Gas Liquid Chromatography and High Performance Liquid Chromatography

8/13/2019 Gas Liquid Chromatography and High Performance Liquid Chromatography

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Gas Liquid Chromatography (GLC)Mobile phase is an inert gas. (eg. Nitrogen, Helium,Hydrogen)

Stationary Phase is a small amount of liquid held on afinely divided inert solid support.The inert solid is used to support the stationary liquidphase, and is packed into a steel coil kept in a thermos

tatted oven to maintain the temperature.


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GLC


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GLCThe sample should be volatile.Sample is injected into the coil through a rubber

septum.The injector is contained in an oven whosetemperature can be controlled. It is hot enough so thatall the sample boils and is carried into the column as a

gas.The column is of 2-3m and is coiled so that the gas cantravel a distance before going into the detector.


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GLCThe temperature of the column oven must be highenough to vaporize the sample which is carried out bythe inert gas.During the passage of the gas, components of thesample is distributed between the two phases.One of the three things may happen to a particularmolecule molecule in the column:

Condense on the stationary phase.Dissolve on the liquid on the surface of the stationaryphase.Remain on the gas phase


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GLC After leaving the column the carrier gas and thesample is passed to another oven with a detector.

The most common detector used is the flameionization detector.The detector measures the retention time of thecompounds in the sample.


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GLCRetention time is the time taken by a gas to travel fromthe injection septum to the detector.

Different compounds have different boiling points andhence have different velocity at which they reach thedetector.Factors affecting retention time:

Boiling point of a compoundSolubility in liquid phaseTemperature of the column


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GLCThe detector amplifies the signal and sends theinformation into a chart recorder.The chart shows a retention time against time graph whichis known as a chromatogram .Chart shows peaks which are compared to the alreadyexisting database of gases. Area under the peak is proportional to the amount ofsubstance present in it.Gases will have the same retention time given that theconditions used in the analysis are same as the conditionsused in the original database.


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GLC


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GLCThe amount of each component can be determined byexpressing it as a percentage of the sum of areas underall peaks.For example, for a mixture with three compounds A, Band C:

% of A= (Peak area or height of A)

Sum of areas of A, B, Cx100


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GLCCompuonds having similar retention time may haveoverlapping spikes.

Different solvents can be used to obtain different velocities for this condition.

Spike is not comparable to the ones in database.It may be a new compound and can be identified by

connecting the output of the GLC to the input of a massspectrometer.


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GLC


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GLCTesting for steroids from urine sample of athletes.Testing fuels in formula one race.

Analyzing blood samples to determine percentage ofdissolved oxygen, nitrogen, carbon-dioxide and carbonmonoxide.


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HPLCUses partition to separate and identify thecomponents in the mixture.

Mobile phase is a liquid.High pressure, about 20-200 atm, is used to pass liquidthrough the column as liquid is more viscous.The stationary phase is a non-volatile liquid.


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HPLCStationary phase consists of a long-chainhydrocarbon liquid bonded onto a solid support which is packed tightly into a column.

The tiny solid particles in thecolumn have a very large surfacearea over which partitioning canoccur resulting in separation.


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HPLCThe molecules coming off the column are detected byan ultraviolet spectrophotometer.

organic compounds absorb UV light of various wavelengths.


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HPLCThe retention time depends upon:the pressure used (because that affects the flow rate ofthe solvent)the nature of the stationary phase (not only whatmaterial it is made of, but also particle size)the exact composition of the solvent

the temperature of the column


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HPLC


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HPLC

Gas Liquid Chromatography and High Performance Liquid Chromatography

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