Gas Chromatography- Mass Spectrometry

1. 01-Jul-20
GC-MS and LC-MS
Dr Hamayun Khan
GC-MS
Gas chromatography–mass spectrometry (GC-MS) is an analytical method that combines
the features of gas-chromatography and mass spectrometry to separate and identify
different substances within a test sample.
OR/
GC-MS is an instrumental technique, comprising of a gas chromatograph coupled to a mass
spectrometer by which complex mixtures of chemicals may be separated, identified &
quantified.
In order to a compound to be analyzed by GC-MS it must be sufficiently volatile &
thermally stable.
Principle
The sample solution is injected into the GC inlet where it is vaporized & swept onto a
chromatographic column by the carrier gas (usually helium). The sample flows through the
column & compounds comprising the mixture of interest are separated by virtue of their
relative interaction with the coating of the column (stationery phase) & the carrier gas
(mobile phase).
The difference in the chemical and physical properties of different molecules in a mixture
will separate the molecules as the sample travels the length of the column. The molecules
take different amounts of time (called the retention time) to come out of (elute from) the
gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize,
accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer
does this by breaking each molecule into ionized fragments and detecting these fragments
using their mass to charge ratio.
INSTRUMENTATION
GC-MS comprise following major blocks
a) the Gas chromatograph
b) Interface
c) the Mass spectrometer
d) A data system is necessary to handle results obtained during a sample run
A) THE GAS CHROMATOGRAPH
Carrier gas

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Split/Splitless GC-MS inlets
Samples are introduced to the column via an inlet. This inlet is typically injection through
a septum. Once in the inlet, the heated chamber acts to volatilize the sample. In a split
system, a constant flow of carrier gas moves through the inlet. A portion of the carrier gas
flow acts to transport the sample into the column. Another portion of the carrier gas flow
gets directed to purge the inlet of any sample following injection (septum purge). Yet
another portion of the flow is directed through the split vent in a set ratio known as the
split ratio.
In a splitless system, the advantage is that a larger amount of sample is introduced to the
column. However, a split system is preferred when the detector is sensitive to trace
amounts of analyte and there is concern about overloading the column.
Oven
The outer part of the GC is a very specialized oven. The column is heated to move the
molecules through the column. Typical oven temperatures range from 40° C to 320° C.
Column
The gas chromatograph in GC-MS utilizes a capillary column (30 meter thin tube) which
most widely are those in which the stationary phase has been chemically bonded to the
fused silica.
B) INTERFACE
Initial concerns for GC-MS were the significant differences in pressure-the GC gas exiting
the system is around one atmosphere (760 torr) whereas the MS operates at a vacuum of
around 10-5–10-6 torr. The pressure incompatibility problem between GC and MS was solved
by inserting an Interface. The GC/MS interface is the section of the instrument starting at the
column exit in the gas chromatograph and extending to the entrance to the ion source of the
mass spectrometer. There are many interfaces like jet, Electrospray, thermospray, direct
electrical ionization, moving wire or belt interface.
Jet Interface
The operation of this separator is based on a
diffusion principle. These jet separators work
well at the higher carrier gas flow rates (10 to
40 mL/min) The eluate from the GC is sprayed
through a nozzle at position A, and shoots toward an orifice in the wall of an adjoining
chamber on its way to the ion source. Across the gap between points A and B, there is
a tremendous expansion of the gases. Compounds that have high diffusivity will diffuse at
right angles (a process known as effusion) much more than those having a lower diffusivity.
The carrier gas is almost always a small molecule with a high diffusion coefficient,
whereas the organic molecules have much lower diffusion coefficients.
C) MASS SPECTROMETER
In general a mass spectrometer consists of:
 an ion source,
 High-vacuum system
 a mass-selective analyzer, and
 an ion collector
Ionization Techniques
 Electron impact (El)
 Chemical ionization (CI)
Mass Analyzer:
The most common type of mass analyzer associated with a gas chromatograph (GC) is:
 the quadrupole mass analyzer, others include:
 Time of Flight Analyzer
 Ion Trap Analyzer
D) Computer System
The data from the mass spectrometer is sent to a computer and plotted on a graph called
a mass spectrum.
APPLICATIONS
It is used:
1. For metabolite profiling
2. In toxicity assessment or toxicology e.g. A specific lesion in liver or kidney can be
profiled.
3. In human dosimetry.
4. For detection of illegal drugs like narcotics- marijuana, cocaine, opioids, oxycodone
and oxymorphone .
5. In sports anti doping laboratories to test athletes urine samples for prohibited
performance enhancing drugs. e.g : anabolic steroids.
6. In detection of lipophilic compounds in diverse plant tissues
7. In analysis of biologically important aromatic amines.
8. For the quantitative analysis of acidic phytohormones and related compounds

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9. In identification of volatile components in samples
10. In analysis of pesticides in foodstuffs, for example the determination of pyrethroid
residues in vegetable samples
11. For tracking organic pollutants in the environment.
12. In criminal forensics to help link a criminal to a crime. Accelerant is significant
evidence in a fire investigation because it suggests that the fire was set intentionally.
LC-MS
LC-MS is a hyphenated technique, which combines the separating power of High
Performance Liquid Chromatography (HPLC), with the detection power of mass
spectrometry.
Hyphenated technique:
The technique developed from the coupling of a separation technique and an on-line
spectroscopic detection technology is known as hyphenated technique.
Principle of LC-MS
Typical LC-MS system is combination of HPLC with MS using interface (ionization
source). The sample is separated by LC, and the separated sample species are sprayed into
atmospheric pressure ion source, where they are converted into ions in the gas phase. The
mass analyzer is then used to sort ions according to their mass to charge ratio and detector
counts the ions emerging from the mass analyzer and may also amplify the signal
generated from each ion. As a result, mass spectrum (a plot of the ion signal as a function
of the mass-to-charge ratio) is created, which is used to determine the masses of particles
and of molecules, and to elucidate the chemical structures of molecules.
INSTRUMENTATION:
1) HPLC Constitutes the LC Part:
a) Solvent System(mobile Phase)
b) Pumps
c) Mixer and degassers
d) Injector
e) Column
2) Mass Spectrometer
a) Ion Sources (function as interface)
i) Electrospray ionization.
ii) Atmospheric Pressure Chemical Ionization.
iii) Atmospheric pressure photoionization
b) Mass Analyzers
i) Quadrupole
ii) Time-of-flight
iii)Ion trap
iv) Fourier transform-ion cyclotron resonance (FT-ICR)

