GAS – LIQUID GAS – LIQUID CHROMATOGRAPHYCHROMATOGRAPHY

BYBYM. GLORY HEPSIBAHM. GLORY HEPSIBAHM.PHARM (PH. ANALYSIS)M.PHARM (PH. ANALYSIS)BHARAT COLLEGE OF PHARMACYBHARAT COLLEGE OF PHARMACY

Contents Contents 1)1) Introduction Introduction 2)2) HistoryHistory3)3) Principle Principle 4)4) Advantages of GLCAdvantages of GLC5)5) Physical Components Physical Components 6)6) Instrumentation of GLCInstrumentation of GLC7)7) Method to Carry out GLCMethod to Carry out GLC8)8) Derivatisation of Sample Derivatisation of Sample 9)9) Different Parameters involved in GLCDifferent Parameters involved in GLC10)10) Applications of GLCApplications of GLC11)11) Conclusion Conclusion

INTRODUCTION :INTRODUCTION :

GLC is one of the newest & largely developed GLC is one of the newest & largely developed technique between a gaseous mobile phase & a Liquid technique between a gaseous mobile phase & a Liquid phase immobilized on the surface of an inert solid.phase immobilized on the surface of an inert solid. In all forms mobile phase is liquid but in GLC, In all forms mobile phase is liquid but in GLC, mobile phase is gas such as He and Stationary phase mobile phase is gas such as He and Stationary phase is high Boiling point liquid absorbed onto a solid.is high Boiling point liquid absorbed onto a solid.

HistoryHistory 1903 - Mikhail semenovich Tswelt1903 - Mikhail semenovich Tswelt 1941 – Martin – Liquid – liquid chromatogram1941 – Martin – Liquid – liquid chromatogram 1944 – Martin – Paper chromatography 1944 – Martin – Paper chromatography 1947 – Fritz Prior – gas – solid chromatography1947 – Fritz Prior – gas – solid chromatography 1950 - Laid the foundation for GC and Later 1950 - Laid the foundation for GC and Later

developed GLC.developed GLC.

Principle:Principle:

The Components of vapourized sample are The Components of vapourized sample are fractionated as a consequence of being partitioned fractionated as a consequence of being partitioned between a gaseous mobile phase & a liquid between a gaseous mobile phase & a liquid stationary phase which is non – volatile liquid.stationary phase which is non – volatile liquid.

Retention Volume – Retention time X Flow rate.Retention Volume – Retention time X Flow rate.

Advantages of GLCAdvantages of GLC

Strong separating power Strong separating power Sensitivity is quite high Sensitivity is quite high Gives good precision & accuracy Gives good precision & accuracy Speed of analysis is fact Speed of analysis is fact Cost of low & life is longCost of low & life is long

Physical Components Physical Components

Auto SampleAuto Sample Inlets Inlets

Auto samples:Auto samples:

Automatic insertion provides better reproducibility Automatic insertion provides better reproducibility and time optimization and time optimization

Classified as : Classified as :

a) Liquida) Liquid

b) Static – head Space by syringe b) Static – head Space by syringe

technology technology

c) Dynamic – head space by transfer – c) Dynamic – head space by transfer –

line technologyline technology

d) SPMEd) SPME

Gas Chromatograph with a head Space samplerGas Chromatograph with a head Space sampler

Inlets :Inlets :

Column Inlet provides the means to introduce a Column Inlet provides the means to introduce a sample into a continuous flow of carrier gas. sample into a continuous flow of carrier gas. Types of inlets are Types of inlets are

a)a) S/SL (Split/ Split less) injectors S/SL (Split/ Split less) injectors

b)b) On column inletOn column inlet

c)c) PVT injectors PVT injectors

d)d) Gas source inlet or Gas switching Valve Gas source inlet or Gas switching Valve

e)e) P/T (Purge & Trap) SystemP/T (Purge & Trap) System

f)f) SPME (Solid Phase micro- extraction)SPME (Solid Phase micro- extraction)

