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Edit Comment Close Premium member Presentation Transcript Gas Liquid Chromatography: Gas Liquid Chromatography Srinivas Nandyala I/II M.Pharm Department of Pharmacology A.K.R.G College of Pharmacy.What is Chromatography?: What is Chromatography? A technique for separating the components of a mixture on the basis of difference in their affinity for a stationary phase and mobile phase.General classification of chromatographic methods: General classification of chromatographic methods Column chromatography Paper chromatography Thin layer chromatography Gas chromatography High pressure liquid chromatography Ion exchange chromatography Gel filtration chromatography Super critical fluid chromatographySlide 4: History Russian Scientist Mikhail Semenovich Tswett is credited for the discovery of chromatography. (1903) German student Fritz Prior is credited for developing gas chromatography. (1947)Slide 5: The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950)Today: Today Estimate : 2,00,000 gas chromatographs are currently used through out the world. More than 30 manufactures. 135 different models. Cost: Rs75,000-20,00,000. Improvements: computers, automatic control open tubular columns.What is Gas Chromatography?: What is Gas Chromatography? It is a technique where by the components of a mixture in the gaseous state are separated as the sample passes over a stationary liquid or solid phase and a gaseous mobile phase. A/C to stationary phase G.C classified into two types. G.S.C-Gas Solid Chromatography G.L.C-Gas Liquid ChromatographySlide 8: Gas Liquid Chromatography A technique used to separate and identify the components of chemical compounds using a gaseous mobile phase.Gas Liquid Chromatography : Gas Liquid Chromatography In GLC the components of vaporize samples are fractionated due to partition between a gaseous mobile phase and a liquid stationary phase held in column. The stationary phase is non volatile liquid held as a thin layer on a solid support like diatomaceous earth. Principle : It works based on the principle of partition.Instrumentation: Instrumentation Tank of carrier gas Flow regulator and flow meter Injection port Column Temperature controlled device Detector Microprocessor/recorderInstrumentation: InstrumentationSlide 13: 13 The Mobile Phase (Carrier Gas) An inert gas such as He or N 2 Function is to transport sample vapors through column No chemical interaction with sample Typical parameters Column inlet pressure: 10-50 psi (above ambient) Flow rate: 25-50 mL/min (packed column) Precise control of carrier gas flow rate is critical to obtaining reproducible retention times. A soap bubble flow meter Attach tubing to outlet of column or detectorSlide 14: 14 Sample Injection Sample is injected using a syringe into a flowing stream of hot mobile phase High temperature (at least 50 o C above boiling point of sample) causes vaporization of sample Introduces a narrow plug of sample vapor onto the column Various designs For packed columns, inject 1 to 5 L of sample For capillary columns, a split valve is used to introduce a small fraction of sample onto column A flash vaporization injectorColumns : Columns Column is heart of GC,which decides the separation efficiency. It is made up of glass or copper. Columns are two types based on it’s use: Analytical column: Length 1-2 mts, outer diameter 3-6 mm. Preparative column: Length 3-6 mts, outer diameter 6-9 mm.Columns are two types based on nature: : Columns are two types based on nature: Packed column: Capillary columns:(Golay column) Micro packed columns: Open tubular columns: wall coated open tubular (WCOT) column porous layer open tubular (PLOT) column Support coated open tubular (SCOT) columnsSlide 17: 17 Packed Columns Diatomaceous earth (diatoms) is a good inert solid support in packed column GLC. The support is coated with a thin layer of stationary phase. This is then packed into a coiled glass or metal tube. A Diatom Diameter 0.1 to 0.5 mm Packed Columns for GCSlide 18: 18 A Capillary Column for GLC Fused silica tubing d c 0.3 mm d f 1 m L = 15 - 60 m Polydimethylsiloxane (silicone) Good for retaining and separating nonpolar solutes by boiling point A thin layer of nonvolatile stationary phase is coating on the inner wall of the tubing (WCOT)Slide 19: 19 Columns for Gas Chromatography Selection of appropriate column geometry and dimensions may be critical to a successful separationSlide 20: 20 Column Oven Precise control of column temperature (0.1 o C) Column temperature should be slightly below the boiling points of the solutes (but above the dew point; i.e., no condensation) For complex mixtures with a broad range of boiling points, use programmed temperature increase the column temperature during the run Precise control of oven temperature is critical to obtaining reproducible retention times.Slide 21: 21 Detectors Generate an electrical signal proportional to solute concentration or mass flow rate Ideal characteristics High sensitivity Low volume of carrier gas Rapid response time Non destructive technique Applicable to wide range of samples Easy to use Stable ,predictable responseSlide 22: May be universal or selective Universal (responds to wide range of solutes) Thermal conductivity detector (TCD) Simple, inexpensive, rugged Modest sensitivity For greater sensitivity or selectivity Flame Ionization detector (FID) Responds to most organic compounds High sensitivity and wide dynamic range (9 orders of magnitude) Electron capture detector (ECD) Selective and very sensitive for halogenated organicsSlide 23: 23 Detectors for