromatography in Env Microbio at Warwick. Environmental microbiologists

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MiBTP Masterclass Chromatography

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  1. MiBTPMasterclassChromatography 5/12/2012 Hendrik Schäfer and Yin Chen

  2. Overview • Introductions • What is chromatography • Principles, glossary • Applications of chromatography • Different kinds of chromatography

  3. Chromatography in EnvMicrobio at Warwick • Environmental microbiologists • Interested in microbial metabolism of trace gases (methylated sulfur compounds, methylated amines, methyl halides, methane and organic pollutants • Main applications of chromatography • Gas chromatography • Thin layer chromatography • HPLC • Protein purification

  4. What is your interest in chromatography? • Experience in using chromatography? • Plans to use chromatography?

  5. Chromatography • Separation of mixtures of compounds into their components based on their different partition coefficients in chromatography media • Can be preparative or analytical technique • Wide range of chromatography techniques, largely governed by the same principles

  6. Basic principles of chromatography • stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas) • Mobile phase flows through the stationary phase and carries the components of the mixture with it • Different components travel at different rates

  7. Main platforms • Thin layer chromatography (TLC) • Gas chromatography (GC) • Ion chromatography (IC) • High performance liquid chromatography (HPLC) • Fast protein liquid chromatography (FPLC)

  8. Thin layer chromatography (TLC) • Uses a thin layer of e.g. silica gel (stationary phase) on a support plate • Pencil start line drawn • Mixture of compounds applied on line using syringe or pipette • Plate put into solvent Beaker TLC plate Start line Solvent

  9. Thin layer chromatography (TLC) • As solvent migrates up plate, mixtures are separated • TLC plate removed, solvent front marked • Distances travelled by compounds are measured, Rf values determined • Rf value = distance of component/distance of solvent Beaker TLC plate Solvent front Start line Solvent

  10. Example TLC of Spinach pigments http://www.austincc.edu/biocr/1406/labm/ex7/prelab_7_2.htm

  11. Example: inorganic sulfur compounds

  12. Gas chromatography • Separation of volatile analytes using a column or capillary (stationary phase) using a gas stream (mobile phase) • Range of system designs with variation in • Inlets • Carrier gas • Column/capillary materials packings/film • Detectors

  13. General GC system

  14. GC components • Carrier gas system: • Carrier gas has to be inert, typically Helium, Nitrogen, Hydrogen used • Pneumatic controls to control pressures • Often in-line gas purifiers to remove water, oxygen, hydrocarbons • Injector: • Injection port with septum • Often with auto-sampler • Split injection systems can allow restricting theamount of sample getting onto the system • Separation system • Columns packed with material or capillaries with liquid polymer film coating on inside • Temperature controlled oven

  15. Injector

  16. Columns Packed Capillary Length 15-100 m Inner diameter tenths mm-1mm Different types Coated with liquid stationary phase (wall coated open tubular, WCOT) Lined with material which adsorbs stationary phase (support coated open tubular, SCOT) Wall coated with porous layer of polymer (PLOT) • Length 1.5 to 10m • Inner diameter 2-4 mm • Packed with inert material coated with liquid stationary phase

  17. GC system components II • Detectors • Monitor the carrier gas eluting from column, changes in electric signal in response to changes in compounds eluting with carrier • Most common ones FID, TCD, ECD, FPD, NPD, MSD) • Recording device • Computer/software or integrator to record trace and integrate peak areas

  18. Detector

  19. Chromatogram terminology

  20. Detectors - TCD • Thermoconductivity detector • First GC detector • Splits carrier gas stream and passes it over to matched filaments • Sample pass over one of the filaments and dilute the carrier, changing the conductivity of filament due to change in temperature • Difference in conductivity between sample/reference filament provides the signal • All compounds that have different thermal conductivity than the carrier are detected

  21. GC Detectors - FID • Flame ionisation detector • Most widely used • Consist of hydrogen/air flame and collector plate which is heated to avoid condensation of water from flame • Effluent from column flows through tiny jet and is ignited and burnt • Normally produces ions that will allow flow of electrons through flame which are collected at an electrode • Signal approx proportional to number of C atoms in analytes • Responds to C-H bond containing molecules

  22. GC detectors - ECD • Electron Capture Detector • Invented by James Lovelock • Basis for measurement of halogens in atmosphere! • Uses a radioactive beta emitter to ionise carrier gas, giving current between a pair of electrodes • In absence of organic speciesstanding current is constant, but the current decreases in the presence of those organic molecules that tend to capture electrons (particularly good for halogenated compounds)

  23. GC detectors - FPD • Flame photometric detector • Combustion of sample in H2/air flame produces optical emission from P and S compounds • A photomultiplier tube equipped with a filter to select only desired wavelength detects this light emission and generates signal

  24. GC detectors - MSD • Mass selective detector • Ionisation of sample molecules e.g. by bombarding with electron beam or by chemical ionisation • Compounds typically generate charged ions or fragment into characteristic charged ions • Ions pass through mass filter allowing to define a range of masses • Mass filter scans through mass range and counts all ions • Abundance of ions per scan is plotted over time, giving a total ion chromatogram • Alternatively, specific ions may be selected and monitored

  25. Sample types • Liquid • Most applications use liquid injection, usually 1µl volume • Analytes are extracted using particular solvents and then injected • Alternatively, samples may be derivatised first (e.g. FAME analysis) to make samples volatile and allow better separation • Headspace analysis • The 'headspace' is the gas space in a chromatography vial above the sample • analysis of volatiles and semi-volatile organics in solid, liquid and gas samples

  26. Headspace • G= gas phasereferred to as headspace, lies above the sample • S = sample phaseContains compounds of interest

  27. Column selection

  28. Capillaries - Sulfur Compounds on Rtx®-1

  29. Columns for specific applications • Check relevant papers • Browse websites of Agilent, Thames Restek etc and find application guides for specific types of compounds • Talk to representatives

  30. References • Materials obtained from • www.gchelp.tk • http://teaching.shu.ac.uk • www.thamesrestek.co.uk

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