gas chromatography

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Chromatography BY VENKATA NAVEEN KASAGANA & SWATHI SREE KARUMURI {M-PHARM -PHARMACEUTICS} S.B. COLLEGE OF PHARMACY SIVAKASI TAMIL NADU INDIA E- MAIL:naveen.kasagana@gmail.com

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A good of sophisticated chromatographic techniques of separation have been put forward:

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Gas Chromatography

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( a ) Gas-Solid Chromatography (GSC), ( b ) Gas-Liquid Chromatography, (GLC), Gas chromatography fundamentally is a separation technique that not only essentially provides identification of a compound but also caters for quantitative estimation after due calibration. Gas chromatography makes use, as the stationary phase , a glass or metal column filled either with a powdered adsorbent or a non-volatile liquid coated on a non-adsorbent powder. The mobile-phase consists of an inert-gas loaded with the vapourised mixture of solutes flowing through the stationary phase at a suitable temperature.

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In the course of the passage of the vapour of the sample through the column, separation of the components of the sample occurs in two ways, namely : due to adsorption effects- i.e., when the prepared column consists of particles of adsorbent only, and ( b ) due to partition effects- i.e., when the particles of adsorbent are coated with a liquid that forms a stationary phase.

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There are, in fact, three theories that have gained virtually wide recognition and acceptance in describing a gas chromatographic separation, namely : Plate theory, ( b ) Rate theory, ( c ) Random walk theory.

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GLC has a much greater application in the field of pharmaceutical Analysis The principal advantages of GC are enumerated below, namely : • It has high frequency of separation and even complex mixtures may be adequately resolved into constituents . • It has a very high degree of sensitivity in detection of components i.e., only a few mg of sample is enough for complete analysis. • Speed of analysis is quite rapid. Gives reasonably good accuracy and precision . The technique is fairly suitable for routine analysis because its operation and related calculations do not require highly skilled personnel , and The overall cost of equipment is comparatively low and its life is generally long.

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INSTRUMENTATION A gas chromatograph essentially comprises of six vital components, namely : ( a ) Carrier Gas Regulator and Flow Meter, ( b ) Sample Injection System, ( c ) Separation Column, ( d ) Thermal Compartment, ( e ) Detectors, ( f ) Recording of Signal Current, and ( g ) Integrator.

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CARRIER GAS PRESSURE REGULATOR AND FLOW METER The various carrier gas used in GC along with their characteristic features are stated below : H 2 : It has a distinctly better thermal conductivity and lower density. Demerits are its reactivity with unsaturated compounds and hazardous explosive nature . He : It has an excellent thermal conductivity, low density, inertness and it permits greater flow rates. It is highly expensive. N 2 : It offers reduced sensitivity and is inexpensive, and Air : It is employed only when the atmospheric O 2 is beneficial to the detector separation.

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SAMPLE INJECTION SYSTEM The sample injection system is very important and critical because GC makes use of very small amounts of the samples. A good and ideal sample injection system should be the one where the sample must not — ( i ) be decomposed at the point of injection, ( ii ) create pressure surges, and ( iii ) undergo fractionation, condensation or adsorption of components during the course of transfer to the column.

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( a ) Liquid Samples : They are usually injected by hypodermic syringes through a self-sealing silicon-rubber septum into a preheated-metal-block flash evaporator. The sample is vapourised as a ‘ plug ’ and carried right into the column by the respective carrier gas. ( b ) Solid Samples : These are either dissolved in volatile liquids (solvents) or temporarily liquefied by exposure to infra-red heat. ( c ) Gas Samples : They are best handled and injected by gas-light syrings or a gas-sampling valve, usually termed as a stream-splitter .

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SEPARATION COLUMN It is also known as the ‘ chromatographic column ’ . In reality the heart of a GC is the column duly packed or capillary in which the separation of constituents is materialized. The packed-column is usually a tubing having an internal diameter of 4.0 mm and made up of stainless-steel, copper, cupronickel or glass either bent in U-shape or coiled. Its length varies from 120 cm to 150 M .

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There are two general types of column, packed and capillary . Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth ) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types: Wall-coated open tubular (WCOT) Support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns.

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THERMAL COMPARTMENT A precise control of the column temperature is must. Whether it is intended to maintain an invariant-temperature or to provide a programmed-temperature. Importantly, the temperature of the column oven must be controlled by a system that is sensitive enough to changes of 0.01°C and that maintains an accurate control to 0.1°C. In normal practice, an air-bath chamber surrounds the column and air is circulated by a blower through the thermal compartment. More recently, programmes are also available that features both in linear and non-linear temperature

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Detectors A number of detectors are used in gas chromatography. The most common are flame ionization detector (FID) and the thermal conductivity detector (TCD) . Both are sensitive to a wide range of components, and both work over a wide range of concentrations. TCD is non-destructive, it can be operated in-series before an FID (destructive)

