SEMINAR-1

COURSE TITLE:ADVANCED SPECTRAL ANALYSIS

COURSE CODE: PCS 512
M.PHARM, 2nd Semester
Date : //2024
Topic :Gas Chromatography-Mass spectroscopy
Presented by:
Sarangi .H
M.PHARM, 2nd Sem
Register number : 23PHPY057004
Department of Pharmaceutical Chemistry
CONTENTS

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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

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Introduction

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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

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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

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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

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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

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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

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Molecular jet separator

Advantage
 Simple and inexpensive
 Inert and efficient

Disadvantage
 Plugging problems at capillary restrictor
 Rate of diffusion is MW dependent

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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

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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

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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

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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
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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

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Analytical information obtained from GC-
MS

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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

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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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 REFERENCES
1. MF.ahromi,Juan Boo Liang et.al. Lovastatin Production by
Aspergillus terreus Using Agro-Biomass as Substrate in Solid State
Fermentation.Journal of biomedicine and biotechnology,2012.
doi: 10.1155/2012/196264
2. https://www.slideshare.net/slideshow/fermentation-
230209775/230209775#50

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