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 • 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 What is your interest in chromatography? • Experience in using chromatography? • Plans to use chromatography? 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 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 Main platforms • • • • • Thin layer chromatography (TLC) Gas chromatography (GC) Ion chromatography (IC) High performance liquid chromatography (HPLC) Fast protein liquid chromatography (FPLC) 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 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 Example TLC of Spinach pigments http://www.austincc.edu/biocr/1406/labm/ex 7/prelab_7_2.htm Example: inorganic sulfur compounds 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 General GC system 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 the amount of sample getting onto the system • Separation system – Columns packed with material or capillaries with liquid polymer film coating on inside – Temperature controlled oven Injector Columns Packed Capillary • Length 1.5 to 10m • Inner diameter 2-4 mm • Packed with inert material coated with liquid stationary phase • 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) 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 Detector Chromatogram terminology 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 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 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) 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 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 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 Headspace • G= gas phase referred to as headspace, lies above the sample • S = sample phase Contains compounds of interest Column selection Capillaries - Sulfur Compounds on Rtx®-1 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 References • Materials obtained from – www.gchelp.tk – http://teaching.shu.ac.uk – www.thamesrestek.co.uk