Presentation on theme: "Supporting & Servicing Excellence"— Presentation transcript:
1 Supporting & Servicing Excellence GC-MS Gas Chromatography-Mass Spectrometry An Hybrid technique which couples the powerful separation potential of gas chromatography with the specific characterization ability of mass spectroscopy.
2 Overview GC History What is GC Key Components Separation Process GC TheoryCarrier GasInjectorsColumns
3 GC History Development of GC (1941) by Martin and Synge Theory of Capillary GC (1957) by GolayCapillary GC Instruments (1977)Fused Silica Capillary Columns (1979)
4 What is GC? GC is a Separation Technique Sample is usually a complex mixture we require to separate into constituent components.Why: usually to quantify some or all components e.g. Pharmaceuticals, Environmental pollutants, etcOccasionally as a qualitative tool
5 What is the sample? Usually a mixture of several components Sample usually introduced as a liquidComponents of interest (analytes) usually in low concentrations (<1% to ppb levels)Samples dissolved in volatile solvent
6 Comaparison: GC & HPLC HPLC non-volatile samples thermally unstable compoundsmacromoleculesinorganic and ionic samplesMore complex interface to Mass Spec .GCvolatile & thermally stablerapid analysisgood resolutioneasily interfaced to Mass Spec
7 Key components of GC Hardware to introduce the sample Technique to separate the sample into componentsHardware to detect the individual components.Data Processing to process this information.
9 Separation ProcessSample is introduced into system via hot, vaporising injector.Typically 1ul injectedFlow of “Carrier Gas” moves vaporised sample (i.e. gas) onto columnColumn is coated with wax type material with varying affinity for components of interestComponents are separated in the column based on this affinity.Individual analytes are detected as they emerge from the end of the column through the Detector.
10 Example Chromatogram (Capillary) Detector ResponseInject PointTime
11 Analysis of Halogenated Pesticides 410711681213914151517 1831622ppb in Water
13 GC Step by Step Carrier Gas Injector Column Detectors Capillary Stationary PhaseDetectorsMass Spectrometer
14 Carrier Gas Inert Helium Choice dictated by detector, cost, availabilityPressure regulated for constant inlet pressureFlow controlled for constant flow rateChromatographic grade gases (high purity)
15 Column Types Capillary Columns Length: 10m to 100m Diameter: 180um, 250um, 320um & 530um I.dPacked ColumnsLength: <2mDiameter: 1/8” & ¼” OD
16 Typical column flow rates Capillary Column Flow250 um 1 ml/min320 um 1.5 ml/min530um up to 2.0 ml/min
17 Purpose of InjectionDeposit the sample into the column in the narrowest band possibleThe shorter the band at the beginning of the chromatographic process - tall narrow peaksGives maximum resolution and sensitivityTherefore type of injection method and operating conditions is critical in obtaining precise and accurate results
19 Cross Section of PTV Injector Modern Temperature Programmable Injector (Varian 1079)Programmable Temperature Vapourising Injector
20 Split & Splitless Injection Most common method of Injection into Capillary ColumnsMost commonly misunderstood also!Same injector hardware is used for both techniquesElectronically controlled Solenoid changes Gas Flow to determine Injector function.
