gas chromatography

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Chromatography
BY VENKATA NAVEEN KASAGANA SWATHI SREE
KARUMURI M-PHARM -PHARMACEUTICS S.B. COLLEGE OF
PHARMACY SIVAKASI TAMIL NADU INDIA E-MAILnaveen.k
asagana_at_gmail.com
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Gas Chromatography
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(a) Gas-Solid Chromatography (GSC), (b)
Gas-Liquid Chromatography, (GLC),
Gas chromatography fundamentally is a separation
technique that not only essentially provides
identification of a compound but also caters for
quantitative estimation after due calibration.
Gas chromatography makes use, as the stationary
phase, a glass or metal column filled either with
a powdered adsorbent or a non-volatile liquid
coated on a non-adsorbent powder. The
mobile-phase consists of an inert-gas loaded with
the vapourised mixture of solutes flowing through
the stationary phase at a suitable temperature.
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• In the course of the passage of the vapour of the
  sample through the column, separation of the
  components of the sample occurs in two ways,
  namely
• due to adsorption effects-i.e., when the prepared
  column consists of particles of adsorbent only,
  and
• (b) due to partition effects-i.e., when the
  particles of adsorbent are coated with a liquid
  that forms a stationary phase.

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• There are, in fact, three theories that have
  gained virtually wide recognition and acceptance
  in describing a gas chromatographic separation,
  namely
• Plate theory,
• (b) Rate theory,
• (c) Random walk theory.

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• GLC has a much greater application in the field
  of pharmaceutical
• Analysis
• The principal advantages of GC are enumerated
  below, namely
• ?
• It has high frequency of separation and even
  complex mixtures may be adequately resolved into
  constituents.
• ?It has a very high degree of sensitivity in
  detection of components i.e., only a few mg of
  sample is enough for complete analysis.
• ?Speed of analysis is quite rapid.
• Gives reasonably good accuracy and precision.
• The technique is fairly suitable for routine
  analysis because its operation and related
  calculations do not require highly skilled
  personnel, and
• The overall cost of equipment is comparatively
  low and its life is generally long.

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INSTRUMENTATION A gas chromatograph essentially
comprises of six vital components, namely (a)
Carrier Gas Regulator and Flow Meter, (b) Sample
Injection System, (c) Separation Column, (d)
Thermal Compartment, (e) Detectors, (f) Recording
of Signal Current, and (g) Integrator.
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CARRIER GAS PRESSURE REGULATOR AND FLOW
METER The various carrier gas used in GC along
with their characteristic features are stated
below H2 It has a distinctly better thermal
conductivity and lower density. Demerits are its
reactivity with unsaturated compounds and
hazardous explosive nature. He It has an
excellent thermal conductivity, low density,
inertness and it permits greater flow rates. It
is highly expensive. N2 It offers reduced
sensitivity and is inexpensive, and Air It is
employed only when the atmospheric O2 is
beneficial to the detector separation.
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SAMPLE INJECTION SYSTEM The sample injection
system is very important and critical because GC
makes use of very small amounts of the samples.
A good and ideal sample injection system should
be the one where the sample must not (i) be
decomposed at the point of injection, (ii) create
pressure surges, and (iii) undergo fractionation,
condensation or adsorption of components during
the course of transfer to the column.
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(a) Liquid Samples They are usually injected by
hypodermic syringes through a self-sealing
silicon-rubber septum into a preheated-metal-block
flash evaporator. The sample is vapourised as a
plug and carried right into the column by the
respective carrier gas.
(b) Solid Samples These are either dissolved in
volatile liquids (solvents) or temporarily
liquefied by exposure to infra-red heat. (c) Gas
Samples They are best handled and injected
by gas-light syrings or a gas-sampling valve,
usually termed as a stream-splitter.
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• SEPARATION COLUMN
• It is also known as the chromatographic column.
  In reality the heart of a GC is the column duly
  packed or capillary in which the separation of
  constituents is materialized.
• The packed-column is usually a tubing having an
  internal diameter of 4.0 mm and made up of
  stainless-steel, copper, cupronickel or glass
  either bent in U-shape or coiled.
• Its length varies from 120 cm to 150 M.

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• There are two general types of column, packed and
  capillary .
• Packed columns contain a finely divided, inert,
  solid support material (commonly based on
  diatomaceous earth) coated with liquid stationary
  phase. Most packed columns are 1.5 - 10m in
  length and have an internal diameter of 2 - 4mm.
• Capillary columns have an internal diameter of a
  few tenths of a millimeter.
• They can be one of two types
• Wall-coated open tubular (WCOT)
• Support-coated open tubular (SCOT).
• Wall-coated columns consist of a capillary tube
  whose walls are coated with liquid stationary
  phase.
• In support-coated columns, the inner wall of the
  capillary is lined with a thin layer of support
  material such as diatomaceous earth, onto which
  the stationary phase has been adsorbed.
• SCOT columns are generally less efficient than
  WCOT columns. Both types of capillary column are
  more efficient than packed columns.

