Interfacing Gas Chromatography

with Mass Spectroscopy

GCMS
 GC/ Mass Spectrometry
 GC equipment can be directly interfaced with rapid-scan
Mass Spectrometers
 GC-MS is a sophisticated instrumental technique that
produces, separates, and detects ion in the gas phase.
 GCMS - Introduction
 After separation in the GC column, analyte species have to be
transported to the mass spectrometer to be ionised, mass filtered
and detected.
 The column outlet needs to be connected to the ion source of the
mass spectrometer and different strategies had been implemented,
all of which need to fulfil the following conditions:
 Analyte must not condense in the interface
 Analyte must not decompose before entering the mass
spectrometer ion source
 The gas load (dictated by the mobile phase gas flow rate)
entering the ion source must be within the pumping capacity of
the mass spectrometer
 The GC/MS Interface
Transports the effluent from the gas chromatograph to the mass
spectrometer
 Capillary Columns
The flow rate is usually small enough (do not exceed 2.0 mL/min) to
feed directly into the ionization chamber of the Mass Spectrometer
 Macrobore and Packed Columns
Higher flow rates will require the use of vapour concentrator devices
(like the Bieman concentrator) or jet separator interfaces. (Why?)
 At flow rates above around 2mL/min. even the most efficient two
stage vacuum systems will struggle to attain the required level of
vacuum (~10-5 to 10-6 torr) to carry out the analysis with the
required sensitivity. Further, filaments will have much reduced
lifetimes at compromised vacuum levels.
 The GC/MS Interface
 Capillary Columns
 Insert exit end of column into ion source
 Under normal operating conditions, the mass spectrometer can
handle the entire effluent of the column
 Must heat the capillary column to prevent condensation
 Surface of columns must be inactive
 Macrobore and Packed Columns
 Effluent must be reduced before entering ion source
 Splitting the effluent results in a loss of sensitivity
 Enrichment devices are used
 Jet Separators are most common
The GC/MS Interface
 The GC/MS Interface
 Jet Separators
 Two capillary tubes aligned with a small space between them. (1mm)
 A vacuum is created between the two tubes using a rotary pump
 The GC effluent enters the vacuum region, those molecules which
continue in the same direction enter the second capillary tube and
continue to the ion source
 Increase momentum of
heavier analyte molecules
so that 50% or more go
into the skimmer
 Lighter helium molecules
are deflected by vacuum
and pumped away
 The surface of the
separator must be inactive
 Principle of GC-MS

Block diagram of mass spectroscopy

 Basic Mass Spec.Theory
 Mass Spec. is a Microanalytical Technique used to obtain
information regarding structure and Molecular weight of an analyte.
 Mass spectroscopy data that provides structural information
tends to be unreliable and thus will only be used to verify a possible
structure or in the event that the other spectral techniques are
unsuccessful.
 In principle each Mass Spectrum is unique and can be used as a
“fingerprint” to characterise the compound
 The fingerprints are compared with a library to identify the
compounds
 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
 The inlet transfers the sample into the vacuum of the mass
spectrometer. In the source region, molecules undergo electron
/chemical ionisation, neutral sample molecules are ionized and then
accelerated into the mass analyzer.
 The mass analyzer is the heart of the mass spectrometer. This
section separates ions, either in space or in time, according to their
mass to charge ratio.
 After the ions are separated, they are detected which produces a
signal proportional to ions detected
 the signal is transferred to a data system for analysis.
 Basic GCMS Theory(2)
 All mass spectrometers also have a vacuum system to maintain
the low pressure, which is also called high vacuum, required for
operation.

 High vacuum minimizes ion-molecule reactions, scattering, and

neutralization of the ions.

 In some experiments, the pressure in the source region or a part

of the mass spectrometer is intentionally increased to study these
ion-molecule reactions. Under normal operation, however, any
collisions will interfere with the analysis.
 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 +ve charged species with an odd number of
cation electrons
Lighter cations formed by the decomposition of
Fragment
the molecular ion.
ions
These often correspond to stable carbcations.
 Typical Mass Spectrum
O C H3
• Characterized by sharp, narrow
H3 C C N
peaks N C
CH
• X-axis position indicates the C C
m/z ratio of a given ion (for O N N
H
singly charged ions this Typical sample: isolated
corresponds to the mass of the compound (~1 nanogram)
ion 194
Mass Spectrum
• Height of peak indicates the
relative abundance of a given 67 109

ion (not reliable for

quantitation) 55
82

• Peak intensity indicates the

42

ion’s ability to desorb or “fly” 94 136 165

(some fly better than others) 40 60 80 100 120

Mass (amu)
140 160 180 200
 Parts of a Mass Spec

Sample introduction
Source (ion formation)
Mass analyzer (ion sep.) - high vac
Detector (electron multiplier tube or microchannel
plate)

