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Gas chromatography by Dr. Anurag Yadav
1. Presenter Dr Anurag Yadav
Morderator Dr Avinash S S
Column Chromatography
1 Dr Anurag Yadav
2. Capsule:
“Chromatography is a technique for
separating mixtures into their components in
order to analyze, identify, purify, and/or
quantify the mixture or components.”
Supporting
medium
planar column
MECHANISM
ION
EXCHANGE
PARTITION
ADSORPTION
AFFINITY
SIZE
EXCLUSION
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4. The father of modern
gas chromatography is
Nobel Prize winner John
Porter Martin, who also
developed the first liquid-
gas chromatograph.
(1950)
First separated
compound was fatty
acid.
History
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7. Gas chromatography
Gas chromatography is a type of column chromatographic
technique that can be used to separate volatile organic
compounds.
Mobile phase: inert gas:
nitrogen, helium, hydrogen → carrier gas.
Stationary phase: liquid/solid.
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8. Types of Gas chromatography
GSC : Packed columns filled with solid sorbent (stationary
phase) support particles.
GLC : Support particles coated with thin liquid layer
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10. How a Gas Chromatography Machine
Works
First, a vaporized sample is
injected onto the
chromatographic column.
Second, the sample moves
through the column through the
flow of inert gas.
Third, the components are
recorded as a sequence of
peaks as they leave the10 Dr Anurag Yadav
12. Chromatographic Separation
Deals with both the stationary phase
and the mobile phase.
Mobile – inert gas used as carrier.
Stationary – liquid coated on a solid or a
solid within a column.
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13. Sample to be separated is converted into vapour
And mixed with gaseous M.P
Component more soluble in the S.P → travels slower
Component less soluble in the S.P → travels faster
Components are separated according to their
Partition Co-efficient
Criteria for compounds to be analyzed by G.C
1.VOLATILITY:
2.THERMOSTABILITY:
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14. Chromatographic Separation
Chromatographic Separation
In the mobile phase, components of the sample
are uniquely drawn to the stationary phase and
thus, enter this phase at different times.
The parts of the sample are separated within
the column.
Compounds used at the stationary phase reach
the detector at unique times and produce a
series of peaks along a time sequence.
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16. Chromatographic Separation
(continued)
The peaks can then be read and analyzed
to determine the exact components of the
mixture.
Retention time is determined by each
component reaching the detector at a
characteristic time.
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17. Chromatographic Analysis
The number of components in a sample is
determined by the number of peaks.
The amount of a given component in a sample is
determined by the area under the peaks.
The identity of components can be determined by
the given retention times.
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19. Instrumentation
Carrier gas (mobile phase) supply: N2, He, H2
Flow control
Injector
Column
Detector
Computer/recorder
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20. Carrier gas supply
Function: to provide carrier gas to chromatographic column
Carrier gas carries sample to column.
Tank, needle valve, flow meter, pressure gauge
Type of carrier gases → depends on column & detector
Capillary columns: H2, He.
Packed columns: N2
TCD, ECD: N2
FID: He
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21. Carrier gas supply
Ideal carrier gases: pure & dry
Impure & moisture: harm the column, ↓performance of
detectors, adversely affect quantification of trace analysis.
Measures:
Tubing (gas source→GC)→uncontaminated.
Molecular sieve beds → ↓moisture, hydrocarbon, oxygen
content.
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22. Requirements of a carrier gas
Inertness
Suitable for the detector
High purity
Easily available
Cheap
Should not cause the risk of fire
Should give best column performance
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23. Flow control
Regulates the carrier gas flow in GC
Constant flow of carrier gas → column efficiency
& reproducible elution time.
Magnitude of carrier gas flow rate depends →
type of column
Packed column – 10-60ml/min
Capillary column – 1-2ml/min
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24. Injection port
The injection port consists of a septum through
which a syringe needle is inserted to inject the
sample.
The sample is injected into a stream of inert gas
usually at an elevated temperature by a
microsyringe.
The vapourized sample is carried into a
column packed with the stationary phase.
