SEMINAR-1
COURSE TITLE:ADVANCED SPECTRAL ANALYSIS
COURSE CODE: PCS 512
[Link], 2nd Semester
Date : //2024
Topic :Gas Chromatography-Mass spectroscopy
Presented by:
Sarangi .H
[Link], 2nd Sem
Register number : 23PHPY057004
Department of Pharmaceutical Chemistry
CONTENTS
2
Introduction
Gas Chromatography-Mass spectroscopy (GC-MS) is one of the
hyphenated techniques which combines two analytical methods to
separate and identify different substances within a test sample
Gas chromatography separates the components of a mixture in time
Mass spectrometer provides information that aids in the
identification and structural elucidation of each component
3
Introduction
4
Introduction
GC detectors are limited in the information that they give
It is usually two-dimensional giving the retention time on the
analytical column and the detector response
Identification is based on comparison of the retention time of the
peaks in a sample to those from standards of known compounds
However, GC alone cannot be used for the identification of
unknowns, which is where hyphenation to an MS works very well
MS can be used as a sole detector, or the column effluent can be
split between the MS and GC detector
5
Principle
The sample solution is injected into the gas inlet where it is
vaporized and swept onto a chromatographic column by the carrier
gas
The sample flows through the column and the compounds are
separated based on their relative interaction with the packing of
column and carrier gas
Latter part of column passes through a heated transfer line and ends
to an ion source where compounds eluting from the column are
converted to ions and detected according to their mass to charge
ratio
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Principle
It separates components of sample
Combines both techniques by removing
pressure incompatibility problem between GC
and MS
Ionise eluted component and separate,identify it
according to it’s mass to charge ratio
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Instrumentation
8
Instrumentation
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Instrumentation-GC
Carrier gas
Acts as mobile phase and determines the efficiency of separation
Eg : Hydrogen, Helium, Nitrogen, Argon
Requirements
Inert
Suitable to detector used
Purity > 99.9%
Cost effective and easily available
Less risk of explosion
10
Instrumentation-GC
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
Oven
Temperature programmable, typically range from 50C-4000C but can
go as low as -250C with cryogenic cooling
2 operation- isothermal programming & linear programming
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Instrumentation-GC
Sample injection port
Sample is made to vaporized rapidly before entering to column
Different injectors are :
Split injector
Splitless injector
PTV injector
On column injector
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Instrumentation-GC
Column
Mainly divided into Packed column and Capillary column
Capillary column – WCOT, SCOT, PLOT, FSOT
GC-MS utilizes capillary column where stationary phase has been
chemically bonded to fused silica
DB-5 is a common trade name
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Instrumentation-GC
Detectors
The main detectors used include
Thermal conductivity detector
Flame ionization detector
Electron capture detector
Nitrogen phosphorous/ Sulfur detector
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Instrumentation - Interfaces
The pressure incompatibility problem between GC and MS was
solved by inserting an interface
Interface joins GC and MS
An ideal interface should
Quantitatively transfer all analyte
Reduce pressure/flow from chromatograph to level that MS can
handle
The major goal of interface is to remove most of the carrier gas-the
majority of effluent
15
Instrumentation-interfaces
Different interfaces include
Molecular jet separator
Permeation interface
Watson-Biemann effusion seperator
Open split interface
Capillary direct interface
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Molecular jet separator
The GC flow is introduced into an evacuated chamber through a
restricted capillary
At the capillary tip a supersonic expanding jet of analyte and carrier
molecules is formed and its core area
sampled into the mass spectrometer
In an expanding jet,high molecular mass
compounds are concentrated in the core flow
Lighter and more diffusive carrier molecules are
dispersed away through collisions
17
Molecular jet separator
Advantage
Simple and inexpensive
Inert and efficient
Disadvantage
Plugging problems at capillary restrictor
Rate of diffusion is MW dependent
18
Permeation 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
It’s a very effective enrichment procedure,but suffers discrimination
effects with more polar analytes
It also produce significant band broadening of chromatographic peaks
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Advantages
