Gas chromatography

Gas chromatography

Dr.Maryam kazemiPhD student of pharmaceuticsShiraz university of medical sciences

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Invention of ChromatographyMikhail TswettRussian Botanist(1872-1919) Mikhail Tswett invented chromatography in 1901 during his research on plant pigments. He used the technique to separate various plant pigments such as chlorophylls, xanthophylls and carotenoids. 3

http://en.wikipedia.org/wiki/Mikhail_TsvetThe method was described on 30 December 1901 at the XI Congress of Naturalists and Physicians (XI ) in St. Petersburg. The first printed description was in 1903, in the Proceedings of the Warsaw Society of Naturalists, biology section. He first used the term "chromatography" in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen botanischen Gesellschaft. In 1907 he demonstrated his chromatogaph for the German Botanical Society.

Tsvet's work was ignored for several decades because of diverse reasons: the tragic events in Russia at the beginning of the 20th century, the fact that Tsvet originally published only in Russian (what made his results inaccessible to western scientists) and an article denying Tsvet's findings. Willstater and Stoll tried to repeat Tsvet's experiments but because they used an aggressive adsorbent (what destroys the chlorophyll's) were not able to do so. They published their results and Tsvet's chromatography method went into oblivion. It was recollected 10 years after his death thanks to German scientist Edgar Lederer and Austrian biochemist Richard Kuhn and the work of Martin and Synge.

4The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950)

5GC-MS Quadri- TOF

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Sample to be separated is converted into vapor And mixed with gaseous M.PComponent more soluble in the S.P travels slowerComponent less soluble in the S.P travels fasterComponents are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by G.C 1.VOLATILITY: 2.THERMOSTABILITY: 7

The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.Principle8

GAS CHROMATOGRAPHYGSC principle is ADSORPTION

GLC principle is PARTITION

Gas - Solid Chromatography (GSC)The stationary phase, in this case, is a solid like silica or alumina. It is the affinity of solutes towards adsorption onto the stationary phase which determines, in part, the retention time. The mobile phase is, of course, a suitable carrier gas.

Most useful for the separation and analysis of gases like CH4, CO2, CO, ... etc.

The use of GSC in practice is considered marginal when compared to gas liquid chromatography. 8

Gas - Liquid Chromatography (GLC)The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas.

GLC is the most widely used technique for separation of volatile species.

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THEORETICAL PLATEAn imaginary unit of the column where equilibrium has been established between S.P & M.PIt can also be called as a functional unit of the column

HETP Height Equivalent to a Theoretical PlateEfficiency of a column is expressed by the number of theoretical plates in the column or HETPIf HETP is less, the column is efficient.If HETP is more, the column is efficient13

HETP= L (length of the column) N(no of theoretical plates)HETP is given by Van Deemter equation HETP= A + B +Cu uA = Eddy diffusion term or multiple path diffusion which arises due to packing of the columnB = Molecular diffusion, depends on flow rateC = Effect of mass transfer, depends on flow rate u = Flow rate14

Efficiency ( No. of Theoretical plates)It can be determined by using the formula n = 16 Rt2 w2n = no. of theoretical platesRt = retention timeW = peak width at baseThe no. of theoretical plates is high, the column is highly efficient

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PRACTICAL REQUIREMENTSCarrier gasFlow regulators & Flow metersInjection devicesColumnsTemperature control devicesDetectorsRecorders & Integrators19

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SyringeInjectorDetector

Carrier Gas CylinderColumnTo Waste or Flow Meter

Flow ControllerTwo-Stage Regulator

Requirements of a carrier gasInertnessSuitable for the detectorHigh purityEasily available CheapShould not cause the risk of fireShould give best column performance

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CARRIER GAS Hydrogen ( H2 ) better thermal conductivity Disadvantage: it reacts with unsaturated compounds & inflammable Helium ( He) excellent thermal conductivity it is expensive Nitrogen ( N2) reduced sensitivity it is inexpensive22

Flow regulators & Flow meters deliver the gas with uniform pressure/flow rate flow meters:- Rota meter & Soap bubble flow meter Rota meterplaced before column inlet it has a glass tube with a float held on to a spring. the level of the float is determined by the flow rate of carrier gas23

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Soap Bubble Meter Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate

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InjectorsSeptum type injectors are the most common.

These are composed of a glass tube where vaporization of the sample takes place.

The sample is introduced into the injector through a self-sealing silicone rubber septum.

The carrier gas flows through the injector carrying vaporized solutes.

The temperature of the injector should be adjusted so that flash vaporization of all solutes occurs. If the temperature of the injector is not high enough (at least 50 degrees above highest boiling component), band broadening will take place. 27

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Carrier GasSyringeVaporization ChamberTo Column

Septum

29Split-splitless Injector

Split-splitless injectors are used for introduction of highly concentrated samples into capillary columns. Sample is volatilized by injection into a heated glass liner. The carrier gas then either sweeps the total sample (Splitless mode) or a portion (Split mode) into the column. The split vent controls the amount of sample entering and the other portion is exhausted. This mode is useful for highly concentrated or dirty samples. It helps in producing narrow band widths. Split-less injection is useful for trace level analysis.

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Head space injectionSimple Definition'Headspace' is the gas space above the sample in a chromatography vial. Volatile sample components diffuse into the gas phase, forming the headspace gas. Headspace analysis is therefore the analysis of the components present in that gas.

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44Gas Chromatographic Columns and Stationary Phases

Packed Columns

These columns are fabricated from glass, stainless steel, copper, or other suitable tubes.

Stainless steel is the most common tubing used with internal diameters from 1-4 mm.

The column is packed with finely divided particles (