Gas chromatography-mass spectrometry (GC-MS) is a hyphenated technique that combines gas chromatography and mass spectrometry. GC is used to separate compounds in a mixture, while MS identifies the compounds based on their mass-to-charge ratios. The document discusses the basic principles, instrumentation, and applications of GC-MS. It explains how the gas chromatograph separates compounds and the mass spectrometer ionizes and detects them, providing both separation and identification capabilities in a single technique.

1. GAS CHROMATOGRAPHY-
MASS SPECTROMETRY
S.BHAGYA
M.PHARMACY
DEPT OF PHARMACOLOGY

2. CONTENTS
 INTRODUCTION
• - HYPHENATED TECHNIQUES
• - GCMS
 BASIC PRINCIPLES
• -GAS CHROMATOGRAPHY
• -MASS SPCTROMETRY
• -GCMS
 INSTRUMENTATION
 APPLICATIONS

3. INTRODUCTION
HYPHENATED TECHNIQUE:
• A hyphenated technique is combination (or) coupling of two different analytical techniques
with the help of proper interface. Mainly chromatographic techniques are combined with
spectroscopic techniques.
• In the chromatography, the pure or nearly pure fractions of chemical components in a mixture
was separated and spectroscopy produces selective information for identification using
standards or library spectra.
• “The coupling of the separation technique and an on-line spectroscopic detection technology
will lead to a hyphenated technique.”
• The term “hyphenation” was first adapted by Hirschfeld in 1980.

4. Advantages of hyphenated techniques:
• Shorted analysis time, Fast and accurate
• Reduction of contamination due to its closed system
• Higher degree of automation
• Higher sample throughput
• Both separation and quantification at same time
• Better reproducibility.
TYPES OF DIFFERENT HYPHENATED TECHNIQUES:
 Gas chromatography-Mass spectrometry (GC-MS)
 Gas chromatography-Infra red spectroscopy
 Gas chromatography-Infra red spectroscopy-Mass spectroscopy
 Gas chromatography-Thin layer chromatography
 Liquid chromatography-Mass spectrometry
 Capillary electrophoresis-Mass spectrometry.

5. GC-MS is an integrated composite analysis Instrument. Combining GC which is excellent in its
ability for separation with mass spectrometry ideal in identification and elucidate structure of
separated component.
 The use of a mass spectrometer as the detector in Gas chromatography was developed during
1950s by ROLAND GOHIKE and Mc LAFFERTY.
 GC= separation; MS= Identification.

6. GCMS
• Gas chromatography–mass spectrometry (GC MS) is an analytical method that combines the
features of gas-chromatography and mass spectrometry to identify different substances
within a test sample.
• Gas chromatography is a technique capable of separating, detecting and partially
characterizing the organic compounds particularly when present in small quantity.
• Mass spectroscopy provides some definite structural information from in small quantity.
• The separation and identification of the components of complex natural and synthetic
mixture are achieved more quickly than any other technique with less sample.
• Compounds that are adequately volatile, small, and stable in high temperature in GC
conditions can easily be analyzed by GC-MS.

7. GAS CHROMATOGRAPHY/MASSSPECTROMETRY(GC-MS)
Gas
Chromatography
Mass
spectrometry
Gas chromatography-
Mass spectrometry.
Separates mixture of
chemicals so each can be
identified individually.
Identifies (detects) chemicals
based on their molecular weight
or mass.
A Chemical Analysis
Technique combining two
instruments to provide for
powerful separation and
identification capabilities.

8. PRINCIPLE
GAS CHROMATOGRAPHY PRINCIPLES:
May be Gas Liquid [GLC] or Gas Solid Chromatography [GSC] but GLC is preferred.
GLC works by partition but GSC works by adsorption.
In GLC the substance to be studied first converted to gas which works as the mobile phase.
MASS SPECTROSCOPY PRINCIPLES:
 Ion Formation
 Ion Detection & Separation.

9. ION FORMATION:
Techniques used to form ions are:
1. Electron Ionization
2. Chemical Ionization
3. Field Ionization
4. Desorption Ionization
i. Secondary Ion Mass Spectroscopy [SIMS]
ii. Plasma Desorption [PD]
iii. Field Desorption [FD]
iv. Fast Atom Bombardment [FAB]
v. Matrix Associated Laser Desorption Ionization [MALDI]
vi.Thermo spray Ionization.
5. Electron spray Ionization.