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• THERMAL COMPARTMENT
• A precise control of the column temperature is
  must.
• Whether it is intended to maintain an
  invariant-temperature or to provide a
  programmed-temperature.
• Importantly, the temperature of the column oven
  must be controlled by a system that is sensitive
  enough to changes of 0.01C and that maintains an
  accurate control to 0.1C.
• In normal practice, an air-bath chamber surrounds
  the column and air is circulated by a blower
  through the thermal compartment.
• More recently, programmes are also available that
  features both in linear and non-linear temperature

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Detectors A number of detectors are used in gas
chromatography. The most common are flame
ionization detector (FID) and the thermal
conductivity detector (TCD). Both are sensitive
to a wide range of components, and both work over
a wide range of concentrations.
TCD is non-destructive, it can be operated
in-series before an FID (destructive)
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• Other detectors are sensitive only to specific
  types of substances, or work well only in
  narrower ranges of concentrations. They include
• discharge ionization detector (DID), which uses a
  high-voltage electric discharge to produce ions.
• electron capture detector (ECD), which uses a
  radioactive Beta particle (electron) source to
  measure the degree of electron capture.
• flame photometric detector (FPD)
• flame ionization detector (FID)
• Hall electrolytic conductivity detector (ElCD)
• helium ionization detector (HID)
• Nitrogen Phosphorus Detector (NPD)
• Infrared Detector (IRD)
• mass selective detector (MSD)
• photo-ionization detector (PID)
• pulsed discharge ionization detector (PDD)
• thermal energy(conductivity) analyzer/detector
  (TEA/TCD)

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Thermal Conductivity
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• IONIZATION DETECTOR
• The general class of ionization detectors
  comprise of the following important detectors,
  namely
• ? Flame ionization detector,
• ? Electron capture detector,
• ? Thermionic detector, and
• ? Photo ionization detector.

conduction of electricity
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GC-MS
GC detector that is also very expensive but very
powerful is a scaled down version of the mass
spectrometer. When coupled to a GC the detection
system itself is often referred to as the mass
selective detector or more simply the mass
detector. This powerful analytical technique
belongs to the class of hyphenated analytical
instrumentation (since each part had a different
beginning and can exist independently) and is
called gas chromatography/mass spectrometry
(GC/MS).
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What kind of info can mass spec gives you?

• Molecular weight
• Elemental composition (low MW with high
  resolution instrument)
• Structural info (hard ionization or CID)

Gas-phase ions are separated according to
mass/charge ratio and sequentially detected
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GC-MS
filament
70 eV e-
To mass analyzer
GC column
anode
Acceleration slits
repeller
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M

• M e-

f1
f2
f4
f3
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The high cost for the pump, ionization source,
mass filter or separator, ion detector, and
computer instrumentation and software has limited
the wide application of this system as compared
to the less expensive GC detectors
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RECORDING OF SIGNAL CURRENT In general, the
signal from a gas chromatograph is recorded
continuously as a function of time by means of a
potentiometric device.
INTEGRATOR An intergrator may be regarded as a
device that essentially facilitates simultaneous
measurement of areas under the chromatographic
peaks in the chromatogram either by mechanical or
electronic means.
GC-COMPUTER SYSTEM Nowadays, a large number of
data-processing-computer-aided instruments for
the automatic calculation of various peak
parameters, for instance relative retention,
composition, peak areas etc., can be conveniently
coupled with GC-systems.
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GC - DERIVATIZATION
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• GC is best for separation of volatile compounds
  which are thermally stable.
• Not always applicable for compounds of high
  molecular weight or containing polar functional
  groups. These groups are difficult to analyze by
  GC either because they are not sufficiently
  volatile, tail badly, are too strongly attracted
  to the stationary phase, thermally unstable or
  even decomposed.
• Chemical derivatization prior to analysis is
  generally done to
• Increase the volatility and decrease the polarity
  of compounds
• Reduce thermal degradation of samples by
  increasing their thermal stability
• Improve separation and reduce tailing
• Increase detector response by incorporating
  functional groups which lead to higher
• detector signals,
• Derivatizing Reagents Common derivatization
  methods can be classified into 4 groups depending
  on the type of reaction applied
• Silylation Acylation Alkylation
  Esterification

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SILYLATION
Silylation produces silyl derivatives which are
more volatile and more thermo stable. It
replaces hydrogen's with a trimethylsilyl group
TMS. Silylation reagents will react with the
water and alcohols first. Care must be taken to
ensure that both sample and the solvents are in
dry state. Pyridine is the most commonly used
solvent. The ease of reactivity of the
functional groups towards silylation follows the
order AlcoholgtPhenolgtCarboxylgtAminegtAmidegtHydroxy
l groups. Ex trimethylchlorosilane TMCS
trimethylsilylimidazole TMSI
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ACYLATION
Acylation reduces the polarity of amino ,
hydroxyl and thiol groups and adds halogenated
functionalities. Acylating reagents targets
functional groups such as carbohydrates and amino
acids. Acyl derivatives are formed with acyl
anhydrides acyl halides and acyl amide reagents.
It increases the volatility and sensitivity of
the compound. Acylation converts active
hydrogen's into esters,thioesters and amides.
Ex trifluoroacetoic anhydride,
pentafluoropropionic anhydride
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ALKYLATION
Replaces active hydrogens with an alkyl group.
The principle reaction employed for preparation
of these derivatives is nucleophilic
displacement. Used to modify carboxylic acids
and phenols. Can be used alone to form
esters,ethers amides or used in conjunction
with acylation or silylation. Ex
dialkylacetals , tetrabutylammonium hydroxide
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APPLICATIONS OF GLC IN PHARMACEUTICAL
ANALYSIS Gas liquid chromatography (GLC) or gas
chromatography (GC) finds its abundant
applications in the accurate and precise analysis
of plethora of official pharmaceutical substances
covering a wide range as enumerated below (i)
Assay of Drugs, (ii) Determination of specific
organic compounds as impurities in official
pharmaceutical substance, (iii) Determination
of related substances in official drugs, (iv)
Determination of water in drug.
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REFERENCES http//www.chem.harvard.edu/mass/tuto
rials/magnetmovie.html http//www.shu.ac.uk/schoo
ls/sci/chem/tutorials/chrom/chrom2.htm http//www
.wfu.edu/chemistry/courses/index.html223 http//
www.chem.vt.edu/chem-ed/ms/ms-intro.html http//e
n.wikipedia.org/wiki/GC-MS
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THANK YOU