Gas chromatography and mass spectroscopy

GAS CHROMATOGRAPHY AND MASS SPECTROSCOPY

1. GC-MS
GAS CHROMATOGRAPHY -MASS SPECTROSCOPY
DIVISION OF QUALITY MANAGEMENT INSTRUMENTATION (QMI )
CSIR-IIIM,JAMMU
PRESENTED BY-MONIKA KAFALIYA
M.SC MICROBIOLOGY 4
TH
SEM
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2. CONTENT
INTRODUCTION
HISTORICAL BACKGROUND OF GC -MS
PRINCIPLE
INSTRUMENTATION
•GAS CHROMATOGRAPHY
•INTERFACE
•MASS SPECTROMETER
WORKING
LIMITATIONS
APPLICATIONS
CONCLUSION
REFERENCE
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3. INTRODUCTION
Gas chromatography - Mass spectrometry is the synergistic
combination of two analytical method to separate and identify different
substances within a test sample.
It is a common type of chromatography used in analytical chemistry for
separating, detecting and analyzing compound that can be vaporized
without decomposition.
Mass spectroscopy provides some definite structural information form 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.
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4. GAS
CHROMATOGRAPHY
MASS SPECTROMETRY
GAS
CHROMATOGRAPHY
MASS SPECTROMETRY
Separate mixture of
chemical 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
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5. •Rolando Gohlke and Mc Lafferty introduce use of MS as detector of GC in
1950s.
•Miniaturized computers has helped in the simplification of instrument.
•In 1968,Finnigan Instrument Corporation delivered the first quadrupole GC-MS.
•By the 2000s computerized GC-MS instrument using quadrupole technology
had become essential.
•In 2005 GC tandem MS–MS have been introduced.
HISTORICAL BACKGROUND OF GC -MS 5

6. PRINCIPLE
The sample solution is injected into the GC inlet where it is vaporized and
swept onto a chromatographic column by the carrier gas (usually He).
The sample flows through column (stationary phase) and compounds are
separated by virtue of their relative interaction with the coating of the column
and the carrier gas (mobile phase).
Latter part of column passes through a heated transfer line and ends at
entrance to ion source where compounds eluting from the column are
converted to ions and detected according to their mass to charge m/z ratio.
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8. INSTRUMENTATION LAYOUT
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GAS
CHROMATOGRAPHY
•Carrier Gas
•Pneumatic control
•Injector
•Column
•Oven
DATA SYSTEM
INTERFACE
❖Jet Interface
❖Direct Capillary
infusion interface
❖Watson- Biemann
effusion separator
MASS SPECTROMETER
➢Ion source
➢High-vacuum
system
➢Mass analyzer
➢Ion collector

9. GAS CHROMATOGRAPHY
1.Carrier Gas
➢Served as mobile phase supplied in the steel tank under high
pressure.
➢At pressure of 40-80psi this passes into flow controllers.
➢Example- He,N2,H2 and Ar can also be used.
❖Requirements-
✓Inert
✓Column requirements
✓Detectors
✓Purity –better than 99.995%
✓Cost effective & freely available
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10. Pros and Cons of the most commonly
used carrier gases
CARRIER GAS PROS CONS
Hydrogen (H
2) •High diffusivity and linear
velocities
•Gets good separation efficiencies
•Short analysis and run time (results
in cheap operational cost)
•Flammable
•Not completely inert (e.g. reacts
with some compounds at high
temperature)
Helium (He) •Inert (safe) and non-flammable
•Gives high resolution
•Expensive , not easily available
Nitrogen (N
2) •Cheap and easily available •Not suited for use in temperature
programmed GC analysis
•Lower or poor separation resolution
•Long analysis and run time
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11. 2.Pneumatic Control :
•Gas supply is regulated to the correct pressure and then
fed to the required part of instrument.
•Older instruments- manual pressure control via regulators.
•Modern GC instruments- Electronic Pneumatic pressure
controller.
3.Oven :
•Temperature programmable , typically range from 5°C-
400°C but can go as low as -25°C with cryogenic cooling.
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12. 4.Sample Injection Port :
➢Sample is made to vaporized rapidly
before entering to column.
➢Various kind of injectors :
✓Packed column injectors
✓Split injection
✓Splitless injection
✓Programmed split/splitless injection
✓Programmed on-column injector
5.Column :
➢Two kinds of column used :
✓Packed column
✓Capillary column
➢Gas chromatograph GC-MS utilizes
capillary column.
➢Here, stationary phase has been
chemically bonded to the fused silica,
e.g., DB-5
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13. DIFFERENCE
❖PACKED COLUMN ❖CAPILLARY COLUMN
•Less commonly used, having diameter of 2-
3mm and length of 0.5-10m.
•Consist of long capillary tubing 10-100m in
length and diameter of 0.10mm-0.53mm.
•Manufactured from steel or glass, internal
wall is treated to avoid catalytic effect with
the sample.
•Made up from stainless steel & coil.
•They can withstand a carrier gas flow rate
within range 10-40ml/min.
•Shorter columns- for fast analysis
•Larger columns- for high resolution separation
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14. 6.Detectors :
✓Simple and reliable
✓Sensitive to electronegative groups (halogens)
✓Largely non-destructive
✓Limited dynamic range 10
2
✓Mass sensitive detectors
- Thermal Conductivity Detector (TCD)
- Flame Ionization Detector (FID)
- Electron Capture Detector (ECD)
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15. INTERFACE
▪Pressure incompatibility problem between GC and MS was solved
by inserting an interface.
▪Mainly three types of interfaces are there commercially available :
✓Jet/Orifice separator
✓Direct capillary infusion interface
✓Watson- Biemann effusion separator
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16. MASS SPECTROMETER
“Mass spectrometry is a technique used for measuring the
molecular weight and determining the molecular formula of an
organic compound.”
In general a mass spectrometer consists of
▪An ion source
▪High-vacuum system
▪A mass-selective analyzer
▪An ion collector
▪Data system
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17. Ion source :sample introduction/ionization method
Ionization MethodTypical Analytes Sample
Introduction
Mass Range Method Highlights
Chemical
Ionization (CI)
Relatively small,
volatile
GC or liquid/solid
probe
Upto 1000 Daltons Soft method,
molecular ion
peak [M+H]
+
Electron Impact
Ionization (EI)
Relatively small,
volatile
GC or liquid/solid
probe
Upto 1000 Daltons Hard method,
versatile, provides
structure info
Electrospray
Ionization (ESI)
Peptides,
proteins,
nonvolatile
Liquid
chromatography
Upto 200000 Daltons Soft method, ions
often multiply
charged
Fast Atom
Bombardment
(FAB)
Carbohydrates,
organometallics,
peptides,
nonvolatile
Sample mixed in
viscous matrix
Upto 6000 Daltons Harder than ESI or
MALDI
Matrix Assisted
Laser Desorption
Ionization (MALDI)
Peptides,
proteins,
nucleotides
Sample mixed in
solid matrix
Upto 500000 Daltons Soft method, very
high mass
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18. Mass Selective Analyzer
•They deflect ions down a curved tubes in a magnetic fields
based on their kinetic energy determined by the mass, charge
and velocity.
•The magnetic field is scanned to measure different ions.
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Mass Analyzers
Quadrupole Ion Trap Time of flight

