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.
Overview
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GC History
What is GC
Key Components
Separation Process
GC Theory
Carrier Gas
Injectors
Columns
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)
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
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
Comaparison: GC & HPLC
HPLC
GC
• non-volatile samples
•volatile & thermally stable
• thermally unstable compounds
•rapid analysis
• macromolecules
•good resolution
• inorganic and ionic samples
• More complex interface to Mass
Spec .
•easily interfaced to Mass Spec
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.
Basic Block Daigram!
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.
Example Chromatogram (Capillary)
:2
WI
:4
WI
c:\star\examples\level4.run
File: c:\star\examples\level4.run
Channel: A = TCD Results
Last recalc: 25/07/1993 18:35
3.210
mVolts
4.463
1.474
500
5.320
2.038
0.541
Detector
Response
0.754
2.853
1.113
750
5.562
250
0
Inject
Point
-87
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5
Minutes
Time
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Analysis of Halogenated Pesticides
-HCH
-HCH
-HCH
Heptachlor
-HCH
Aldrin
Heptachlor epoxide
Endosulfan I
4,4’-DDE
Dieldrin
Endrin
4,4’-DDD
Endosulfan II
4,4’-DDT
Endrin aldehyde
Endosulfan sulfate
Methoxychlor
Endrin ketone
4
10
7
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11
6
8
12
13
9
14
1
5
3
15
16
2
2ppb in Water
17 18
Chromatogram
GC Step by Step
• Carrier Gas
• Injector
• Column
– Capillary
– Stationary Phase
• Detectors
– Mass Spectrometer
Carrier Gas
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Inert
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Helium
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Choice dictated by detector, cost, availability

Pressure regulated for constant inlet pressure

Flow controlled for constant flow rate
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Chromatographic grade gases (high purity)
Column Types
Capillary Columns
Length: 10m to 100m
Diameter: 180um,
250um, 320um &
530um I.d
Packed Columns
Length: <2m
Diameter: 1/8” & ¼” OD
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
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
Splitless injector Design
Graphite/Viton Seal
•Reduced Sample Contact
“Unique”Dual Split Vent design
•Improved Precision
Large Internal
Volume
•Minimum
Solvent Tailing
•More Efficient Sweep
Shortened Capillary Guide
•Minimal Cold Spots
•Minimal Upswept Volume
Cross Section of PTV Injector
Modern
Temperature
Programmable
Injector
(Varian 1079)
Programmable
Temperature
Vapourising
Injector
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.
Split Injection
• Mechanism by which a portion of the injected solution is discarded.
• Only a small portion (1/1000 - 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
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
On Column Injection
• All of the sample is transferred to the column
• Needle is inserted directly into column or into
insert directly above column
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Trace analysis
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Thermally labile compounds e.g Pesticides, Drugs
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Wide boiling point range
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High molecular weight
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
Columns
Material of Construction
• Metal (1957)
• Glass (1959)
• Fused Silica (1979)
• Aluminium Clad (1984)
• Inert Metal (1990)
Capillary Column Characteristics
• Length (10M - 50M)
• Internal Diameter (0.1mm - 0.53mm)
• Liquid Stationary Phase
• Film Thickness (0.1um - 5um)
• Polarity (Non-polar - Polar)
Stationary Phases
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Choice of phase determines selectivity
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Hundred of phases available
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Many phases give same separation
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Same phase may have multiple brand names
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Stationary phase selection for capillary columns much simpler
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Like dissolves like
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Use polar phases for polar components
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Use non-polar phases for non-polar components
Column Bleed
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Bleed increases with film thickness
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Polar columns have higher bleed
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Bleed is excessive when column is damaged or degraded
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Avoid strong acids or bases
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Adhere to manufacturer’s recommended temperature limits
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Avoid leaks
Choosing a Column
• Internal Diameter
• Film Thickness
• Length
• Phase
Internal Diameter, Smaller ID’s
• Good resolution of early eluting compounds
• Longer analysis times
• Limited dynamic range
ID Effects - larger ID’s
• Have less resolution of early eluting compounds
• Shorter analysis times
• Sufficient resolution for complex mixtures
• Greater dynamic range
Film Thickness
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Amount of stationary phase coating
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Affects retention and capacity
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Thicker films increase retention and capacity
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Thin films are useful for high boilers
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Standard capillary columns typically 0.25µm
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0.53mm ID (Megabore) typically 1.0 - 1.5µm
Column Capacity
The maximum amount that can be injected without significant peak
distortion
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Column capacity increases with :
film thickness
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temperature
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internal diameter
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stationary phase selectivity
If exceeded, results in :
peak broadening
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asymmetry
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leading
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?-
Length effects - programmed analysis
• Retention more dependant on temperature
• Marginally increases analysis times
• Run conditions should be optimised
Summary - Effect of ID, Film
Thickness, and Length
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ID
Film Thickness
Length
Choice based on
 Gain in resolution is
capacity and resolution • Thick film for low
not double
Use 0.25mm for MSDs
boilers
 Isothermal: tR  L
Use 0.32mm for
• Thin film for high
 Programmed: tR is
split/splitless & DI
boilers
more dependent on
Use 0.53mm for DI &
• Thicker films for larger
temperature
purge & trap
ID's
Detectors
Overview
• Basic Mass Spectrometry Theory
• Types of Ionisation
- Electronic Ionisation
- Chemical Ionisation
• Interpretation of Mass Spectra
• Ion Trap Theory
• Components of the Ion Trap
Ion Trap Mass Spectrometry
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.
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
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
Types of Ionisation
• Electron impact ionisation
• Chemical Ionisation
Definition of Terms
Molecular The ion obtained by the loss of an electron from
ion
the molecule
The most intense peak in the MS, assigned 100%
Base peak
intensity
M+
Symbol often given to the molecular ion
Radical
cation
+ve charged species with an odd number of
electrons
Lighter cations formed by the decomposition of
Fragment
the molecular ion.
ions
These often correspond to stable carbcations.
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
Electron Ionisation
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.
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
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
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]+
– C3H5+ + M  [M + C3H5]+
M+29 Adduct
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
EI vs CI for Cocaine analysis
• EI Spectrum of Cocaine
• Extensive Fragmentation
• Molecular Ion is Weak at m/z 303
Methane CI of Cocaine
Pseudomolecular Ion and Fragment Ions
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
Interpretation of Mass Spectra(1)
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
Interpretation of Mass Spectra(3)
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).
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.
Isotope Patterns 2,Chloropropane
•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.
1,Bromopropane
• 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.
ION TRAP THEORY
• 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
Electron Ionization Happens Inside the Ion Trap
Filament
Filament
Gate
Gate
Ring electrode
Ring Electrode
Trapped
IonsIons
Trapped
CARRIER
Analytes + He
GAS
Carrier Gas
Mass Spectrum of Toluene
Mass Spectrum of Caffeine
Mass Spectrum of Glycerin
Mass Spectrum of Cholesterol
Mass Spectrum of Aspirin