10. Ion sources:
• The ion source is the part of the mass spectrometer that ionizes the material under analysis
(analyte).
• The ions are then transported by magnetic or electric fields to the mass analyzer.
• Molecular ions are formed when energy of the electron beam reaches 10-15 eV.
• Fragmentation of the ion reaches only at higher bombardment energies at 70 eV.

11. IONIZING AGENTS IN MASS SPECTROSCOPY

12. IONIZATION METHODS IN MASS SPECTROSCOPY

13. ELECTRON IMPACT
• In the Electron Impact (EI) process,
electrons are emitted from a heated
filament (usually made of tungsten or
rhenium) and are accelerated across the
source by using an appropriate potential
(5-100V) to achieve the required electron
energy (sufficient to ionize the molecule).

14. CHEMICAL IONIZATION
• A reactive gas like methane is introduced along
with the sample into the ionization chamber.
When the reactive gas bombards with the
electron beam it undergoes ionization to
produce ions which further react with neutral
molecules to form chemically reactive species
that interact with the sample molecules to
produce positive ions.

15. FIELD IONIZATION
• The analyte molecules exposed to a very high
electrostatic field of about 10 -10V/cm, which is
about 1000 times higher than that of a typical
MALDI ion source.
• Thin metal wire is used as anode.
• The anode and cathode is placed very closely to
the ionization chamber.
• When the sample is introduced between the
electrodes with of high potential the sample
ionizes and produce electrons.

16. FAST ATOM BOMBARDMENT
• Argon gas is ionized by hot filament and
focused beam that bombards the
sample.
• Beam impinges the sample a series of
molecular reactions occur and analyze
in mass spectroscopy analyzer.
• Example: Insulin, Amino glycosides.

17. MATRIX ASSISTED LASER DESORPTION/IONIZATION
• MALDI is an LIMS method of vaporizing and
ionizing the sample molecules are dispersed
in solid matrix such as Nicotinic acid.
• A UV laser pulse ablates the matrix which
carries some of the large molecules into the
gas phase in an ionized form so they can be
extracted into the Mass Spectrometer.

18. PLASMA DESORPTION
• This technique was first developed by Macfarlane and Torgerson in 1976.
• It was first used for the ionization of thermo labile and non volatile molecules including
peptides, proteins, nucleotides etc.
• In this technique, californium252 is used to ionize the sample.
• The sample is dissolved in a suitable solvent and deposited on a thick nickel foil.
• The sample foil is then positioned closed to californium 252 source.
• When fission occurs two fragments having kinetic energy up to 200MeV are released.
• These fragments are travel in opposite directions one moves towards the sample and other
moves towards fission fragment detector.

19. • The fragment penetrating into the sample imparts sufficient energy to the sample and
thereby causes volatilization and ionization in sample.
• The resulting samples get desorbed from the surface of the sample into the gas phase.
• The gaseous ions are then detected and recorded.

20. ELECTRON SPRAY IONIZATION
• ESI consist of very fine needle and series of
skimmers.
• A Sample solution is sprayed into source
chamber to form droplets.
• When droplets carry the charge exit the
capillary end, as the solvent evaporates , the
droplets disappear leaving highly charged
analyte molecules.

21. ION SEPARATION AND DETECTION:
• Mass analyzer also called Ion separator
• After ionization , the gaseous ions enter the mass analyzer where they are separated according to
their m/e ratio.
• It plays a important role in the instrument’s accuracy and mass range.
Types of instruments used are-
i. Direct Focusing Type
a) Single Focusing
b) Double Focusing
ii. Quadrapole MassAnalyzer
iii. Magnetic sector Mass Analyzer
iv. Fourier Transfer Ion cyclotron Resonance
v. Time Of Flight MassAnalyzer.

22. QUADRAPOLE MASS ANALYZER
• In a Quadrapole mass analyzer, a set of four
rods are arranged parallel to the direction.
Here a DC current and radio frequency RF is
applied to generate oscillating electrostatic
field in between the rods.
• Based on this only m/z is been determined
and stable oscillation takes place. And ion
travels in Quadrapole axis with cork screw
type of trajectory.

23. MAGNETIC SECTOR MASS ANALYZER
• In magnetic sector analyzers ions are
accelerated through a flight tube, where the
ions are separated by charge to mass ratios.
As moving charges enter a magnetic field, the
charge is deflected to a circular motion of a
unique radius in a direction perpendicular to
the applied magnetic field.
• Basically the ions have certain m/e value will
have unique path radius, if similar ions passed
through magnetic field they will be deflected
in same degree.