21 Split InjectionMechanism by which a portion of the injected solution is discarded.Only a small portion (1/ /20) of sample goes through the columnUsed for concentrated samples (>0.1%)Can be performed isothermallyFast injection speedInjector and septa contamination not usually noticed
22 Splitless InjectionMost of the sample goes through the column (85-100%)Used for dilute samples (<0.1%)Injection speed slowShould not be performed isothermallySolvent focusing is importantControlled by solenoid valveRequires careful optimisation
23 On Column Injection All of the sample is transferred to the column Needle is inserted directly into column or into insert directly above columnTrace analysisThermally labile compounds e.g Pesticides, DrugsWide boiling point rangeHigh molecular weight
24 Large Volume Injection To enhance sensitivity in Envoirnmental applications.Uses 100µL syringe: Inject up to 70 µlVery slow injection with injector temperature a few degrees below solvent boiling point, split open, flow at about 150 mls/ minSolvent vents out of split vent, thus concentrating the analytesClose splitFast temperature ramp to top column temperature +20°CColumn programming as per sample requirements
28 Stationary Phases Choice of phase determines selectivity Hundred of phases availableMany phases give same separationSame phase may have multiple brand namesStationary phase selection for capillary columns much simplerLike dissolves likeUse polar phases for polar componentsUse non-polar phases for non-polar components
29 Column Bleed Bleed increases with film thickness Polar columns have higher bleedBleed is excessive when column is damaged or degradedAvoid strong acids or basesAdhere to manufacturer’s recommended temperature limitsAvoid leaks
30 Choosing a ColumnInternal DiameterFilm ThicknessLengthPhase
31 Internal Diameter, Smaller ID’s Good resolution of early eluting compoundsLonger analysis timesLimited dynamic range
32 ID Effects - larger ID’s Have less resolution of early eluting compoundsShorter analysis timesSufficient resolution for complex mixturesGreater dynamic range
33 Film Thickness Amount of stationary phase coating Affects retention and capacityThicker films increase retention and capacityThin films are useful for high boilersStandard capillary columns typically 0.25µm0.53mm ID (Megabore) typically µm
34 Column CapacityThe maximum amount that can be injected without significant peak distortionColumn capacity increases with :-film thicknesstemperatureinternal diameterstationary phase selectivityIf exceeded, results in :-peak broadeningasymmetryleading
35 Length effects - isothermal analysis Retention more dependant on lengthDoubling column length doubles analysis timesResolution a function of Square Root of LengthGain 41% in resolutionIs it worth the extra time and expense?-
36 Length effects - programmed analysis Retention more dependant on temperatureMarginally increases analysis timesRun conditions should be optimised
37 Summary - Effect of ID, Film Thickness, and Length
41 Basic Mass Spec.TheoryMass Spec. is a Microanalytical Technique used to obtain information regarding structure and Molecular weight of an analyteDestructive method ie sample consumed during analysisIn all cases some form of energy is transferred to analyte to cause ionisationIn principle each Mass Spectrum is unique and can be used as a “fingerprint” to characterise the sampleGC/MS is a combination technique that combines the separation ability of the GC with the Detection qualities of Mass Spec.
42 Basic GCMS Theory(1) Sample injected onto column via injector GC then separates sample moleculesEffluent from GC passes through transfer line into the Ion Trap/Ion sourceMolecules then undergo electron /chemical ionisationIons are then analysed according to their mass to charge ratioIons are detected by electron multiplier which produces a signal proportional to ions detected
43 Basic GCMS Theory(2)Electron multiplier passes the ion current signal to system electronicsSignal is amplifiedResult is digitisedResults can be further processed and displayed
44 Types of IonisationElectron impact ionisationChemical Ionisation
45 Definition of Terms Molecular ion The ion obtained by the loss of an electron from the moleculeBase peakThe most intense peak in the MS, assigned 100% intensityM+Symbol often given to the molecular ionRadical cation+ve charged species with an odd number of electronsFragment ionsLighter cations formed by the decomposition of the molecular ion. These often correspond to stable carbcations.
46 Electron Ionisation(1) Sample of interest vaporised into mass specEnergy sufficient for Ionisation and Fragmentation of analyte molecules is acquired by interaction with electrons from a hot Filament70 eV is commonly usedSource of electrons is a thin Rhenium wire heated electrically to a temp where it emits free electrons
48 Electron IonisationThe physics behind mass spectrometry is that a charged particle passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e. In an electron impact mass spectrometer, a high energy beam of electrons is used to displace an electron from the organic molecule to form a radical cation known as the molecular ion. If the molecular ion is too unstable then it can fragment to give other smaller ions. The collection of ions is then focused into a beam and accelerated into the magnetic field and deflected along circular paths according to the masses of the ions. By adjusting the magnetic field, the ions can be focused on the detector and recorded.