Instrumentation of GLCInstrumentation of GLC

1)1) Carrier gas supply Carrier gas supply

2)2) Flow regulators & Flow metersFlow regulators & Flow meters

3)3) Injection devices Injection devices

4)4) Columns Columns

5)5) Temperature control devicesTemperature control devices

6)6) Detectors Detectors

7)7) Recorder & Integrators Recorder & Integrators

Schematic Diagram of GLCSchematic Diagram of GLC

Carrier GasCarrier Gas

Choice of Carrier Gas depends on, their efficiency Choice of Carrier Gas depends on, their efficiency of chromatographic separation of chromatographic separation

different gases used are different gases used are

HH22, He, N, He, N22 & Ar& Ar

As carrier gas is compressible, gases are stored As carrier gas is compressible, gases are stored under high pressure in cylindersunder high pressure in cylinders

Requirements of Carrier GasRequirements of Carrier Gas

InertnessInertnessSuitable to the detectors used Suitable to the detectors used High Purity High Purity Easily available & less risk of explosion Easily available & less risk of explosion CheapCheapShould give best column performance consistent Should give best column performance consistent with the required speed of analysis with the required speed of analysis

Flow regulators & flow meters Flow regulators & flow meters

Flow regulators – to deliver the gas with uniform Flow regulators – to deliver the gas with uniform pressure or flow rate.pressure or flow rate. Flow meters – to measure flow rate of carrier gas.Flow meters – to measure flow rate of carrier gas.

Two types of flow meters are Two types of flow meters are

a) Rota metera) Rota meter

b) Soap bubble meterb) Soap bubble meter

Soap bubble meterSoap bubble meterRota meter Rota meter

Sample injection devices Sample injection devices

Sample injectors used are depends upon their nature Sample injectors used are depends upon their nature i.e., Solid, Liquid & gases. i.e., Solid, Liquid & gases.

Solid : dissolved in a suitable solvent & then injected Solid : dissolved in a suitable solvent & then injected through a septum.through a septum.

Liquids – injected through loop or septum devicesLiquids – injected through loop or septum devices

Gases – injected through valve devices.Gases – injected through valve devices.

Columns:Columns:

Columns are generally made up of glass or stainless Columns are generally made up of glass or stainless steel.steel.

Classified as :Classified as : Based on its useBased on its use

a)a) Analytical column Analytical column

b)b) b) Preparative columnb) Preparative column Based on its nature:Based on its nature:

a)a) Packed columnsPacked columns

b)b) Open tubular or capillary or Golay columnsOpen tubular or capillary or Golay columns

c)c) Scot (support coated open tubular columns)Scot (support coated open tubular columns)

A gas chromatography oven, open to show a capillary column

Temperature Control DevicesTemperature Control Devices Pre Pre – – HeatersHeaters Thermo – statically controlled Thermo – statically controlled

devicesdevices

Two types of OperationsTwo types of Operations : :

Isothermal ProgrammingIsothermal ProgrammingLinear ProgrammingLinear Programming

Detectors :Detectors :

Heart of an apparatus Heart of an apparatus Detect the difference between a pure carrier gas and an Detect the difference between a pure carrier gas and an

eluted component.eluted component.

Different detectors used are :Different detectors used are :

1)1) Kathorometer or thermal conductivity detector (TCD)Kathorometer or thermal conductivity detector (TCD)

2)2) Flame Ionization Detector Flame Ionization Detector

3)3) Argon Ionization detectorArgon Ionization detector

4)4) Electron capture detector Electron capture detector

Thermal Conductivity Detector (TCD) or Thermal Conductivity Detector (TCD) or KathorometerKathorometer

Thermal Conductivity of some gases are :

Gases: H2 He N2 Methane Hexane

T.C’s : 32.7 33.9 5.2 6.5 3.0

Thermal Conductivity Detector (TCD) or Kathorometer

Flame ionization detectorFlame ionization detector

The whole detectors is enclosed in its own oven which is hotter thatn the column The whole detectors is enclosed in its own oven which is hotter thatn the column temperature that slops any thing condensing in the detectors temperature that slops any thing condensing in the detectors

Argon Ionization DetectorsArgon Ionization Detectors

Argon irradiation Argon + e` collision met stable state of Ar

↑ In current Detector ionization

Electron Electron CaptureCapture Detector Detector

It is highly sensitiveIt is highly sensitive It can even detect nanogram quantities It can even detect nanogram quantities

Recorders & Integrators :Recorders & Integrators :

Recorder Recorder : : To Record responses obtained after To Record responses obtained after amplification from detectors.amplification from detectors.

Integrators Integrators : : Improved version of recorders with some Improved version of recorders with some data processing capabilities. data processing capabilities.

