Gas chromatography

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GAS CHROMATOGRAPHY

• M.PRASAD NAIDU
• Msc Medical Biochemistry,
• Ph.D Research scholar

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INTRODUCTION

• 2 types
• 1. GSC 2. GLC
• GSC is not widely used b/cos limited no of
  stationary phases available.
• Adsorption is the principle
• GSC is used only in case where there is less
  solubility of solutes in stationary phase, which
  are rare.
• GLC only

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Principle

• Partition is the principle
• Stationary phase liquid which is coated on to a
  solid support
• Mobile phase Gas
• Components are separated according to their
  partition coefficients
• Partition coefficient is the ratio of solubility
  of a substance distributed between two immiscible
  liquids at a constant temp.

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Criteria for compounds to be analysed by GC

• 2 important criteria are
• 1. Volatility unless a compound is volatile, it
  cannot be mixed with mobile phase.
• 2. Thermostability
• All the compounds will not be in the form of
  vapour.(solids liquids)
• Hence to convert them to a vapour form, they have
  to be heated.
• At that temp the compounds have to be
  thermostable
• If they are not thermostable, the compounds
  cannot be analysed by GC, since they will be
  decomposed.

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Practical requirements

• Carrier gas
• Flow regulators and flow meters
• Injection devices
• Columns
• Temparature control devices
• Detectors
• Recorders and integrators

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Carrier gas

• The choice of carrier gas determines the
  efficiency of chromatographic separation
• Eg H2, He, N2, Ar
• H2 better thermal conductivity, low density
• Used in thermal conductivity detector / FID
• Demerits
• 1. it reacts with unsaturated compounds
• 2. inflammable
• He excellent thermal conductivity, but expensive
• Used in thermal conductivity detector.
• N2 inexpensive but has reduced sensitivity

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Requirements of a carrier gas

• Inertness
• Suitable to the detector used
• High purity
• Easily available
• Cheap
• Less risk of explosion or fire hazards
• Should give best performance
• Consistent with the required speed of analysis
• Compressible, gases are stored under high
  pressure in cylinders
• N2, He are the most commonly used

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Flow regulators flow meters

• As carrier gases are stored under high pressure,
  flow regulators are used to deliver the gas with
  uniform pressure or flow rate
• Flow meters? to measure the flow rate of carrier
  gas
• 1. Rotameter
• 2. Soad bubble meter

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Injection devices

• Gases can be introduced by valve devices
• Liquids can be injected through loop or septum
  devices
• Most GC instruments have a high quality rubber
  septum
• Solid samples are dissolved in a suitable solvent
  and injected through a septum

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Columns

• Glass / stainless steel
• Stainless steel columns ? long life can be
  easily handled without the fear of fragility
• But some samples react with them
• Hence in such cases, glass columns are used Eg
  steroids
• Glass columns are inert but highly fragile and
  are difficult to handle

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Types of columns

• A) depending on its use
• 1. analytical column 1-1.5m of length outer
  diameter of 3-6mm
• They are packed columns are made up of glass or
  stainless steel
• Demerit Only small quantity of sample can be
  loaded
• 2. Preparative columns larger large amount of
  sample can be loaded
• 3-6m of lengthy, outer diameter 6-9mm

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B) depending on its naturei. Packed column
Stationary phase Nature Temp
Polydimethyl siloxane Non-polar -600-3200C
Poly(diphenyl) dimethyl siloxane Non-polar bonded phase -600-3200C
Polycyano propyl phenyl dimethyl siloxane Intermediate polarity Upto 280C
Polyalkylene glycol Polar 30-220
Polyethylene glycol Polar 50-280
PEG modified with Nitroterephthalic acid Polar bonded phase 60-200
Poly bis cyano propyl siloxane Very polar non-bonded phase Upto 250
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ii) Open tubular columns / capillary columns /
Golay columns

• Long capillary tubing of 30-90m
• 0.025-0.075cm internal diameter
• Stainless steel coiled
• The inner wall is coated with the st.
• phase liquid (0.5-1µ thin film)
• these columns offer least resistance to flow of
  carrier gas
• More efficient than packed columns (
  offers more resistance)
• Demerit more sample cannot be loaded

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iii) SCOT columnssupport coated open tubular
columns

• An improved version of Golay or capillary columns
• A support material is deposited (1µ) on the inner
  wall then coated with a thin film of liquid
  phase
• Have a low resistance to flow of carrier gas
• Advantage more sample load

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Temparature control devices

• Preheaters converts the sample into its vapour
  form mix them with mobile phase or carrier gas
• Preheaters are present along with injecting
  devices
• Thermostatically controlled oven
• In GC partition is the principle
• Since partition coefficient as well as solubility
  of a solute depends upon temp, temp maintenance
  in a column is highly essential for efficient
  separation
• Hence column injecting devices should be
  maintained at a particular temp.

