3 Metabolomics Goal – The analysis of everything in anything biological Reality – The analysis of anything in everythingEffectively targeted analysis, or, broad analyses where many compounds are present, but, many at levels too low for detection in the sample matrix.
4 Volatility: implications If something enters the gas phase (headspace) you can sample it from air – instantly separating it from the non-volatile material – big advantageVolatility also impacts on analysis options Gas Chromatography for volatiles/semi-volatiles Liquid Chromatography – HPLC for non-volatilesSome compounds are chemically modified (derivatized) to make them volatile e.g. acids
5 Gas Chromatography (GC) Sampling, injection, separationVolatile compounds
6 Analytical Gas Chromatography Injection portWhere the sample gets inHot to ensure compoundsvolatilise and enter columnDetectorWhere the compoundsleaving the columnAre monitored.Carrier gasEnters injector andtransports compoundsthrough systemGas used typically HeliumColumnWhere the compounds in thesample are separated
7 Sampling Options Sample from headspace (air above sample) or Solvent extractGas Phase -HeadspaceSampleGas Phase -HeadspaceSampleSolventSAMPLE
8 Gas Chromatography: Column Typically long and thin 25m x 0.25mmCoated with a gum which forms the stationary phaseThe gum itself can be polar or non-polar to alter partitioning of compounds between the gum and gas phaseInjector EndDetector endWallGumStartAs temperature increases, compounds move….Dependent on partition with gum (polarity) and volatilityGAS FLOW
9 Detection: Electron Impact Mass Spectrometry Compounds enter a high vacuum region where they are bombarded by high energy electrons that cause compounds to fragment. Fragmentation patterns are dependent on the structure of the compound. Ions are guided to the analyser where an electric field separates them on the basis of their mass and they are detected.
11 Chromatogram: Change in signal over time recording compounds arriving at detector FusedpeaksOverloadedpeakBaselineResolvedpeakIntensityTimeLater peaks areLess volatileHigher boiling point
12 Spectrum: Cross section of signal at a specific chromatographic time With GC this is the mass spectrumIntensityMass (m/z)
13 Example of Tea analysis LinaloolE-2-hexenalHexanalMe-SalicylateTea blenders try to produce two teas with identical aroma profiles (QC).Overall good match, except a branched ester.Questiondoes it smell?what is it?where does it come from?These affect significance of result.New BlendOriginal BlendBoiling Point of compounds increases
14 Solvent Extraction of beverage: ageing study AgedFreshChange interpeneprofileAppearanceor increasein terpeneoxidationproductDCM shaken with the beverage and the organic fraction analysed by GC.Profile shows volatiles appearing, or disappearing on storage.
15 Fatty acid profilingFatty acid profile of sample compared with that of standard (mix of 36 saturated and unsaturated FA).What fatty acids are there and in what proportions.Lipid can be fractionated (polar vs. non-polar) and “sub-profiles” determined.Used in product authentication or diet impact studies.StandardSampleC12C14C16C18C20C22C24Fatty acid methyl esters produced by derivatization of lipid: transesterification with trimethyl sulfonium hydroxide in methanol
16 Fit Spectra from sample Library spectra: C11 acid ester
17 Liquid Chromatography High performance liquid chromatography (HPLC) Non-volatiles
18 High Performance Liquid Chromatography (HPLC) InjectorPUMPOperates at1 – 5,000psiColumnDetectorSolventReservoirTubing, fittings etc have to bedesigned to cope with highpressures
19 Sample ExtractsCompounds extracted from matrix and may be concentrated or fractionatedExtraction method depends on the compound – particularly its polarity – is it water or fat soluble – use water or organic solvents (e.g. hexane) respectively
20 Separation Injector end Detector endSolvent FlowCompounds are retained on the column to different extents. This depends on the affinity of the compound for the column packing(stationary phase) relative to its affinity for the solvent. Plus the competition of the solvent molecules for the sites where the analyte is absorbed.Essentially dependent on the polarity of the compound and the stationary and mobile (solvent) phases
21 IsocraticSolvent composition remains the same throughout chromatogram. Later peaks are broader than earlier peaks.InjectionSolventfrontThe solvent font is the time at whichun-retained molecules arrive at theend of the column/detector
22 Gradient: solvent composition changes during run allowing analytes with very different polarities to be chromatographed in one run% MeOH inWater increased from10% to 60% over 2 rampsseparated by an isocratic phaseHPLC SignalTime
23 Isocratic vs. GradientGradient: wider range of analytes with different polarities analysed in one runGradient: more expensive equipmentGradient: longer run times since column has to re-equilibrate to initial starting conditions before next runGradient may help resolve peaks that are not separated by isocratic runs
24 Stationary and solvent phases Silica particles a few microns across typically surface treated to alter propertiesSurface treatments polar or non-polarSolvent phase usually opposite polarity to surfacePolarity driven partitioning between solvent and surface of column particles
25 DetectionInOutLightdetectorOptical properties of compounds Light passed through windows on a cell through which the solvent stream passes Absorbance of UV or visible light Fluorescence emission of light at a certain wavelength after excitation by photons of a different wavelengthMass spectrometry The eluent stream is heated in a stream of gas to vaporise it. An electric charge is applied across the vapour to ionise the compounds.
26 Identification of compounds Optical detection: Like GC need comparison with authentic standards: retention timedetectors set to work at a single wavelength have a degree of selectivity (only compounds that absorb at that wavelength detected), but give little evidence for identificationdetectors can produce a spectrum, additional proof of identification, quality of confirmation depends on complexity of optical spectrumSampleStandardIntensityIntensityWavelengthWavelength
27 Compounds in a chromatogram after one size and 3 polarity based purification steps Objective: purification of an unknown for identification. But, still a significant number of peaks – and hence compounds in sample (40L of bacterial broth now in a volume of 1mL).Active compound detected by separate bioassay.
28 LC-MS ESI and APCI ESI APCI Probe Charged molecules enter vacuum 4kV applied to probeProbeChargedmoleculesenter vacuumregion of MSSourceESIDESOLVATIONREGIONAPCISourceProbeChargedmoleculesenter vacuumregion of MS4kV applied toCorona Pin to ionisemoleculesCorona pin
29 Singularly charged small molecules With ESI and APCI you get limited mass information, spectra depends on conditions usedIdentification difficult – no libraries of spectra for comparison.Isotope Peaks
30 ESI of Horse heart Myoglobin Mwt = 16951.48 Lots of charge per molecule mass spec is a mass/charge analyser. Work out original mass by reversing maths+15+14+13+12+11+10
31 Overview Difficult to analyse everything at once – true metabolomics GC – good for volatiles. Combined with mass spectrometry can give information for identificationLC – good for non-volatiles. Limited information for identification of compounds even with mass spectrometry.