Gas Liquid Chromatography (GLC)

Mobile phase is an inert gas. (eg. Nitrogen, Helium,

Hydrogen) Stationary Phase is a small amount of liquid held on a finely divided inert solid support. The inert solid is used to support the stationary liquid phase, and is packed into a steel coil kept in a thermos tatted oven to maintain the temperature.

GLC

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

septum. The injector is contained in an oven whose temperature can be controlled. It is hot enough so that all 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 can travel a distance before going into the detector.

GLC
The temperature of the column oven must be high

enough to vaporize the sample which is carried out by the inert gas. During the passage of the gas, components of the sample is distributed between the two phases. One of the three things may happen to a particular molecule molecule in the column:
Condense on the stationary phase.

Dissolve on the liquid on the surface of the stationary

phase. Remain on the gas phase

GLC
After leaving the column the carrier gas and the

sample is passed to another oven with a detector. The most common detector used is the flame ionization detector. The detector measures the retention time of the compounds in the sample.

GLC
Retention time is the time taken by a gas to travel from

the injection septum to the detector. Different compounds have different boiling points and hence have different velocity at which they reach the detector. Factors affecting retention time:
Boiling point of a compound

Solubility in liquid phase

Temperature of the column

GLC
The detector amplifies the signal and sends the

information into a chart recorder. The chart shows a retention time against time graph which is known as a chromatogram. Chart shows peaks which are compared to the already existing database of gases. Area under the peak is proportional to the amount of substance present in it. Gases will have the same retention time given that the conditions used in the analysis are same as the conditions used in the original database.

GLC

GLC
The amount of each component can be determined by

expressing it as a percentage of the sum of areas under all peaks. For example, for a mixture with three compounds A, B and C: % of A= (Peak area or height of A) x100 Sum of areas of A, B, C

GLC
Compuonds having similar retention time may have

overlapping 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 mass spectrometer.

GLC

GLC
Testing for steroids from urine sample of athletes.
Testing fuels in formula one race. Analyzing blood samples to determine percentage of

dissolved oxygen, nitrogen, carbon-dioxide and carbon monoxide.

HPLC
Uses partition to separate and identify the

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

HPLC Stationary phase consists of a long-chain

hydrocarbon liquid bonded onto a solid support which is packed tightly into a column.
The tiny solid particles in the column have a very large surface area over which partitioning can occur resulting in separation.

HPLC
The molecules coming off the column are detected by

an ultraviolet spectrophotometer. organic compounds absorb UV light of various wavelengths.

HPLC
The retention time depends upon: the pressure used (because that affects the flow rate of the solvent) the nature of the stationary phase (not only what material it is made of, but also particle size) the exact composition of the solvent the temperature of the column

HPLC

HPLC