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Supporting & Servicing Excellence

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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 Theory Carrier Gas Injectors Columns

3 GC History Development of GC (1941) by Martin and Synge
Theory of Capillary GC (1957) by Golay Capillary 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, etc Occasionally as a qualitative tool

5 What is the sample? Usually a mixture of several components
Sample usually introduced as a liquid Components 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 compounds macromolecules inorganic and ionic samples More complex interface to Mass Spec . GC volatile & thermally stable rapid analysis good resolution easily interfaced to Mass Spec

7 Key components of GC Hardware to introduce the sample
Technique to separate the sample into components Hardware to detect the individual components. Data Processing to process this information.

8 Basic Block Daigram!

9 Separation Process Sample is introduced into system via hot, vaporising injector. Typically 1ul injected Flow of “Carrier Gas” moves vaporised sample (i.e. gas) onto column Column is coated with wax type material with varying affinity for components of interest Components 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 Response Inject Point Time 

11 Analysis of Halogenated Pesticides
4 10 7 11 6 8 12 13 9 14 1 5 15 17 18 3 16 2 2ppb in Water

12 Chromatogram

13 GC Step by Step Carrier Gas Injector Column Detectors Capillary
Stationary Phase Detectors Mass Spectrometer

14 Carrier Gas Inert Helium
Choice dictated by detector, cost, availability Pressure regulated for constant inlet pressure Flow controlled for constant flow rate Chromatographic grade gases (high purity)

15 Column Types Capillary Columns Length: 10m to 100m
Diameter: 180um, 250um, 320um & 530um I.d Packed Columns Length: <2m Diameter: 1/8” & ¼” OD

16 Typical column flow rates
Capillary Column Flow 250 um 1 ml/min 320 um 1.5 ml/min 530um up to 2.0 ml/min

17 Purpose of Injection Deposit the sample into the column in the narrowest band possible The shorter the band at the beginning of the chromatographic process - tall narrow peaks Gives maximum resolution and sensitivity Therefore type of injection method and operating conditions is critical in obtaining precise and accurate results

18 Splitless injector Design
Graphite/Viton Seal Reduced Sample Contact “Unique”Dual Split Vent design Improved Precision More Efficient Sweep Large Internal Volume Minimum Solvent Tailing Shortened Capillary Guide Minimal Cold Spots Minimal Upswept Volume

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 Columns Most commonly misunderstood also! Same injector hardware is used for both techniques Electronically controlled Solenoid changes Gas Flow to determine Injector function.

21 Split Injection Mechanism by which a portion of the injected solution is discarded. Only a small portion (1/ /20) of sample goes through the column Used for concentrated samples (>0.1%) Can be performed isothermally Fast injection speed Injector and septa contamination not usually noticed

22 Splitless Injection Most of the sample goes through the column (85-100%) Used for dilute samples (<0.1%) Injection speed slow Should not be performed isothermally Solvent focusing is important Controlled by solenoid valve Requires 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 column Trace analysis Thermally labile compounds e.g Pesticides, Drugs Wide boiling point range High molecular weight

24 Large Volume Injection
To enhance sensitivity in Envoirnmental applications. Uses 100µL syringe: Inject up to 70 µl Very slow injection with injector temperature a few degrees below solvent boiling point, split open, flow at about 150 mls/ min Solvent vents out of split vent, thus concentrating the analytes Close split Fast temperature ramp to top column temperature +20°C Column programming as per sample requirements

25 Columns

26 Material of Construction
Metal (1957) Glass (1959) Fused Silica (1979) Aluminium Clad (1984) Inert Metal (1990)

27 Capillary Column Characteristics
Length (10M - 50M) Internal Diameter (0.1mm mm) Liquid Stationary Phase Film Thickness (0.1um - 5um) Polarity (Non-polar - Polar)

28 Stationary Phases Choice of phase determines selectivity
Hundred of phases available Many phases give same separation Same phase may have multiple brand names Stationary phase selection for capillary columns much simpler Like dissolves like Use polar phases for polar components Use non-polar phases for non-polar components

29 Column Bleed Bleed increases with film thickness
Polar columns have higher bleed Bleed is excessive when column is damaged or degraded Avoid strong acids or bases Adhere to manufacturer’s recommended temperature limits Avoid leaks

