column chromatography mechanism of separation gas chromatography in detail

1. Presenter Dr Anurag Yadav
Morderator Dr Avinash S S
Column Chromatography
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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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3. Contents
Gas Chromatography:
 Definition
 Types of Gas chromatography
 Basic principle
 Instrumentation
 Practical consideration
 Applications
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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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5. 5 Dr Anurag Yadav

6. Column
chromatography
GC LC
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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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9. Basic principle
 GSC: adsorption
chromatography
 GLC : partition
chromatography
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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

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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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15. Separation
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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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18. Peaks and Data
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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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25. Injection techniques
 Split
 Splitless
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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

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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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52. Resolution
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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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60. 60 Dr Anurag Yadav

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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63. Split Injection Default /
Development
Conditions
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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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65. 65 Dr Anurag Yadav