19. Quadrupole Analyzer :
Also known as “Hewlett-Packard” or “Mass Selective Detector”.
In quadrupole mass analyzer a set of four rods are arranged
parallel to the direction.
Only m/z is been determined and stable oscillation takes place.
Ions travels in quadrupole axis with cork screw type of trajectory.
It functions as a mass filter.
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20. Ion Trap Mass Analyzer :
The ion trap mass analyzer operates by similar principles where it consists of circular ring
electrode and two end caps that form a chamber.
AC or DC power along RF potential is applied between the cups and the ring electrode.
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Ions entering into
chamber
Trapped by
electromagnetic fields
Ions oscillate in concentric
trajectories
Resonant Ejection

21. Time-of-flight Analyzer :
The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through
the same potential.
Then it measures the time take to reach the detector.
If the particles all have the same charge, the kinetic energies will be identical and
their velocities will depend upon only on their masses.
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22. DATA HANDLING
▪Mass spectrometers employ computer control of same
functions and also use a computerized display and
output.
▪It is used to identify and measure the concentration of
one or more analytes in a complex mixture.
▪Advantages-
➢Amount of data generated is very large
➢It stores every sec for Upto 90min.
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MASS
CHROMATOGRAMS
SELECTED ION
MONITORING

23. WORKING
Switch on the instrument (Gas Chromatography /
Head Space)
Open the Realtime Analysis (Method development
and Batch maker)
Method Development
Will do the method selection ; Download initial
parameters
Create Batch File by save as the last file run
Check the parameters and the injection volume for
the given sample
Checking the Blank and Washing solution for GC-MS
, afterwards run the sample orderly.
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24. Post Run Analysis
Open the post run analysis
First check the peaks.
Label the peaks by giving the
parameters (area /height/depth)
Register it to spectrum and search
the expected components in the
library.
Make Reports and submit.
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25. LIMITATIONS?
Only compounds with vapour pressure exceeding about 10
-10
torr
can be analyzed by gas chromatography-mass spectrometry (GC-
MS).
Determining positional substitution on aromatic ring is often difficult.
Certain isomeric compound cannot be distinguished by mass
spectrometry.
Non-volatile matrices require additional preparation (extraction ,
outgassing , etc.)
Atmospheric gases are challenging (CO
2,N
2,O
2,Ar,CO,H
2O).
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26. APPLICATIONS 26
▪Analysis of Natural Products and Traditional Herbal Medicine
▪Identification of Metabolite
▪Bio analysis / Bioequivalence Studies
▪Dissolution Testing
▪Method Development / Validation
Impurity Profiling
Manufacturing / QA / QC
Analysis of amino acid
Determination of Pesticides
Biotechnology
Biochemical analysis

27. 27
➢Braun R., Introduction To Instrumental Analysis, Second Edition, Pharma Med Press,
Hyderabad, Page no. 251-270.
➢Chatwal G.R., Anand S.K., Instrumental method of Chemical Analysis, Himalaya
Publishing House, Fifth Edition-2012,New Delhi, Page no. 420-449.
➢Gohlke R.S.(1959), Analytical Chemistry, 36, Page no. 759-764.

28. THANK YOU
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