The powerpoint contains princple instrumentation and application of Gas chromatography in form of animation.

1. GAS CHROMATOGRAPHY
Mr. SHINDE G.S
PRAVARA RURAL COLLEGE OF PHARMACY, PRAVARANAGAR
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

2. Contents :-
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 Introduction
 Principle
 Instrumentation
 Working
 Evaluation
 Applications
 Reference

3. Ganesh Shashikant Shinde
Pravara Rural college of pharmacy, pravaranagar

4.  The father of modern
gas chromatography is
Nobel Prize winner
John Porter Martin,
who also developed the
first liquid-gas
chromatograph. (1950)
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5. INTRODUCTION:-
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 Gas chromatography – “It is a process of
separating component(s) from the givencrude
drug by using a gaseous mobilephase.”
 It involves a sample being vaporized and injected
onto the head of the chromatographic column.
The sample is transported through the columnby
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.

6. Twomajor types:
• Gas-solid chromatography:
Here, the mobile phase is a gas while the stationary
phase is asolid.
Used for separation of low molecular gases, e.g.,
air components, H2 S, CS2 ,CO2 ,rare gases, CO
and oxides of nitrogen .
• Gas-liquid chromatography:
The mobile phase is a gas while the stationary
phase is a liquid retained on the surface as an inert
solid by adsorption or chemicalbonding.
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7. Principles:
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 The principle of separation in GC is“partition.”
 The mixtureof component to be separated is converted
to vapourand mixed with gaseous mobilephase.
 The component which is more soluble in stationary
phase travel slower and eluted later. The component
which is less soluble in stationary phase travels faster
and eluted outfirst.
 No two components has same partition coefficient
conditions. So thecomponentsare separated according
to their partitioncoefficient.
 Partition coefficient is “the ratio of solubility of a
substancedistributed between two immiscible liquidsat
a constanttemperature.’

8. Instrumentation:-
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 Carriergas
- He (common), N2, H2, Argon
 Sample injectionport
- micro syringe
 Columns
 Detectors
 Thermal conductivity (TCD)
 Electron capture detector(ECD)
 Flame Ionization detector(FID)
 Flame photometric(FPD)

9. Gas chromatography Video Demonstration

10. 10

11. Carrier gas:-
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 The cylinder/ gas tank is fitted with a pressure
controller to control the pressure of gas, a pressure
gauge that indicates the pressure, a molecular sieve to
transfer filtered drygas and a flow regulatortoensurea
constantrateof flow of mobile phase to thecolumn.
 It should meet the followingcriteria:
Should be chemicallyinert
Should be cheap and readilyavailable
Should beof high qualityand notcauseany fire
accidents
Should give best possibleresults
Should be suitable for the sample to beanalyzed and
for thedetector

12.  Hydrogen, helium, nitrogen and
carbon dioxide are commonlyused.
 Hydrogen has low density andbetter
thermal conductivity. However, it
reacts with unsaturated compounds
and is inflammable and explosive in
nature.
 Nitrogen is inexpensive but itgives
reduced sensitivity.
 He is the most preferredgas.
 Inlet pressure ranges from: 10-50psi
-Flow rate : 25-150 mL/minfor
packed columns
-Flow rate: 2-25 mL/min foropen
tubularcolumn
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13. Sampling unit:-
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 Sampling unitor injection port is
attached to the columnhead.
 Since the sample should be in
vapourized state, the injectionport
is provided with an oven that helps
to maintain its temperature at
about 20-500 C above the boiling
point of thesample.
 Gaseous samples may be
introduced by useof agas tight
hypodermic needle of 0.5-10 ml
capacity.
 For Liquid samples , microsyringes
of 0.1-100µL capacity may beused.
Microsyringe

14. Injections of samplesinto
capillarycolumns:-
a. Split injections- it splits
the volume of sample
stream into twounequal
flows by means of a
needle valve , andallow
thesmallerflow to passon
to the columns and the
bigger part is allowed to
be vented to the
atmosphere. This
technique is not suitable
when highest sensitivity is
required.
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15. b. Splitless injectors- They allow all of the
sample to pass through the column for
loading. Sample should beverydilute toavoid
overloading of thecolumn and a high capacity
column such as SCOT or heavily coated
WCOT columns should beused.
c. On column injectors: A syringe with a very
fine quartz needle is used. Aircooled to -200c
below the b.p. of the sample. After then the
warmer air is circulated to vaporize the
sample.
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16. d. Automatic injectors: For
improving the reproducibility and
if a large number of samples areto
be analyzed or operation is
required without an attendant,
automatic injectors areused.
• The solid samples are introduced
as a solution or in a sealed glass
ampoule, crushed in the gas
stream with the helpof a gas tight
plunger, and the sample gets
vapourized and flows intocolumn
under the influence of carriergas.
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17. Column unit:-
 Columnsare of different shapes and sizes that includes: “U”
tube typeorcoiled helix type.
 They are mainly made of copper, stainless steel, aluminium, Glass,
nylonand othersynthetic plastics.
Support material:-
 it’s main function is to provide mechanical supportto the liquid phase.
An ideal supportshould havea large surface area, chemically
inert, should get uniformly wet with liquid phase, should be
thermostable.
 Commonly used solid phases are: diatomaceous earth or kieselguhr,
glass beads, porous polymers,sand,etc.
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18. Liquid phase:-
It should have the followingrequirements:
It should be non-volatile
Should have high decomposition temperature
Should be chemicallyinert
Should posses lowvapourpressure atcolumn
temperature
Should be chemically and structurally similarto
thatof the solute i.e., polar for polarsolute.
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19. Examples of different liquid phases:
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CATEGORY EXAMPLES
Non-polar hydrocarbon phases Paraffin oil (nujol), siliconoil,
silicon rubber gum (used for
high temp of about4000
Polar compounds (having polar
groups like -CN, -CO and –OH)
Polyglycols (carbowaxes)
Liquids having hydrogen
bonding
Glycol, glycerol, hydroxy acids