Sample

+
_

Ionizer Mass Analyzer Detector

 Mass Spectrometer Schematic

Rough pumps
High Vacuum System Rotary pumps
Turbo pumps
Diffusion pumps

Ion Mass Data

Inlet Detector
Source analyzer System

Vapor MALDI TOF Microch plate PC’s

HPLC ESI Quadrupole Electron Mult. UNIX
GC FAB Ion Trap Mac
Solids probe EI/CI Mag. Sector
FTMS
 Sample Introduction/Sources

Volatiles
• Probe/electron impact (EI),Chemical ionization (CI)
• GC/EI,CI
Involatiles
• Direct infusion/electrospray (ESI)
• HPLC/ESI
• Matrix Assisted Laser Adsorption (MALDI)
Elemental mass spec
• Inductively coupled plasma (ICP)
• Secondary Ion Mass Spectrometry (SIMS)
– surfaces
 Types of Ionisation
• Electron Impact Ionisation (hard ionization)
Gas-phase molecules enter source through heated
probe or GC column
70 eV electrons bombard molecules forming M+*
ions that fragment in unique reproducible way to
form a collection of fragment ions
• Chemical Ionisation (soft ionization)
Higher pressure of methane leaked into the source
(mtorr)
Reagent ions transfer proton to analyte
 The excess energy in MH+ usually not enough to
give extensive fragmentation
 Electron Impact Ionization Source

Electron Collector (Trap)

Positive
Ions
+
Repeller Neutral Inlet __
Molecules
+ + + + + + to
+
Analyzer

e- e- e-
_ Electrons
70 eV e-
Filament Extraction (Acceleration
Plate Slits)

18
 Electron Ionisation
 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
 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
than 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
• 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
• 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
 Chemical ionisation
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
• The excess energy in MH+ is the difference in proton
affinities between methane and M, usually not enough
to give extensive fragmentation
EI vs CI for Cocaine analysis
• In EI Extensive Fragmentation
• Molecular Ion is Weak at m/z
303

• In CI Pseudomolecular
Ion at m/z 304
 Electron Impact (EI)
Electron Ionization (EI) is the most common
ionization technique used for mass spectrometry. EI
works well for many gas phase molecules, but it
does have some limitations.

Although the mass spectra are very reproducible

and are widely used for spectral libraries, EI causes
extensive fragmentation so that the molecular ion is
not observed for many compounds. Fragmentation is
useful because it provides structural information for
interpreting unknown spectra.
 Chemical Ionization
Chemical Ionization (CI) is a “soft” ionization
technique that produces ions with little excess energy.
As a result, less fragmentation is observed in the mass
spectrum.

Since this increases the abundance of the molecular

ion, the technique is complimentary to 70 eV EI.

CI is often used to verify the molecular mass of an

unknown.
 Only slight modifications of an EI source region are
required for CI experiments.
 Chemical Ionization
In Chemical Ionization the source is enclosed in a
small cell with openings for the electron beam, the
reagent gas and the sample.

The reagent gas is added to this cell at approximately

10 Pa (0.1 torr) pressure.

This is higher than the 10-3 Pa (10-5 torr) pressure

typical for a mass spectrometer source

The reagent gas in the CI source is ionized with an

electron beam to produce a cloud of ions. The reagent
gas ions in this cloud react and produce adduct ions like
CH5+ ,which are excellent proton donors.
 Chemical Ionization
When analyte molecules (M) are introduced to a
source region with this cloud of ions, the reagent gas
ions donate a proton to the analyte molecule and
produce MH+ ions.

The energetic of the proton transfer is controlled

by using different reagent gases.