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Interfacing LC and MS
o It was difficult to couple liquid chromatography to a mass spectrometer because of the
necessity to remove the solvent. However, it was made possible with the use of interface
which connected the LC with MS.
o Earlier LC/MS systems used interfaces that involved techniques for evaporating solvent
and splitting the flow from LC columns to admit eluted compounds into the high vacuum
of the spectrometer. These approaches were successful only for a very limited number of
compounds. The introduction of atmospheric pressure ionization (API) techniques
greatly expanded the number of compounds that can be successfully analyzed by
LC/MS. Interfaces involving sources at vacuum (such as thermospray, particle-beam
and continuous-flow fast atom bombardment) are still in use, but API interfaces are by
far the most widely used, being the most suitable for LC/MS coupling.
o The commonly used interfaces are:-
 Electrospray ionization (ESI)
 Atmospheric pressure chemical ionization (APCI)
 Atmospheric pressure photoionization(APPI)
 Thermospray ionization (TSI)
1. Electrospray ionization (ESI):
In ESI, the LC eluent is sprayed (nebulized)
into a chamber at atmospheric pressure in the
presence of a strong electrostatic field and
heated drying gas. The capillary through which
the eluent passes has a high voltage potential
across its surface, and small, charged droplets
are expelled into the ionization chamber. The
charged droplets are subjected to a counter flow of a drying gas (usually nitrogen) that
evaporates solvent molecules from the droplets. Thus, the charge density of each droplet
increases until the electrostatic repulsive forces exceed the surface tension of the droplet
(the Rayleigh limit), at which point the droplets break apart into smaller droplets. This
process continues until solvent-free sample ions are left in the gas phase. These ions are
attracted to and pass through a capillary sampling orifice into the mass analyzer.
 Electrospray is especially useful for analyzing large biomolecules such as proteins,
peptides and oligonucleotides, but can also analyze smaller molecules like benzo-
diazepines.
2. Atmospheric pressure chemical ionization (APCI):
In APCI, the LC eluent is sprayed through a heated
(typically 250°C–400°C) vaporizer at atmospheric pressure.
The heat vaporizes the liquid. The resulting gas-phase
solvent molecules are ionized by electrons discharged from
a corona needle. The solvent ions then transfer charge to the
analyte molecules through chemical reactions (chemical
ionization). The analyte ions pass through a capillary
sampling orifice into the mass analyzer.
APCI is applicable to a wide range of polar and nonpolar
molecules. Since it involves high temperatures, APCI is
less well-suited than electrospray for analysis of large biomolecules that may be thermally
unstable. APCI is used with normal-phase chromatography more often than electrospray is
because the analytes are usually nonpolar.
3. Atmospheric pressure photoionization
Atmospheric pressure photoionization (APPI) for LC/MS is a relatively new technique. As
in APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates
photons in a narrow range of ionization energies. The range of energies is carefully chosen
to ionize as many analyte molecules as possible while
minimizing the ionization of solvent molecules. The resulting
ions pass through a capillary sampling orifice into the mass
analyzer.
Mass Analyzers
Although in theory any type of mass analyzer could be used
for LC/MS. The following four types are used most often each
has advantages and disadvantages depending on the
requirements of a particular analysis.
 Quadrupole
 Time-of-flight
 Ion trap
 Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS)

5. 01-Jul-20
Detectors:
Once an ion is separated, isolated, or fragmented by the mass spectrometer, it is necessary
to convert the abundance of that ion into an electric signal, which can be read by a data
station. Most modern-day instruments rely on:
 Electron multiplier detectors.
 Microchannel plate detectors (MCP)
Applications of LC-MS
LC-MS is most widely used in food, pharmaceutical and chemical industries for
quantitative and qualitative analysis
Molecular weight determination:
 It is used to determine the molecule weight of chemical substance, pharmaceutical
substances, proteins, etc.
Structural determination/elucidation:
 Used for structural determination e.g. structural determination of ginsenosides
(sapnins).
Pharmaceutical applications:
 It is used to determine the pharmacokinetic profile of drug, drug metabolites/
degradation product, impurities and chiral impurities. The separation and detection of
chiral impurities in pharmaceuticals are of great importance because the D-isomer of a
drug can have different pharmacological, metabolic and toxicological activity from the
L-isomer.
 Used in Bioequivalence and bio-avaiability studies.
 Detection of degradation Products for certain drugs which are difficult to be detected by
other methods (UV) e.g. Salbutamol.
Clinical and biochemical applications:
 MALDI-TOF MS is used quantification of DNA, gene expression analysis, DNA and
RNA sequencing.
 Used for detection of trimipramine and thioridazine.
Applications in Food sciences and poultry:
 Used to identify aflatoxins (toxic metabolic product in certain fungi) in foods.
 To determine vitamin D3 in poultry fed supplements, etc.