20. Types of columns:-
• There are two general types ofcolumns:
1. Packed columns:- In GLC, they are denselypacked
with finely divided, inert, solid supportmaterial
( diatomaceous earth) coated with liquidstationary
phase.
In GSC, thecolumnsare packed with
adsorbents or porouspolymers.
 Length- 1.5 - 10m
 internal diameter- 2 - 4mm.
1. Capillary columns-
 length ranges from 10-100m
 inner diameter is usually0.1-0.5mm.
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21. • It is mainly of twotypes:
Wall-coated columns - consist of a capillary tube
whosewallsarecoated with liquid stationary phase.
Support-coated columns- the inner wall of the
capillary is lined with a thin layer of support material
such as diatomaceousearth, ontowhich the stationary
phase has been adsorbed. It is also known as PLOT
(porous-layer open tubularcolumns).
SCOTcolumnsaregenerally lessefficient than WCOT
columns. Both types of capillary column are more
efficient than packedcolumns.
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22. 22

23. Equilibrium of the column:-
• The packed columns areequilibrated before introduction of
the sample. This is done by allowing continuous flow of
heated carrier gas through the columns for a specific
duration of time (24hrs) at prescribedtemperature.
• Ideally prepared and conditioned columns show a zero base
lineon the recorder upon passage of carriergas alone.
Column temperature:-
• This can be controlled by jackets equipped with vapours of a
boiling liquid, electrically heated metal blocks or circulating
air baths.
• Compounds of low B.P- eluted at lowertemperature
• Compounds of high B.P- boils at highertemperature
resulting in broader and shallower peaks, require
temperature programming.
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24. Detectors:-
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 The eluted solute particles along with the
carrier gas exit from the column and enter
thedetector.
 Thedetectorthen produces electrical signals
proportional to the concentration of the
components of solute.
 The signals areamplified and recorded as
peaks at intervals on thechromatograph.

25. Properties of an ideal detector:
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 Sensitive
 Operate at high T (0-400 °C)
 Stable and reproducible
 Linearresponse
 Wide dynamicrange
 Fast response
 Simple (reliable)
 Nondestructive
 Uniform response to allanalytes

26. I. Thermal conductivitydetector:-
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 “TCD is based upon the fact that the heat lost
from a filament depends upon the thermal
conductivity of the stream of surrounding gasas
well as its specificheat.”

27. When only carrier gas flows heat loss to
metal block is constant, filament Tremains
constant.
When an analyte species flows past the
filament generally thermal conductivity
changes, thus resistance changes which is
sensed by Wheatstone bridgearrangement.
The imbalance between control andsample
filament temperature is measured and a
signal is recorded.
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29. • Advantages :-
Simple and inexpensive
Durable and posses long life
Accurate results
Non-selective, hence known as universaldetectors
• Disadvantages:-
Low sensitivity
Affected by fluctuations in temperature andflow
rate.
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30. II. Electron capturedetector:-
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 Molecules of compounds, which posses affinity for electrons,
differ in their electron absorbing capacities. This difference is
utilized in this detectorfor identificationof the compounds.
 Working- A foil made up of a radioactive metal like Ni63 (β-
emitter) is placed inside a Teflon coated cell which also
containsacathodeand an anode.
 In the absence of organic species, the produced electrons
migrate towards positive electrode and produce a certain
constant standingcurrent.
 When a sample/eluent is present itcaptures theelectrons,
elutes from column, there is adrop in this constantcurrent.
 The potential across twoelectrodes is adjusted tocollectall
the ions and a steady saturation current, is therefore,
recorded.