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Types of operations

• Isothermal programming same temp is maintained
  throughout the process of separation
• Linear programming in which the oven is heated
  linearly over a period of time
• This is required when a sample has a mixture of
  low bp high bp compounds
• Separation of complex mixtures

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Detectors

• Heart of the apparatus
• Requirements of an ideal detector
• Applicability to wide range of samples
• High sensitivity to even small conc
• Rapidity of response
• Linearity i.e., less response to low conc vice
  versa
• Response should be unaffected by temp, flow rate
  or characteristics of carrier gas
• Non destructive to the sample in case of
  preparative work
• Simple easy to maintain
• inexpensive

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Types of detectors

• Katharometer / Thermal Conductivity Detector
  (TCD)
• Flame Ionization Detector (FID)
• Argon Ionization Detector (AID)
• Electron Capture Detector (ECD)
• Nitrogen Phosphorous Detector (NPD)

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Katharometer / TCD

• Principle is based upon thermal conductivity
  difference b/n carrier gas that of component
• TCD has 2 platinum wires of uniform size which
  form part of Wheatstone bridge
• Through one of them, pure carrier gas always
  flows through the other the effluents of the
  column passes
• 2 Pt wires are heated electrically
• When pure carrier gas passes through both or
  them, there is no diff in temp or resistance
  hence baseline is recorded
• When a component emerges from the column, it
  alters the thermal conductivity resistance of
  the wire
• Hence this produces a diff in resistance
• So conductivity b/n wires, which is amplified
  recorded as a signal.

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TCD

• The thermal conductivities of some carrier gases
• H232.7 He33.9 N25.2 CH46.5 C6H123.0
• Advantages
• Applicable to most compounds
• Linearity is good
• Sample is not destroyed used in preparative
  scale
• Simple, easy to maintain inexpensive
• Disadvantages
• Low sensitivity
• Affected by fluctuations in temp flow rate
• Response is only relative not absolute
• Biological samples cannot be analysed

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FID

• Based upon the electrical conductivity of carrier
  gases
• At normal temp pressure, gases act as
  insulators, but become conductive if ions are
  present
• H2 is the carrier gas used in FID
• If the carrier gas is either N2/Ar, it can be
  mixed with H2
• Anode Ag gauze placed over the burner tip
• Cathode burner tip made up of Pt capillary
• When pure carrier gas alone passes, there is no
  ionization no current flows
• When a component emerges, no. of ions are
  produced b/cos of ionization by the thermal
  energy of the flame
• This causes a potential diff causes a flow of
  current which is amplified recorded as a signal

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Advantages of FID

• Extremely sensitive background noise is low
• µg quantities can be detected
• Stable insensitive to small changes in the flow
  rate of carrier gas water vapour
• Responds to most of the org compounds
• Linearity is excellent

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Argon Ionization Detector (AID)

• AID depends on the exitation of Ar atoms to a
  metastable state, by using radioactive energy.
• This is achieved by irradiating the carrier gas
  with either a- or ß- particles
• a- particles can be obtained from radium-D
• ß- particles can be obtained from Sr90/ H3
• These high E particles ionize the Ar atoms
  hence they are exited to metastable state
• These molecules collide with the effluent
  molecules and ionizes them
• These ions when reach the detector will cause an
  increase in current
• Thus the components are detected

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AID

• Advantages
• Responds to most of the org compounds
• Sensitivity is very high
• Disadvantages
• Response is not absolute it is relative
• Linearity is poor
• Sensitivity is affected by water is much
  reduced for halogenated compounds
• The response varies with the temp of the detector
  
• High temp like 2400C, voltages of 1000V or less
  are usually necessary

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Electron capture detector (ECD)

• ECD has 2 electrodes
• Column effluent passes b/n them
• One of the electrode is treated with a radio
  active isotope which emits electrons as it
  decays.
• These emitted electrons produce 2o electrons
  which are collected by the anode, when a PD of
  20V is applied b/n them
• When carrier gas alone flows through, all the 2o
  electrons are collected by the vely polarised
  electrode
• Hence a steady baseline is recorded
• Effluent molecules which have affinity for
  electrons, capture these e- when they pass
  through the electrodes
• Hence the amount of steady state current is
  reduced
• This diff is amplified recorded as output signal

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ECD

• The carrier gas used in this detector depends
  upon the e- affinity of the compounds analysed
• For compounds with high e- affinity, Ar is used
• For low e- affinity , N2, H2, He or CO2 can be
  used
• Advantages highly sensitive (10-9)
• Disadvantage ECD can be used only for compounds
  with e- affinity
• Halogenated compounds, pesticides etc can be
  detected by ECD

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Comparision of the sensitivity of detectors
Detector Katharometer/ TCD FID AID ECD
Min. detectable conc v/v 10-6 10-11 10-11 10-12
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Recorders integrators

• Recorders to record the responses
• They record the baseline all peaks obtained
  with respect to time
• Retension time for all the peaks can be found out
  from such recordings, but the area of individual
  peaks cannot be known
• Integrators improved version of recorders with
  some data processing capabilities
• Can record the individual peaks with Rt, height
  width of peaks, peak area, of area , etc
• Int provide more information on peaks than
  recorders

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THANK YOU