GAS CHROMATOGRAPHY

GAS CHROMATOGRAPHY • In gas chromatography (GC), the sample is injected onto the head of a chromatographic column and immediately vaporized. • The components of vaporized sample are fractionated as a consequence of partition between the mobile gaseous phase and the stationary phase held in the column. • The mobile phase is inert, it does not inter act with the sample, it only carries the sample at elevated temperature when it leaves the stationary phase thus. It is also called the carrier gas.

GC methods can either be: a- Gas solid chromatography GSC • The stationary phase is a solid and retention of substances occurs as a result of adsorption b-Gas liquid chromatography GLC • The stationary phase is a liquid supported on an inert solid matrix. • The components of the sample have finite solubility in the stationary phase thus they distribute themselves between this phase and the gas. • Elution is accomplished by forcing the inert carrier gas through the column. The rates of which the various components move along the column depends on their tendency to dissolve in the stationary phase.

Types of samples for GC: 1- Any compound that can be volatilized without decomposition, such as low molecular weight hydrocarbons, aldehydes, ketones and esters. 2- Samples that can be converted to volatile compounds, such as fatty acids which can be converted to methyl esters which is more volatile also amino acids can be converted to fluoroamide ester derivative. 3-The sample may be organic or inorganic, but not ionic. 4-The molecular weight ranges from 2 to 1000.

Advantage of GC • High sensitivity (10-9- 10-12 gm.) • High accuracy. • High speed (short time of analysis). Limitation of GC • The sample must be volatile and thermally stable below 4000C. • Dirty and biological samples such as blood, soil and tissues require clean up. • GC cannot identify the compounds surely ,it must be connected to another instrument such as mass-spectrometer for accurate identification of compounds.

Instrument for gas chromatography A gas chromatographic instrument is formed of FIVE components: • Carrier gas supply with its regulator, purifier, desiccant and flow meter. • Sample injection system. • Column and thermostating system. • Detector. • Integrator or computer data station.

1-Gas supply system: This includes: a-carrier gas. b-gases used for detector. • The most commonly used carrier gases are nitrogen, helium, hydrogen and argon. • They are supplied in cylinders or produced by electrical generators fitted with pressure regulator and manometer.

The carrier gas must be : • Pure (99.999% purity), N.B. • impure gases will cause damage of the stationary phase, noise and unidentified peaks or signals. • To ensure the purity of the gases, usually the gases are passed through filters to remove impurities then moisture filter and lastly oxygen filter since oxygen will destroy the stationary phase and it gives signal in case of electron capture detector. • Inert( will not interact with the sample).

Dry as traces of water at high temperature will hydrolyze the sample and the stationary phase. • The pressure of the gas is adjusted according to its viscosity and the length of the column, to obtain the suitable flow rate. The choice of the gas depends on the detector used: • For flame ionization detector, the carrier gas can be nitrogen ,helium, argon or hydrogen. The detector gas is air and hydrogen. • For electron capture detector the carrier gas is nitrogen and no other gases are needed for detector. • For thermal conductivity detector low viscosity, high thermal conductivity gas is needed as carrier gas and which passes through the detector these are helium and hydrogen.

Column and Thermostating System: • The fundamental part of a gas chromatograph is the thermostating oven. • It is the place where the chromatographic column is put and the sample is separated into its components. • The optimum column temperature depends upon the boiling point of the sample and the degree of separation required. • There are two types of columns :Packed and Capillary.

Packed columns: • They are fabricated from glass, metal or teflon and about two meter length and 0.5 cm diameter. • They are uniformly packed with packing material consists of finely divided, uniform spherical solid inert support coated uniformly with very thin layer of stationary liquid phase. • The carrier gas flow ranges from 30–50 ml/min.

Capillary or open tubular columns: • These are capillary tubes made of glass or stainless steel or fused silica. • The inner wall is coated with thin film of liquid stationary phase. • Since there is no resistance for the gas flow as the tube is open. The length of the tube can be from 15 - 100 meter. • The flow rate of carrier gas is reduced to 1 ml/min.

Stationary liquid phases: Desirable properties for immobilized, liquid phase in GLC include: 1- Low volatility; ideally the boiling point of the liquid should be at least 2000C higher than the maximum operating temperature for the column. 2- Thermally stable 3-Chemically inert 4-To have a reasonable retention time in the column, a species must show some degree of solubility with the stationary phase. • Generally, the polarity of the stationary should match that of the sample components. When the match is good, the order of elution is determined by the boiling point of the eluate.