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32. Advantages:-
 Highly selective
 Highly sensitive for thedetectionof compounds like
halogens, quinones, peroxides, nitrites,etc.
 It is non-destructive
 More sensitive than TCD andFID.
Disadvantages:-
 Least sensitive tocompoundswhose molecules have
negligible affinity forelectrons.
 Carriergas used should beof pure form like pure
nitrogen.
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33. III.Flame ionizationdetector:-
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 This employs hydrogen flame that is maintained
in a small cylindrical jet made up of platinum or
quartz.
 Effluent from the column with helium or nitrogen
as carrier gas are fed into the hydrogen flame, gets
ignited and undergoes pyrolysis to produceions.
 Fordetection of these ions, twoelectrodes are
used that provide a potentialdifference.
 The ions produced are repelled by the positive
electrode which hit the collector plate. The
current produced indoing so is amplified and fed
to an appropriaterecorder.

34. Flame ionization detector
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35. IV.Flame photometricdetector:-
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 It is a selective detector that is responsive to
compounds containing sulphur orphosphorous
 The detection principle is the formation of
excited sulphur (S2*) and excited hydrogen
phosphorous oxide species (HPO*) in areducing
flame.
 A photomultiplier tube measures the
characteristic chemiluminescent emission from
these species. The optical filter can be changed
toallow the photomultiplier toview light of 394
nm for sulphur measurement or 526 nm for
phosphorus.

36. Flame photometric detector
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 Applications:
-For detectionof
heavy metals like
chromium,
selenium, tin, etc,
in organometallic
compounds.
-Also for analysis
of pesticides, coal,
hydrogenated
products as wellas
air andwater
pollutants.

37. Working:-
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• Fill the syringe withsample.
• Record the setting i.e., column temperature,
detector temperature and injection port
temperature.
• Introduce sample into the injection port by
completely inserting the needle into the rubber
septum. Note down the injection time.
• The samplegetsvapourized due to highertemperatureof
injection port and is swept intocolumn bycarriergas.
• This samplecomponents now get distributed between
the gas and stationary liquid phase depending upon
their solubilizing tendencies.
• Thecomponents with minimal solubility move faster
and those with maximum solubility travelslowly.
• Thecomponents leaving thecolumn activatedetector
and recorder to give aplot.

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39.  The components that slowly leave the column give a
bell shaped curveof shorterpeak while theonewhich
travels faster gives a bluntly pointed curve of larger
peak.
 In abovegraph, thecomponent that first emergesout
of thecolumn is component 4 followed by 2,5,3 and 1.
 Thearea under thecurve is determined in order to
obtain the percentage compositionof the mixture.
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40. Evaluation :-
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 HETP (height equivalent to theoretical plate)- It is the
distance on the column in which equilibrium is attained
between the solute in thegas phase and thesolute in liquid
phase. Larger the numberof theoretical plates/ smaller the
HETP, the moreefficient thecolumn is forseparation.
HETP = Length of column/n ; Where n = number oftheoretical
plates
 Retention Time: defined as theabsolute time taken bya
sample toshow maximum peak after injecting.
 Retention Volume: defined as thevolume of gas required to
eluteabout half of thesolute through thecolumn.
VR = tR ×F
F =average volumetric flow rate (mL/min) ,estimated by
using soap bubble meter (some gases dissolving in soap
solution)

41. Applications:-
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 Qualitative Analysis – by comparing the retention time or
volume of the sample to the standard / by collecting the
individual components as they emerge from the
chromatograph and identifying these compounds byother
methods like UV, IR, NMR.
 Quantitative Analysis- area under a single component
elution peak is proportional to thequantityof thedetected
component/response factor of the detectors. It is doneby:
I. Direct comparison method-
A(sample) / A(std) = α C(sample)/C(std)
Where, α is the response factordetermined forevery pure
compound under givenconditions.

42. II. Calibration curve- a graph is plotted by taking
peak areas on Y axis and concentration of
standard compound on X axis. Concentrationof
unknown sample is then determined byplotting
its peak area on samegraph.
III. internal standard method- A known
concentration of internal standard, which has
similar retention characteristics as that of
sample is added to both reference standardand
testsample.
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43.  Pharmaceutical applications-
Qualitycontrol and analysis of drug products like
antibiotics (penicillin), antivirals (amantidine),
general anesthetics (chloroform, ether),
sedatives/hypnotics (barbiturates), etc.
Assayof drugs – purityof a compound can be
determined for drugs like:
Atropine sulphate
Cloveoil
Stearicacid
In determining the levels of metabolites in bodyfluids
like plasma, serum, urine,etc.
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44.  Miscellaneous:
analysis of foods like carbohydrates, proteins, lipids,
vitamins, steroids, drug and pesticides residues,trace
elements.
Pollutants like formaldehyde, carbonmonoxide,
benzene, DDT etc.
Dairy productanalysis like milk, butter–fordetection
of aldehydes, milk sugars, ketones and fattyacids.
Separation and identification of volatile materials,
plastics, natural and synthetic polymers, paints,and
microbiological samples.
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