To ensure rapid & complete solute
volatilization temp of injector → 30-50 degree
celsius>column temp
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27. Split Injection Advantages &
Disadvantages
Advantages
1.Simple to Use
2.Rugged Design
3.Narrow analyte band
on column
4.Protects column from
involatile sample
components
5.Easy to Automate
Disadvantages
1.Not suitable for ultra-
trace analysis
2.Suffers from
Discrimination
3.Liner geometry
dictates injector
settings
4.Analytes susceptible
to thermal
degradation
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28. Splitless Injection Advantages &
Disadvantages
Advantages
1.Simple to Use
2.Rugged Design
3.Excellent for trace
analysis
4.Less Risk of Analyte
Discrimination than
Split Mode
5.Easy to Automate
Disadvantages
1.Need to carefully
optimise conditions
2.Risk of backflash
3.Analytes susceptible
to thermal
degradation
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31. Common problems of injection port
Backflash
Septum leak
Adsorption of components from sample onto septum
Septum heated→Decomposition products → leak into column
→ ghost peak (chromatogram)
Measures
Back flash: Septum purge, small injector volume, larger vol
injector liners.
Teflon coated low leak septum is used
Inner surface is purged continuously with carrier gas
Septum should be replaced → 100 injection31 Dr Anurag Yadav
32. Columns & its types
Packed column Capillary (open tubular)
column
1 - 4mm ID; 1 - 5 m length
Glass/stainless steel coil
Packed solid particles either
porous/non-porous coated
with thin (1 μm) film of
liquid
0.1 - 0.5 mm I.D. (ID); 10 -
150 m length
Thin fused-silica.
Inner wall coated with thin
(0.1-5 μm) film of liquid
(stationary phase)
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33. Capillary (open tubular) column
3 layers
1. Polyamide coating – strong water proof barrier
2. Thin fused-silica- minimize chemical reactivity, uniform
surface for stationary phase
3. Stationary phase
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34. Stationary phase
Polymer – inner surface of fused silica layer
Thickness, uniformity, chemical nature → influences the
separation of components in sample.
Mc stationary phase silicon polymer used → polysiloxane.
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35. GC Columns : Composition of
stationary phase
100% dimethyl polysiloxane: non polar; for drugs and amino
acid derivatives
Polyethylene glycol : Polar ; for acids, ketones and alcohols.
Disadvantages: high susceptibilty of structural damage by
oxygen at high temperatures.
GSC: PLOT: Polystyrene, aluminium oxide, molecular sieve
Separation: partition/adsorption
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36. Selection criteria for capillary column
Stationary phase – close to polarity of solutes.
Column diameter : small diamter(0.25mm)→ when sample
overloading is not a problem.
Film thickness: thin → high boiling point solutes (TG, steriods)
thick → low boiling point solutes
Column length: 30mts → most application
15mts → simple samples (<10 components)
60mts →complex samples
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37. Temperature control
Opertionally temp control → injector, column, detector →
thermostatted chamber
-Directly → heating of column, Injector & detector
-Column temp maintained at constant level →isothermal
operation
-Varied with function of time → temperature programmed
operation →mc in clinical application →solute separation
in a wide range boiling point → sharp & distinct
chromatographic peak in less time.37 Dr Anurag Yadav
39. Detection Systems
The detector is the device located at the end of the
column which provides a quantitative measurement
of the components of the mixture as they elute in
combination with the carrier gas.
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40. Types of Gas Chromatography
Detectors
Non-selective
Responds to all compounds present in carrier
gas stream except the carrier gas itself
Selective
Responds to range of compounds with a
common physical or chemical characteristic
Specific
Responds to a single specific compound only
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41. Detectors can also be grouped into concentration or
mass flow detectors
Concentration Dependent
The response of such Gas Chromatography detectors is
proportional to the concentration of the solute in the
detector such as TCD. Dilution of sample with makeup
gas will lower detector response.
Mass Flow Dependent
Signal is dependent on the rate at which solute
molecules enter the detector such as FID. Response of41 Dr Anurag Yadav
42. Desirable characteristics of
detectors
Reproducible response to changes in eluent
composition in carrier gas stream
High sensitivity
Large linear dynamic range
Low noise
Small volume to avoid peak broadening and
resultant loss of resolution
Preferably non – destructive
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43. Types of Detectors in GC
To measure the separated analytes as they elute from the
column.
Universal unit → detect most analytes
Thermal conductance detector (TCD)
Mass spectrometer (MS)Selective detectors → detect
specific substances
Flame Ionization Detector (FID) → hydrocarbon
Electron capture detector (ECD) → electronegative groups
Commonly used ones are43 Dr Anurag Yadav
44. Flame Ionization Detector (FID)
Mc detector used for clinical analysis
Compounds that produce ions when burned in an H2-air
flame → organic cation → releases electron → detected
by collector electrode → generation of current.