30 Choosing a Column Internal Diameter Film Thickness Length Phase

31 Internal Diameter, Smaller ID’s
Good resolution of early eluting compounds Longer analysis times Limited dynamic range

32 ID Effects - larger ID’s
Have less resolution of early eluting compounds Shorter analysis times Sufficient resolution for complex mixtures Greater dynamic range

33 Film Thickness Amount of stationary phase coating
Affects retention and capacity Thicker films increase retention and capacity Thin films are useful for high boilers Standard capillary columns typically 0.25µm 0.53mm ID (Megabore) typically µm

34 Column Capacity The maximum amount that can be injected without significant peak distortion Column capacity increases with :- film thickness temperature internal diameter stationary phase selectivity If exceeded, results in :- peak broadening asymmetry leading

35 Length effects - isothermal analysis
Retention more dependant on length Doubling column length doubles analysis times Resolution a function of Square Root of Length Gain 41% in resolution Is it worth the extra time and expense?-

36 Length effects - programmed analysis
Retention more dependant on temperature Marginally increases analysis times Run conditions should be optimised

37 Summary - Effect of ID, Film Thickness, and Length

38 Detectors

39 Overview Basic Mass Spectrometry Theory Types of Ionisation
- Electronic Ionisation - Chemical Ionisation Interpretation of Mass Spectra Ion Trap Theory Components of the Ion Trap

40 Ion Trap Mass Spectrometry

41 Basic Mass Spec.Theory Mass Spec. is a Microanalytical Technique used to obtain information regarding structure and Molecular weight of an analyte Destructive method ie sample consumed during analysis In all cases some form of energy is transferred to analyte to cause ionisation In principle each Mass Spectrum is unique and can be used as a “fingerprint” to characterise the sample GC/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 molecules Effluent from GC passes through transfer line into the Ion Trap/Ion source Molecules then undergo electron /chemical ionisation Ions are then analysed according to their mass to charge ratio Ions 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 electronics Signal is amplified Result is digitised Results can be further processed and displayed

44 Types of Ionisation Electron impact ionisation Chemical Ionisation

45 Definition of Terms Molecular ion
The ion obtained by the loss of an electron from the molecule Base peak The most intense peak in the MS, assigned 100% intensity M+ Symbol often given to the molecular ion Radical cation +ve charged species with an odd number of electrons Fragment ions Lighter 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 spec Energy sufficient for Ionisation and Fragmentation of analyte molecules is acquired by interaction with electrons from a hot Filament 70 eV is commonly used Source of electrons is a thin Rhenium wire heated electrically to a temp where it emits free electrons

47 Electron Ionisation

48 Electron Ionisation The 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 technique Differs from EI in that molecules are ionised by interaction or collision with ions of a reagent gas rather that with electrons Common reagent gases used are Methane , Isobutane and Ammonia Reagent 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 Spectrum Form Adduct Ions C2H5+ + M  [M + C2H5]+ M+29 Adduct C3H5+ + M  [M + C3H5]+ M+41 Adduct Molecular Ion by Charge Transfer CH4+ + M  M+ + CH4 Hydride Abstraction (M-1) C3H5+ + M  C3H6 + [M-H]+ Common for saturated hydrocarbons

52 EI vs CI for Cocaine analysis
EI Spectrum of Cocaine Extensive Fragmentation Molecular 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 species The greater the difference in proton affinities between the analyte and reagent gas the more energy transferred to the protonated molecule –more fragmentation

55 Interpretation of Mass Spectra(1)

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

57 Interpretation of Mass Spectra(3)

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.

60 Isotope Patterns 2,Chloropropane

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.  

62 1,Bromopropane

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 ionize Store ions and continue to ionize until the optimum trap capacity is reached Optimum ion time calculated by software Increase the voltage on the Ring Electrode of the ion trap to scan ions out in order from low to high mass This voltage-time relationship called the EI/MS Scan Function Store the mass-intensity information as a mass spectrum

66 Electron Ionization Happens Inside the Ion Trap
Filament Gate Ring electrode CARRIER GAS Trapped Ions

67 Mass Spectrum of Toluene

68 Mass Spectrum of Caffeine

69 Mass Spectrum of Glycerin

70 Mass Spectrum of Cholesterol

71 Mass Spectrum of Aspirin

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