M.Prasad NaiduMSc Medical Biochemistry,

Ph.D,.

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

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

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 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

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