Column temperature and temperature program • The column(s) in a GC are contained in an oven, the temperature of which is precisely controlled electronically. • The temperature of the column can be varied from about 50°C to 250°C. It is cooler than the injector oven, so that some components of the mixture may condense at the beginning of the column. • The rate at which a sample passes through the column is directly proportional to the temperature of the column.

A method which holds the column at the same temperature for the entire analysis is called "isothermal." Most methods, however, increase the column temperature during the analysis, the initial temperature, rate of temperature increase (the temperature "ramp"), and final temperature are called the "temperature program." • A temperature program allows analytes that elute early in the analysis to separate adequately, while shortening the time it takes for late-eluting analytes to pass through the column.

Detectors: • The detector has the function to detect the presence of chemical components in the gas flow. • All detectors measure a relative value; a sample component in the carrier gas compared to the pure carrier gas. • This change is usually represented in the form of an electrical signal as a function of time.

A good detector should comply with the following requirements: 1- Responds rapidly and reproducibly to low concentrations of solutes emerged from the column. 2- Sensitive to very low concentrations of sample, 10-9 to10-12 g of sample could be detected. 3-Accurate and reliable.

The following are some types of GC detectors: 1-Thermal Conductivity Detector TCD The detector has the advantage that it has no destructive effect on the sample.

2-Flame Ionization Detector FID: Most organic compounds, when pyrolyzed at the temperature of hydrogen/air flame, produce ionic intermediates that provide a mechanism by which electricity can be carried through the flame. The FID is highly sensitive, has wide range of linear response but it is destructive for the sample.

3-Electron Capture Detector ECD • A  -ray source (a radioactive substance that emit electrons) such as Ni63. • In the absence of the organic species, an amplifier electron current is formed which runs in the direction of collector electrode and is monitored as a continuous background current. • The moment there are electro-negative components present in the carrier gas, the background current is reduced because these compounds capture electrons. The change in the background current is registered and that is the detector signal. • ECD is a selective and highly sensitive detector for molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups.

QUALITATIVE ANALYSIS • Qualitative analysis by gas chromatography is divided into two parts. • The first is the separation of component or components of interest from each others in the mixture, and the second is the identification of the separated components. Retention Data • The retention volume, time or distance of a peak is a qualitative property of the compound and is constant for a given set of conditions (the same apparatus, temperature and stationary phase).

To identify a specific compound in the mixture, a reference compound and the unknown sample are co-chromatographed under identical conditions (spiking or enrichment technique). • The formation of one peak and the increase of its height indicate that the unknown may be identical with the reference compound, but it is not positive proof. • To confirm the identity of the unknown, other stationary phases of different polarity could be also used and chromatography at variable temperatures might be carried out.

QUANTITATIVE ANALYSIS Quantitative Analysis A-Peak Height Measurement: • The use of the peak height as the quantitative measurement is to be preferred to the peak area because of its simplicity. But this demands; • The conditions of chromatography be constant allover the operation and peak width does not vary during the set of determinations, • The response of the detector must be carefully calibrated over the concentration range of the components. This is done by allowing standard samples to be chromatographed periodically.

B-Peak Area Measurement: • In some cases the peak width is affected with certain factors, e.g. adsorption, overloading of sample, long retention volumes and variation in operating conditions like temperature. • All these factors cause broadening of the peak and measurement of the peak height becomes of little importance. • Therefore, it appears more accurate to determine the peak area. This can be carried out as follows:

By means of a planimeter or counting square numbers on group paper. • Weighting the cut-out peaks if paper thickness is uniform. • By means of some automatic integrating device. • By a geometric method or approximation such as multiplying the peak height by the width at half height. • The first three methods are useful in the determination of asymmetrical peaks.

Calibration: • The calibration involves the determination of chromatograms with pure components of samples. • A quantitative analysis through calibration can be achieved by plotting chromatographed increasing amounts of pure samples, against their peak areas. • The value of the unknown concentration can be deduced from the curve

Quantitative Determination of Atropine by Area Measurement

GAS CHROMATOGRAPHY