M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar
2 types1. GSC 2. GLCGSC is not widely used b/cos limited
no of stationary phases available.Adsorption is the principleGSC is used only in case where there
is less solubility of solutes in stationary phase, which are rare.
GLC only
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.
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.
Carrier gasFlow regulators and flow meters Injection devicesColumnsTemparature control devicesDetectorsRecorders and integrators
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
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
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:
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
Glass / stainless steelStainless steel columns long life &
can be easily handled without the fear of fragility
But some samples react with themHence in such cases, glass columns
are used Eg: steroidsGlass columns are inert but highly
fragile and are difficult to handle
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
Stationary phaseStationary phase NatureNature TempTempPolydimethyl siloxanePolydimethyl siloxane Non-polarNon-polar -60-6000-320-32000CC
Poly(diphenyl) dimethyl siloxanePoly(diphenyl) dimethyl siloxane Non-polar bonded Non-polar bonded phasephase
-60-6000-320-32000CC
Polycyano propyl phenyl dimethyl siloxanePolycyano propyl phenyl dimethyl siloxane Intermediate polarityIntermediate polarity Upto 280CUpto 280C
Polyalkylene glycolPolyalkylene glycol PolarPolar 30-22030-220
Polyethylene glycolPolyethylene glycol PolarPolar 50-28050-280
PEG modified with Nitroterephthalic acidPEG modified with Nitroterephthalic acid Polar bonded phasePolar bonded phase 60-20060-200
Poly bis cyano propyl siloxanePoly bis cyano propyl siloxane Very polar non-Very polar non-bonded phasebonded phase
Upto 250Upto 250
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
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
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.
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
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
Katharometer / Thermal Conductivity Detector (TCD)
Flame Ionization Detector (FID)Argon Ionization Detector (AID)Electron Capture Detector (ECD)Nitrogen Phosphorous Detector
(NPD)
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.
The thermal conductivities of some carrier gases: H2=32.7 ; He=33.9 ; N2=5.2; CH4=6.5; C6H12=3.0 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
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
Extremely sensitive & background noise is low
µg quantities can be detectedStable & insensitive to small changes
in the flow rate of carrier gas & water vapour
Responds to most of the org compounds
Linearity is excellent
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 α- or β- particles
α- particles can be obtained from radium-D β- 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
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
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
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
DetectorDetector KatharoKatharometer/ meter/ TCDTCD
FIDFID AIDAID ECDECD
Min. Min. detectable detectable conc %v/vconc %v/v
1010-6-6 1010-11-11 1010-11-11 1010-12-12
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