The most common reagent gases are methane,

isobutane and ammonia. Methane is the strongest
proton donor commonly used with a proton affinity
(PA) of 5.7 eV. For softer ionization, isobutane (PA
8.5 eV) and ammonia (PA 9.0 eV) are frequently used.
 Mass Spectrometer Schematic

Rough pumps
High Vacuum System Rotary pumps
Turbo pumps
Diffusion pumps

Ion Mass Data

Inlet Detector
Source analyzer System

Vapor MALDI TOF Microch plate PC’s

HPLC ESI Quadrupole Electron Mult. UNIX
GC FAB Ion Trap Mac
Solids probe EI/CI Mag. Sector
FTMS
 Mass Analyzer

After ions are formed in the source region they are accelerated
into the mass analyzer by an electric field.
 The mass analyzer separates these ions according to their m/z
value.

The selection of a mass analyzer depends upon the

resolution, mass range, scan rate and detection limits
required for an application.

Each analyzer has very different operating characteristics and

the selection of an instrument involves important tradeoffs.
 Mass Analyzer
Common Mass Analyzers for Mass Spectrometry
Basic Type Analysis Principle
Magnetic sector Deflection of ions in a magnetic field. Ion
trajectories depend on m/z value.
Double-focusing Electrostatic focusing followed by magnetic field
deflection. Trajectories depend on m/z values.
Quadrupole Ion motion in dc and radio-frequency fields. Only
certain m/z values are passed.
Ion trap Storage of ions in space defined by ring and end
cap electrodes. Electric field sequentially ejects
ions of increasing m/z values.
Ion cyclotron Trapping of ions in cubic cell under influence
resonance of trapping voltage and magnetic field. Orbital
frequency related inversely to m/z value.
Time-of-flight Equal kinetic energy ions enter drift tube. Drift
velocity and thus arrival time at detector depend on mass.
 Quadrapole Mass Filter
The quadrupole mass spectrometer is the most common mass
analyzer.

Its compact size, fast scan rate, high transmission

efficiency, and modest vacuum requirements are ideal
for small inexpensive instruments.

It ‘filter’ and only allow specific ions to pass.

Most quadrupole instruments are limited to unit m/z resolution and

have a mass range of m/z 1000.

Many benchtop instruments have a mass range of m/z 500 but

research instruments are available with mass range up to m/z 4000.
 Quadrupole Mass Analyzer
 A quadrupole mass resonant ion
filter consists of four
non-resonant ion
parallel metal rods with
different charges
_
 The applied voltages +
Detector

affect the trajectory of +

_
ions traveling down the
flight path
 For given dc and ac
Ion
voltages, only ions of a Source

certain mass-to-charge
ratio pass through the
DC and AC
quadrupole filter and all Voltages

other ions are thrown •Only compound with specific m/z ratio will
out of their original path resonate along the field (stable path)
•Achieved by rapidly varying the voltage
 Time of Flight Analyzer
The time-of-flight (TOF) mass analyzer separates ions in time as
they travel down a flight tube.
This is a very simple mass spectrometer that uses fixed voltages and
does not require a magnetic field. The greatest drawback is that TOF
instruments have poor mass resolution, usually less than 500. ( the
older instruments)

These instruments have high transmission efficiency, no

upper m/z limit, very low detection limits, and fast scan
rates.

For some applications these advantages outweigh the low resolution.

Recent developments in pulsed ionization techniques and

new instrument designs with improved resolution have
renewed interest in TOF-MS.
 Time of Flight Analyzer

This picture shows the working principle of a linear time of flight

mass spectrometer.
To allow the ions to fly through the flight path without hitting
anything else, all the air molecules have been pumped out to create
an ultra high vacuum.
Ions of different m/z ratio travel at different velocities.
The difference in arrival times separate two (or more) ions.
 Magnetic Sector Mass Analyzer
ion trajectory
in register
ion trajectory
not in register
(too light)

Ion Detector
Source
N ion trajectory
not in register
(too heavy)
Electromagnet

In the magnetic sector analyzer, separation is based on the deflection of

ions in a magnetic field. The trajectories that ions take depend on their
m/z values. Typically, the magnetic field is slowly changed to bring ions
of different m/z value to a detector.
In the double-focusing mass spectrometer, an electric sector precedes
the magnetic sector. The electrostatic field serves to focus a beam of ions
having only a narrow range of kinetic energies onto a slit that leads to the
magnetic sector. Such instruments are capable of very high resolution.
Magnetic Sector Mass Analyzer
 Ion Trap analyzer
 compact - less expensive than quadropole
 simplest mass detector for use in GC
 ions are created from eluted sample by electron impact or chemical
ionization
 stored in radio-frequency field
 ions injected from the storage area to a detector
 Ejection is controlled so the scanning of mass to charge ratio is
possible Filament
Filament
Gate
Gate