24. ION TRAP MASS ANALYZER
• The ion trap mass analyzer operates by similar
principles where it consists of circular ring
electrode
• Plus two end caps that form a chamber.
Here AC or DC power along RF potential is
applied between the cups and the ring
electrode.
• There the ions entering into the chamber are
trapped by electromagnetic fields and they
oscillates in concentric trajectories. This process
is called resonant ejection.

25. ION DETECTORS
 Detectors plays important role in identifying charged ions.
 Different types of detectors used are:
• - Electron Multiplier Detector
• - Photo Multiplier tube Detector
• - Micro channel plate Detector.

26. Electron Multiplier detector

27. • This is used when positive and negative ions need to be detected on same instrument.
• An electron multiplier is a vacuum-tube structure that multiplies incident charges.
• In a process called secondary emission, a single electron when bombarded on secondary-
emissive material, induce emission of roughly 1 to 3 electrons.
• If an electric potential is applied between this metal plate and yet another, the emitted
electrons will accelerate to the next metal plate and induce secondary emission of still more
electrons.
• This can be repeated a number of times, resulting in a large shower of electrons all collected
by a metal anode, all having been triggered by just one.

28. Photomultiplier detector

29. • These are typically constructed with an evacuated glass housing containing a photocathode,
several dynodes, and an anode.
• Incident photons strike the photocathode material, which is usually a thin vapor-deposited
conducting layer on the inside of the entry window of the device.
• Electrons are ejected from the surface as a consequence of the photoelectric effect. These
electrons are directed by the focusing electrode toward the electron multiplier, where electrons
are multiplied by the process of secondary emission.
• The electron multiplier consists of a number of electrodes called dynodes. Each dynode is held at
a more positive potential, by ≈100 Volts, than the preceding one. A primary electron leaves the
photocathode with the energy of the incoming photon.
• This last stage is called the anode. This large number of electrons reaching the anode results in a
sharp current pulse that is easily detectable.

30. Micro-channel plate (MCP)
• It is a planar component used for detection of single
particles (electrons, ions and neutrons ) and low
intensity impinging radiation (ultraviolet radiation and
X-rays).
• It is closely related to an electron multiplier, as both
intensify single particles or photons by the
multiplication of electrons via secondary emission.
However, because a micro channel plate detector has
many separate channels, it can additionally provide
spatial resolution.

31. Vacuum system
All mass spectrometers need vacuum to allow ions to reach the detector without colliding
with other gaseous molecules or Atoms.

32. PRINCIPLEOF WORKING OF GC-MS AND INTERFACES
PRINCIPLE OF GCMS
The sample solution is injected into the GC inlet where it is vaporized and swept onto a
chromatographic column by the carrier gas (usually helium).
The sample flows through the column and the compounds comprising the mixture of interest
are separated by virtue of their relative interaction with the coating of the column (stationary
phase) and the carrier gas (mobile phase).
The latter part of the column passes through a heated transfer line and ends at the entrance to
ion source where compounds eluting from the column are converted to ions.
Then ions are detected by Mass spectrometer.
The time elapsed between injection and elusion is called “retention time” (tR).

33. Samples:
 Nature: Samples should be organics, must be volatile or semi-volatile thermally stable.
 State: Organic compounds must be in solution for injection into the gas chromatograph. The
solvent must be volatile and organic (for example, hexane or dichloromethane)
 Amount: Depending on the ionization method, analytical sensitivities of 1 to 100 pg per
component are routine.
 Preparation: Sample preparation can range from simply dissolving some of the sample in a
suitable solvent to extensive. Clean up procedures using various forms of liquid chromatography.

34. INTERFACES OF GC-MS
• The pressure incompatibility problem between GC and MS was solved by Inserting an Interface.
• Interface join GC with MS, There are many interfaces like:
 JET SEPARATOR
 PERMSELECTIVE
 MEMBERANE MOLECULAR
 EFFUSION DIRECT INTRODUCTION.

35. Commercially available interfaces are:
• Jet Interface
 Device takes advantage of the differences in diffusibility between the carrier gas and the organic
compound.
 These jet separators work well at the higher carrier gas flow rates (10 to 40 mL/min).
 In these separators, the GC flow is introduced into an evacuated chamber through a restricted
capillary. Light particle dispersed away.