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Other detectors are sensitive only to specific types of substances, or work well only in narrower ranges of concentrations. They include: discharge ionization detector (DID), which uses a high-voltage electric discharge to produce ions. electron capture detector (ECD), which uses a radioactive Beta particle (electron) source to measure the degree of electron capture. flame photometric detector (FPD) flame ionization detector (FID) Hall electrolytic conductivity detector (ElCD) helium ionization detector (HID) Nitrogen Phosphorus Detector (NPD) Infrared Detector (IRD) mass selective detector (MSD) photo-ionization detector (PID) pulsed discharge ionization detector (PDD) thermal energy(conductivity) analyzer/detector (TEA/TCD)

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Thermal Conductivity

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IONIZATION DETECTOR The general class of ‘ ionization detectors ’ comprise of the following important detectors, namely : • Flame ionization detector, • Electron capture detector, • Thermionic detector, and • Photo ionization detector. conduction of electricity

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GC detector that is also very expensive but very powerful is a scaled down version of the mass spectrometer. When coupled to a GC the detection system itself is often referred to as the mass selective detector or more simply the mass detector. This powerful analytical technique belongs to the class of hyphenated analytical instrumentation (since each part had a different beginning and can exist independently) and is called gas chromatography/mass spectrometry (GC/MS). GC-MS

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What kind of info can mass spec gives you? Molecular weight Elemental composition (low MW with high resolution instrument) Structural info (hard ionization or CID) Gas-phase ions are separated according to mass/charge ratio and sequentially detected

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GC-MS To mass analyzer filament 70 eV e- anode repeller Acceleration slits GC column

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M + e- M +* f 1 f 2 f 3 f 4

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The high cost for the pump, ionization source, mass filter or separator, ion detector, and computer instrumentation and software has limited the wide application of this system as compared to the less expensive GC detectors

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RECORDING OF SIGNAL CURRENT In general, the signal from a gas chromatograph is recorded continuously as a function of time by means of a potentiometric device. INTEGRATOR An ‘ intergrator ’ may be regarded as a device that essentially facilitates simultaneous measurement of areas under the chromatographic peaks in the chromatogram either by mechanical or electronic means. GC-COMPUTER SYSTEM Nowadays, a large number of data-processing-computer-aided instruments for the automatic calculation of various peak parameters, for instance : relative retention, composition, peak areas etc., can be conveniently coupled with GC-systems.

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GC - DERIVATIZATION

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GC is best for separation of volatile compounds which are thermally stable. Not always applicable for compounds of high molecular weight or containing polar functional groups. These groups are difficult to analyze by GC either because they are not sufficiently volatile, tail badly, are too strongly attracted to the stationary phase, thermally unstable or even decomposed. Chemical derivatization prior to analysis is generally done to : Increase the volatility and decrease the polarity of compounds; Reduce thermal degradation of samples by increasing their thermal stability; Improve separation and reduce tailing Increase detector response by incorporating functional groups which lead to higher detector signals, Derivatizing Reagents & Common derivatization methods can be classified into 4 groups depending on the type of reaction applied: Silylation Acylation Alkylation Esterification

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SILYLATION Silylation produces silyl derivatives which are more volatile and more thermo stable. It replaces hydrogen's with a trimethylsilyl group {TMS}. Silylation reagents will react with the water and alcohols first. Care must be taken to ensure that both sample and the solvents are in dry state. Pyridine is the most commonly used solvent. The ease of reactivity of the functional groups towards silylation follows the order: Alcohol>Phenol>Carboxyl>Amine>Amide>Hydroxyl groups. Ex: trimethylchlorosilane {TMCS} trimethylsilylimidazole {TMSI}

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ACYLATION Acylation reduces the polarity of amino , hydroxyl and thiol groups and adds halogenated functionalities. Acylating reagents targets functional groups such as carbohydrates and amino acids. Acyl derivatives are formed with acyl anhydrides acyl halides and acyl amide reagents. It increases the volatility and sensitivity of the compound. Acylation converts active hydrogen's into esters,thioesters and amides. Ex: trifluoroacetoic anhydride, pentafluoropropionic anhydride

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ALKYLATION Replaces active hydrogens with an alkyl group. The principle reaction employed for preparation of these derivatives is nucleophilic displacement. Used to modify carboxylic acids and phenols. Can be used alone to form esters,ethers & amides or used in conjunction with acylation or silylation. Ex: dialkylacetals , tetrabutylammonium hydroxide

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APPLICATIONS OF GLC IN PHARMACEUTICAL ANALYSIS Gas liquid chromatography (GLC) or gas chromatography (GC) finds its abundant applications in the accurate and precise analysis of plethora of official pharmaceutical substances covering a wide range as enumerated below : ( i ) Assay of Drugs, ( ii ) Determination of specific organic compounds as impurities in official pharmaceutical substance, ( iii ) Determination of related substances in official drugs, ( iv ) Determination of water in drug.

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REFERENCES: http://www.chem.harvard.edu/mass/tutorials/magnetmovie.html http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/chrom2.htm http://www.wfu.edu/chemistry/courses/index.html#223 http://www.chem.vt.edu/chem-ed/ms/ms-intro.html http://en.wikipedia.org/wiki/GC-MS

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THANK YOU