Precisely control the quantity of sample introduced into GC-MS
Disadvantages
Membrane selectivity based on polarity and MW
Slow to respond
Only a small fraction of analyte actually permeates through
membrane
20
Watson-Biemann effusion separator
Here the effluent gas passes through a porous glass frit situated in
the vacuum chamber
Small molecules traverse the microscopic pores in the tube walls
and are evacuated whereas high molecular mass molecules are
transferred to ion source
Disadvantage
High surface area
High dead volume added
21
Interfaces
The three methods present above are based on enrichment of
analyte of carrier gas by eliminating carrier molecules
Thus enough sample can be introduced into ion source with total
gas flows compatible with the pumping capacity of the system
Among this jet separator is the most extensively used and successful
interface
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Open split interface
Used when sensitivity is not a critical factor,as sample enrichment does not
takes place
Flow splitting occurs at the exit of gas chromatograph,to a capillary
restrictor that limits the flow to ion source to a manageable constant value
The GC column exit is situated close to the restrictor entrance in an open
connector 23
Open split interface
The MS pulls in about The restrictor samples the effluent from the
ml/min through the flow restrictor
If column flow is above that- excess is vented
If flow is below that,He from external source is pulled in
That is,GC column exit and the excess column flow is removed from
the connector by helium
Best for sources that have flows close to ml/min like capillary
columns
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Capillary direct interface
GC-MS interfacing can be done simply by inserting a capillary column
directly into the ion source without splitting
The MS operates under high vacuum,which helps to draw the GC
effluent directly to the ionisation source
A column of length 25-30 m and 220-250 µm is used to give high
pressure so that carrier gas flows with a velocity of 25-35 cm/sec or 1-25
Capillary direct interface
Advantage
Maximum sensitivity in case of low concentration analytes as entire
sample is introduce
Disadvantage
Limits the flow rate and diameter of column that can be used
Risk of overloading when dealt with high concentration
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Instrumentation-Mass spectrometer
27
Working
Vaporized sample introduced into GC inlet
It is swept onto the column by He carrier gas and separates the
mixture into it’s components
Sample components eluted from the column moved to the MS
through a heated transfer line
Mass analyzer sort ions based on m/z ratio
There are numerous different mass analyzer types which determines
the mass resolution
Mass resolution is the ability of the mass analyzer to separate ions
with very small differences in m/z
28
Working
Unit mass resolution instruments can only separate nominal masses
or those down to a single decimal place, whereas high mass
resolution instruments can separate them to four or five decimal
places
The detector counts the ions that emerges from the mass analyzer
The signal is recorded by the acquisition software on a computer to
produce a chromatogram and a mass spectrum for each data point
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Analytical information obtained from GC-
MS
GC-MS data is three-dimensional
The x-axis shows the retention time; the time from sample injection
to the end of the GC run
This can also be viewed as the scan number, which is the number of
data points that have been acquired by the MS across the run
The y-axis is the response or intensity measured by the ion detector
The z-axis is the m/z of the ions across the mass range acquired
30
Analytical information obtained from GC-
MS
31
Analytical information obtained from GC-
MS
The two-dimensional chromatogram is produced by summing the
abundances of all the ions at a single data point and plotting it
against the retention time /scan number to produce a total ion
chromatogram
TIC more comparable to a chromatogram
produced by a GC detector
However each data point in the total ion
chromatogram is a separate mass
spectrum
32
GC-MS
Advantages Disadvantages
Efficient Only compounds with vapor
High resolution pressure >10 torr can be used
Non destructive sample Determining positional
substituents on aromatic ring is
recovery possible
often difficult
Small sample size If MS feed poor-background
Sensitive noise
Certain isomers can’t be
determined
Applications
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35
REFERENCES
1. [Link],Juan Boo Liang [Link]. Lovastatin Production by
Aspergillus terreus Using Agro-Biomass as Substrate in Solid State
[Link] of biomedicine and biotechnology,2012.
doi: 10.1155/2012/196264
2. [Link]
230209775/230209775#50
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