GCThermal conductivity detector: Thermal conductivity detector Element is electrically heated at constant power. Temperature depends on thermal conductivity of surrounding gas. Measure conductivity with respect to a reference. When analyte comes off, filament temperature goes up, resistance goes down. Thermal Conductivity Detector A universal detectorSlide 25: Mechanism: a detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, change in the filament current occurs. The current change is compared against current in reference cell. The difference is measured and a signal is generated. Sensitivity: 5-20 ng. Selectivity: All compounds. Gases: hydrogen, helium. Temperature: 150-250 0 CSlide 26: Flame Ionization Detector For most organic compounds Flame Ionization Detector Column effluent is passed through a H 2 -air flame -produces ions and electrons. Charged particles are accelerated by voltage applied between jet and collector-results in current Number of ions depends on number of reduced carbons in molecule-one molecule of ethane gives twice the signal of one molecule of methane. Less sensitive to non hydrocarbon groups. Insensitive to H 2 o,Co 2 ,So 2Slide 27: Mechanism : compounds are burned in a hydrogen-air flame. carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Sensitivity : 0.1-10 ng. Selectivity : compounds with C-H bonds. Gases : combustion –hydrogen and air, makeup-He,N 2. Temperature : 250-300 0 C.Electron capture detector: Electron capture detector Carrier gas (and analyte) passes over β -emitter, resulting in ionization and electron production. Produce current between electrodes. In the presence of other compounds electrons are captured, causing decreasing in current. Most commonly used for halogenated organics.Slide 29: Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. Sensitivity: 0.1-10 ng. Selectivity: Halogens, nitrates. Gases: Nitrogen or argon. Temperature: 300-400 0 CNitrogen phosphorous detector: Nitrogen phosphorous detector Mechanism: compounds are burned in a plasma surrounding a rubidium bead supplied with hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Sensitivity: 1-10 pg. Selectivity: Nitrogen phosphorous containing compounds. Gases: combustion- hydrogen ,make up – helium. Temperature: 250 -300 0 C.Recorder: Recorder Strip chart recorder used in GLCAdvantages: Advantages Both qualitatitve and quantitative analysis are possible. Instrument is simple ,time of analysis is short. High sensitivity. The method is applicable to about 60% of organic compounds. Very small samples sizes can be used. Analysis can be highly accurate and precise.Factors affecting separation: Factors affecting separation Particle size and surface area. Carrier gas flow rate. Type and amount of stationary phase. Column length. Column diameter. Column temperature.Applications:: Applications: Qualitative Information Identity of eluted solutes may be based on Retention time Spectral information obtained for eluted peak Quantitative Information is based on peak areas Isolation and identification of drugs or metabolites in urine plasma, serum. Analysis of petroleum products, gasoline, waxes etc. Determining various cosmetics and perfumes.Slide 35: Analysis of pesticides. Identifications of plastics and determination of esters, co-polymers. Determination of water in creams, ointments, pastes. Normalization.Slide 36: 36 Applications – Sample Chromatograms Areas of Application Industrial Petrochemicals Consumer goods Pharmaceuticals Environmental Food/Agriculture MedicineSlide 37: Forensic Applications Analysis of bodily fluids (for example: semen or urine) to test for illegal substances. Test fibers and blood recovered at a crime scene. Detect residue from explosives and ignitable fluids/presence of accelerants.Slide 38: 38 Special Topics in GC Gas Chromatography-Mass Spectrometry (GC-MS) Programmed Temperature Gas Chromatography (PTGC)Slide 39: 39 Gas Chromatography – Mass Spectrometry (GC-MS) A powerful analytical tool combining High resolution gas chromatography Mass spectrometric detection Qualitative indentification of solutes High sensitivitySlide 40: 40 Programmed Temperature GC of a Complex MixtureSlide 41: 41 Summary Gas chromatography An important method for separation and analysis of complex mixtures Applicable to volatile compounds Retention depends on Temperature Solute interactions with stationary phase Requirements for successful GC Sample volatility and stability Precise control of carrier gas flow rate Appropriate injector design and injection technique Selection of appropriate column type, geometry and stationary phase Precise control of column oven temperatureSlide 42: 42 Recent Developments in Chromatographic Analysis Using a molecular-beam-based GC-MS method, researchers at Tel Aviv University separate components of complex mixtures of large hydrocarbons and identify them with the aid of their prominent molecular-ion signals, as shown here for C 72 H 146 . Chem. Engr. News, April 16, 2007. Molecular beam method in GC-MS provides molecular weight for large molecules C 72 H 146Reference:: Reference: Instrumental methods of analysis by WILLARD,MERITL,DEAN,SETTLE. Practical pharmaceutical chemistry by A.H.BECKETT AND J.B.STENLAKE. Fundamentals of analytical chemistry by SKOOG,WEST,HOLLER,CROUCH . A textbook of analytical chemistry by ANJANEYULU,CHANDRASEKHAR,MANICKAM. www.wikipedia.comImage Sources: Image Sources Microsoft Clipart Wikipedia.org http://www.und.edu/fasl/natsci/slconline/GC/pen15.html http://teaching.shoe.ic.uk/bjt/chemistry/chrom/gaschrm.htmSlide 45: thank you You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.