36. • Direct Interface
 Most GC-MS interfacing is now done by simply inserting the capillary column directly into the
ion source.
 This gives a helium or hydrogen GC carrier gas velocity of 25 to 35 cm/sec or a flow of about 1 to
2 mL/min.
 In this method capillary column is directly inserted into MS ionization chamber.

37. • Perm selective membrane interface:
It is made of a silicone-rubber membrane that transmits organic non-polar molecules and acts as a
barrier for (non-organic) carrier gases.

38. • Molecular effusion /Watson- Biemann interface:
It is based on the molecular filtering of the gas effluent by means of a porous glass fritted tube.

39. INSTRUMENTATION
GAS CHROMATOGRAPHY
 Gas Chromatography – “It is a process of separating components from the given crude drug
by using a gaseous mobile phase.”
 It involves a sample being vaporized and injected onto the head of the
chromatographic column.
 The sample is transported through the column by the flow of inert, gaseous mobile phase.
The column itself contains a liquid stationary phase which is adsorbed onto the
surface of an inert solid.

40. INSTRUMENTATION PARTS OF GC :
 Carrier Gas
 Sample Injection System
 Oven
 Separation Column
 Detectors
 Amplification
 Recorder.

41. MASS SPECTROSCOPY
 The mass spectroscopy is an technique in which, the compound under
investigation is bombarded with beam of electrons which produce an ionic
molecule or ionic fragments of the original species.
 The mass spectrometer is an instrument in which the substance in gaseous or vapor
state is bombarded with a beam of electrons, to form a positively charged ions
(cations) which are further sorted according to their mass to charge ratio to
record their masses and their abundances.

42. INSTRUMENTATION PARTS OF MS :
 Sample Handling System
 Ionization Chamber
 Ion Separator or Mass Analyzer
 Ion Collector, Detector and
Read Out System
 Vacuum System.

44. INSTRUMENTATION OF GCMS
Parts of GCMS:
1. Pneumatic controls
2. Injector
3. Oven
4. Column
5. Interface
6. Ion Source
7. Mass Analyzer
8. Detector
9. Vacuum System
10. Control Electronics

45. CHROMATOGRAMGENERATEDBYGASCHROMATOGRAPHY
 While the instrument run, the computer
generated a graph from signal called
chromatogram.
 X-axis show the Rt.
 Y-axis show the intensity of the signal.

46. SPECTRUM GENERATED BY MASS SPECTROMETRY
The computer record a graph for each scan
calledspectrum
 The mass spectrum is essentially a
fingerprint for the molecule and can be
used to identify the compound.

47. INTERPRETATION OF RESULTS
• Through GC a CHROMATOGRAM is obtained.
• Through MS a SPECTRUM is obtained.
• GC-MS gives a 3D graph which has both
chromatogram and spectrum to each
separated components in the chromatogram.

48. APPLICATIONS
 Elucidation of organic, inorganic, and biological molecules.
 Impurity profiling of pharmaceuticals.
 Identification of components in Thin layer and Paper chromatography.
 Identification of drugs of abuse & metabolites of drugs of abuse in blood, urine and saliva.
 Testing of the presence of the drugs in blood in race horses and in Olympic athletic (in
forensic GC-MS).
 Analysis of aerosol particles.
 Determination of pesticide residues in food.
 Polymer characterization.
 Drug monitoring and toxicological studies.

49.  Detection of lipophilic compounds in diverse plant tissues.
 Analysis of biologically important aromatic amines.
 Application of human Dosimetry.
 Identification of volatile components.
 For the determination of pyrethoid residues in vegetable samples.
 Environmental and forensic applications.
 Toxicity assessment.
 GC-MS is increasingly used for detection of illegal narcotics marijuana, cocaine, opioids,
oxycodeine and oxymorphine.
 Petrochemical and hydrocarbons analysis.
 Food, beverages and perfume analysis.

50. Advantages:
- High sensitivity
• Excellent detection limits. Typically low ppb to high ppt
- High selectivity
• Identification is based on two parameters not one (retention time and mass spectrum must
match standard) selects analyte of interest with very high confidence.
• Typical analysis takes from 1/2 hour to approx. 1 hour analysis can contain upwards of 80
and more pollutants.
• It can provides sensitive response to most analytes.
• Good Accuracy and Precision.
Disadvantages:
- Higher capital cost.
- Higher maintenance (time, expertise and money)
- For optimum results, it requires analyst of knowledge in both chromatography and mass
spectrometry.