Magnitude of current α mass of carbon material
delivered to detector → used for detection &
quantification of eluting solutes.
Advantages → simple, reliable, sensitive,linearity
excellent.
Dis –Advantage – destroy all the sample.
uses → detects hydrocarbon including fattyacids.
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45. Thermal conductance detector (TCD)
Universal detector → most of the analytes
Difference in thermal conductivity between the carrier gas
and sample gas causes a voltage output
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46. Electron capture detector (ECD)
Selective type of detector – electronegative groups- halogens (F, Cl,Br,
I), peroxides, quinones, & nitro groups
Principle – reaction b/n electronegative groups & thermal electrons
(radioactive source) →Thermal electrons captured on the electrode →
If electron capturing compound is present the number of thermal
electrons on the electrode (standing current) is decreased.
ECD Advantages
Highly sensitive
Easy to use
reliable
Selective
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49. GC-MS
Eluted solutes introduced into a ion source of a
MS, blasted with electrons, which cause them to
turn into positively charged molecular ions and
fragmented ions (ion source).
When these charged particles passed through filter
→ separated according to m/e ratio → ions
collected.
TIC the current generated by all such ions from
analytes is measured, which would be proportional
to the concentration of analyte.
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51. Computer
Regulates mobile phase composition, flow rate, column-
detector temp.
Electronic signals generated by detectors are recorded in the
form of chromatograghic peak at varied function of time
Area, height, retention time,base width of chromatograghic
peak is measured to compute analyte concentration of each
peak.
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53. Asymmetry Factor
Chromatographic peak should be symmetrical about
its centre
If peak is not symmetrical- shows Fronting or Tailing
FRONTING
Due to saturation of S.P & can be avoided by using
less quantity of sample
TAILING
Due to more active adsorption sites & can be
eliminated by support pretreatment,
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54. Practical consideration
Sample extraction – ex: barbiturates
Sample derivatization-
- Clinically relevent compounds are nonvolitile –difficult to
separate, so chemical modification or derivatization is necessary
Chemical reaction – nonpolar –methylation, silylation,
esterification.
Derivatization enhances the specificity & sensitivity of particular
separation.
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55. Applications of GC
Separation & identificaton of lipids, carbohydrates & proteins.
Separation & identificaton of aminoacids in urine by GC-MS for
diagnostic purpose.
Measurement of drugs & other metabolites in biological fluids.
Used for toxiclogical analysis of biological fluid by using ECD
detectors in GC.
Analysis of pesticides in soil, water, food.
Forensic analysis of blood and urine alcohol levels by using PEG-SP IN
GC
GC can be used to identify nitro-compounds in trace quantities.
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57. ADVANTAGES OF G.C
Very high resolution power, complex mixtures
can be resolved into its components by this
method.
Very high sensitivity with TCD, detect down to
100 ppm
It is a micro method, small sample size is
required
Fast analysis is possible, gas as moving phase-
rapid equilibrium57 Dr Anurag Yadav
58. References
Tietz –clinical chemistry text book
Kaplan-technique text book
Keith wilson-technique text book
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61. Split Injection Mechanisms
1.Sample syringe pierces septum
which seals around needle
2.Sample rapidly introduced into
heated inlet
3.Liquid sample volatilises and the
gaseous ‘plasma’ is contained
within a quartz glass liner
4.The sample gas is swept by the
carrier gas through the liner and
EITHER into the GC Column OR
between the liner and inlet body
and down the Split Line
5.% of sample reaching the
column depends upon the relative
flow rates in the column and split
flow line61 Dr Anurag Yadav
62. Split Injection Set-Up Summary
1.Used as the Default Vaporising Injector
2.Primarily used for non-trace analysis of volatile
samples
3.Need to consider gas flows (particularly split flow)
carefully / Don’t forget septum purge flow!
4.Increasing split flow:
a.Improves peak shape
b.Lowers column loading
c.Lowers analytical sensitivity
d.Decreases analyte inlet residence time –therefore
reduces the opportunity for thermal degradation
5.Need to consider Discrimination effects
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64. Splitless Injection Mechanism
1.Same principle as Split Injection
2.DIFFERENCES INCLUDE
3.Initial injector state is SPLITLESS i.e. The
split line flow is turned off
4.All sample reaches the column
5.Sample vapours trapped onto head of
column (solvent and thermal effects)
6.Column temperature programmed to initiate
elution
7.At some point after analyte transfer to the
column the split line is turned on to empty
the injector
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