Method of carrying out GLCMethod of carrying out GLC

Injection of sampleInjection of sample How the column worksHow the column works Packing material Packing material Column temperatureColumn temperature How separation works on the columnHow separation works on the column

Derivatisation:Derivatisation:

Treatment of Sample to improve the process of Treatment of Sample to improve the process of separation by column or detection by detectors.separation by column or detection by detectors.It is of two types :It is of two types :1)1) Pre – column derivatisationPre – column derivatisation2)2) Post – Column derivatisation Post – Column derivatisation Pre treatment of solid support is also required.Pre treatment of solid support is also required.

Different parameters in GLCDifferent parameters in GLC

1)1) Retention timeRetention time

2)2) Retention volumeRetention volume

3)3) Separation FactorSeparation Factor

4)4) Resolution Resolution

5)5) Plate theoryPlate theory

6)6) HETPHETP

7)7) Fronting Fronting

Retention time (RRetention time (Rtt))

It is the difference in time between the point of It is the difference in time between the point of injection and appearance of peak maximainjection and appearance of peak maxima

Retention Volume (VRetention Volume (Vll)) It is the volume of carrier gas required to elute 50% It is the volume of carrier gas required to elute 50% of the component from the column Vof the component from the column V tt = R = Rtt x Flow x Flow rate.rate.

Separation factors :Separation factors :

It is ratio of partition coefficient of the two It is ratio of partition coefficient of the two components to be separated components to be separated

S = Kb/Ka = (Tb – To) / (Ta – To)S = Kb/Ka = (Tb – To) / (Ta – To)

Where, Where,

To = Retention time of unretained substance To = Retention time of unretained substance

Ka,Kb = Partition coefficients of b & aKa,Kb = Partition coefficients of b & a

Ta, Tb = Retention times of b & aTa, Tb = Retention times of b & a

Resolution Resolution

Resolution is a measure of extent of separation ofResolution is a measure of extent of separation of

two components & the Base line separation achieved.two components & the Base line separation achieved.

RRss = 2(Rt = 2(Rt11- Rt- Rt22)/W)/W11+W+W22

Plate Theory : Plate Theory : (functional unit of (functional unit of column)column)

It is an imaginary or hypothetical unit of a It is an imaginary or hypothetical unit of a

column where distribution of solute between column where distribution of solute between stationary phase & mobile phase hasstationary phase & mobile phase has

attained equilibriumattained equilibrium. .

HETPHETP (Height Equivalent to a Theoritical Plate) (Height Equivalent to a Theoritical Plate) If HETP is Less, column efficiency is more.If HETP is Less, column efficiency is more.

HETP = Length of the Column/ No of theoretical PlatesHETP = Length of the Column/ No of theoretical Plates According to Van = Deemter equation According to Van = Deemter equation HETP = A+B/U + CuHETP = A+B/U + CuA = Eddy DiffusionA = Eddy DiffusionB = Longitudinal diffusion B = Longitudinal diffusion C = Effect of Mass transferC = Effect of Mass transferU = flow rate U = flow rate Fronting : It is due to saturation of Stationary Phase & Fronting : It is due to saturation of Stationary Phase &

Can be avoided by using less quantity of sample.Can be avoided by using less quantity of sample.

ApplicationsApplications Qualitative analysis Qualitative analysis Checking the purity of the compound Checking the purity of the compound Quantitative Analysis Quantitative Analysis Detection of steroidal drugsDetection of steroidal drugs Monitoring Hazardous Pollutants Monitoring Hazardous Pollutants Analysis of Commercial Drug Preparations, drug Analysis of Commercial Drug Preparations, drug

samples, Blood urine, etc. samples, Blood urine, etc. Analysis of foodsAnalysis of foods Separation & identification of lipids, proteins Separation & identification of lipids, proteins

flavors, Carbohydrates, colorants etc. flavors, Carbohydrates, colorants etc.

Conclusion Conclusion Gas – Liquid Chromatography is basically a Gas – Liquid Chromatography is basically a

separation process in which the compound of a separation process in which the compound of a vaporized sample are separated fractionated as a vaporized sample are separated fractionated as a consequence of partition between a mobile gaseous consequence of partition between a mobile gaseous phase and stationary phase held in column.phase and stationary phase held in column.

Hence the components are separated according Hence the components are separated according to their partition coefficients. to their partition coefficients.