49 Chemical ionisation Used to confirm molecular weight Known as a “soft” ionisation techniqueDiffers from EI in that molecules are ionised by interaction or collision with ions of a reagent gas rather that with electronsCommon reagent gases used are Methane , Isobutane and AmmoniaReagent gas is pumped directly into ionisation chamber and electrons from Filament ionise the reagent gas
50 Chemical Ionisation(2) First - electron ionization of CH4:CH4 + e- CH4+ + 2e-Fragmentation forms CH3+, CH2+, CH+Second - ion-molecule reactions create stable reagent ions:CH4+ + CH4 CH3 + CH5+CH3+ + CH4 H2 + C2H5+CH5+ and C2H5+ are the dominant methane CI reagent ions
51 Chemical Ionisation(3) Form Pseudomolecular Ions (M+1)CH5+ + M CH4 + MH+M+1 Ions Can Fragment Further to Produce a Complex CI Mass SpectrumForm Adduct IonsC2H5+ + M [M + C2H5]+ M+29 AdductC3H5+ + M [M + C3H5]+ M+41 AdductMolecular Ion by Charge TransferCH4+ + M M+ + CH4Hydride Abstraction (M-1)C3H5+ + M C3H6 + [M-H]+Common for saturated hydrocarbons
52 EI vs CI for Cocaine analysis EI Spectrum of CocaineExtensive FragmentationMolecular Ion is Weak at m/z 303
53 Methane CI of Cocaine Pseudomolecular Ion and Fragment Ions
54 Proton Affinity Proton Affinity Governs CI Susceptibility The higher the affinity the more tightly bound the proton is to the parent speciesThe greater the difference in proton affinities between the analyte and reagent gas the more energy transferred to the protonated molecule –more fragmentation
56 Intepretation of Mass Spectra(2) The MS of a typical hydrocarbon, n-decane is shown above.The molecular ion is seen as a small peak at m/z = 142.Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleave of bonds at successive -CH2- units
58 Interpretation of Mass Spectra(4) The MS of benzyl alcohol is shown above.The molecular ion is seen at m/z = 108. Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl benzenes at m/z = 91. Loss of 31 (-CH2OH) from the molecular ion gives 77 corresponding to the phenyl cation.Note the small peaks at 109 and 110 which correspond to the presence of small amounts of 13C in the sample (which has about 1% natural abundance).
59 Determining Isotope Patterns in Mass Spectra Mass spectrometers are capable of separating and detecting individual ions even those that only differ by a single atomic mass unit.As a result molecules containing different isotopes can be distinguished.This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br, intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained.The intensity ratios in the isotope patterns are due to the natural abundance of the isotopes."M+1" peaks are seen due the the presence of 13C in the sample.
61 Examples of haloalkanes with characteristic isotope patterns. The first MS is of 2-chloropropane.Note the isotope pattern at 78 and 80 that represent the M and M+2 in a 3:1 ratio.Loss of 35Cl from 78 or 37Cl from 80 gives the base peak a m/z = 43, corresponding to the secondary propyl cation.Note that the peaks at m/z = 63 and 65 still contain Cl and therefore also show the 3:1 isotope pattern.
63 The second MS is of 1-bromopropane. Note the isotope pattern at 122 and 124 that represent the M amd M+2 in a 1:1 ratio.Loss of 79Br from 122 or 81Br from 124 gives the base peak a m/z = 43, corresponding to the propyl cation.Note that other peaks, such as those at m/z = 107 and 109 still contain Br and therefore also show the 1:1 isotope pattern.
65 Ionize analytes within the ion trap Use energetic electrons to ionizeStore ions and continue to ionize until the optimum trap capacity is reachedOptimum ion time calculated by softwareIncrease the voltage on the Ring Electrode of the ion trap to scan ions out in order from low to high massThis voltage-time relationship called the EI/MS Scan FunctionStore the mass-intensity information as a mass spectrum
66 Electron Ionization Happens Inside the Ion Trap FilamentGateRing electrodeCARRIER GASTrapped Ions