Ring
Ring electrode
Electrode

Trapped Analytes + He
CARRIER
Trapped Ions Carrier
GAS Gas
Ions
 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
 Resolution of Mass Spectrometers

 The capability of a mass spectrometer to differentiate between

masses is usually stated in terms of its resolution, R, which is
defined as

 where Δm is the mass difference between two adjacent peaks that

are just resolved and m is the nominal mass of the first peak (the
mean mass of the two peaks is sometimes used instead).
 The resolution required in a mass spectrometer depends greatly on
its intended use.
 Resolution of Mass Spectrometers

 For example, to detect differences in mass among ions of the same

nominal mass, such as

 (all ions of nominal mass 28 Da but exact masses of 28.054, 28.034,

28.014, and 28.010 Da, respectively), requires an instrument with a
resolution of several thousand. On the other hand, low-
molecularmass ions differing by a unit of mass or more, such, as

 can be distinguished with an instrument having a resolution smaller

than 50.
 Commercial spectrometers are available with resolutions ranging
from about 500 to 500,000.
 Resolution of Mass Spectrometers

• Low resolution (Unit resolution)

– Quadrupole
– Ion trap

• High resolution
– TOF time of flight (up to 15000)
– Sector instruments (magnet)

• Ultra high resolution

– ICR ion cyclotron resonance (over 30000)
 MS Detectors
 Early detectors used
photographic film
 Today’s detectors (ion
channel and electron
multipliers) produce
electronic signals via 2ry
electronic emission when
struck by an ion
 Timing mechanisms integrate
these signals with scanning
voltages to allow the
instrument to report which
m/z has struck the detector Electron Multiplier
 Need constant and regular
calibration
 Tandem mass spectrometry
(MS/MS)
Tandem mass spectrometry, also called mass spectrometry-mass
spectrometry (MS/MS), is a technique that allows the mass spectrum
of a preselected or fragmented ion to be obtained.
 Tandem mass spectrometry
(MS/MS)
With a tandem mass spectrometer, an ionization source produces
molecular ions and fragment ions. These are then the input to the first
mass analyzer, which selects a particular ion (the precursor ion) and
sends it to the interaction cell.
 In the interaction cell, the precursor ion can decompose
spontaneously, react with a collision gas, or interact with an intense
laser beam to produce fragments, or product ions. These ions are then
mass analyzed by the second mass analyzer and detected by the ion
detector.
 Tandem mass spectrometry
(MS/MS)
Tandem mass spectrometers can produce a variety of different
spectra.
Production spectra are obtained by scanning mass analyzer 2 while
mass analyzer 1 is held constant to act as a mass selector for the
precursor ion.
A precursor-ion spectrum can be obtained by scanning mass
analyzer 1 and selecting a given product ion with mass analyzer 2.
If both mass analyzers are scanned with a small offset in mass
between them, a neutral loss spectrum can be obtained. A neutral loss
spectrum might be used, for example, to identify the m/z values of all
ions losing a common molecule, such as water.
 Tandem mass spectrometry
(MS/MS)
Finally, a complete three-dimensional MS/MS spectrum can be
acquired by recording a product ion spectrum for each selected
precursor ion, that is, by scanning mass analyzer 2 for various
settings of mass analyzer 1.
Tandem mass spectrometry can produce an enormous amount of
information and has proven useful in structural elucidation as well as
in the analysis of mixtures.
Conventional mass spectrometry of mixtures usually requires
chromatographic or electrophoretic separation to present a single
compound at a time to the mass spectrometer
 Interpretation of Mass Spectra(1)

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
 Intepretation of Mass Spectra(2)

The MS of benzyl alcohol. 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.
small peaks at 109 and 110 are correspond to the presence of small
amounts of 13C in the sample (which has about 1% natural abundance).
 Interpretation of Mass Spectra(3)
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.
 Intepretation of Mass Spectra(4)

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.
 Intepretation of Mass Spectra(5)

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
 LCMS Interface
Moving Belt/Wire
The moving-belt interface separates the condensed liquid-phase side of the LC
from the high vacuum of the MS and uses a belt to transport the analytes from
one to the other. The mobile phase of the LC is deposited on a band and
evaporated. The analytes remain on the continuously cycling belt and are
transported from atmospheric pressure into the vacuum of the ion source
through two differentially pumped vacuum locks. A heater in the ion source
evaporates the sample from the belt allowing MS analysis