GAS CHROMATOGRAPHY

GAS CHROMATOGRAPHY:

GAS CHROMATOGRAPHY GUIDED BY: Dr. Nehal Shah M.Pharm, Ph.D. Principal of Dharmaj Pharmacy College PREPARED BY: Chirag Solanki M.Pharm (Q.A.) Roll No 11 cs_apc_09@yahoo.in 1 CHIRAG SOLANKI

Introduction:

Introduction Mobile phase : Gas Stationary phase : solid adsorbent or a liquid coated or bonded on a solid support or on wall of column. solid: GSC – Gas-Solid Chromatography . liquid: GLC – Gas-Liquid chromatography . The sample used in GC separation must be a gas or be capable of being converted to a gas at temperature of column. GC provides both qualitative and quantitative analytical information. 2 CHIRAG SOLANKI

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GC is most widely used for separation and analysis of organic compounds, many complex mixtures. GC cannot be used for analysis of inorganic salts. In GC, volatile components can also be separated and analyzed which cannot be possible with liquid chromatography. 3 CHIRAG SOLANKI

Instrumentation:

Instrumentation Source Injector Pressure regulator Column Detector Recorder 4 CHIRAG SOLANKI

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Temperature programming:

Temperature programming The column sits in an oven. If the temperature is held constant during the entire analysis it is isothermal. If the temperature is varied during the analysis, temperature program is used. With homologues, retention time increases exponentially with the number of carbon. As t R increases, width increases and the height decreases making detection impossible after a few peaks have eluted. Since solubility of a gas in a liquid decreases as temperature goes up, it is possible to reduce retention of material by increasing T Column . 6 CHIRAG SOLANKI

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Factors to consider during temperature programming: :

Factors to consider during temperature programming: Variations in solubility of solutes. Changes in volatility of solutes. Stability of solutes Flowrate changes Stability of stationary phase. Must stay within T min / T max of column. Other factors are found experimentally. 8 CHIRAG SOLANKI

Carrier gas supply:   :

Carrier gas supply : Carrier gas (He, N 2 , Ar ) is contained in compressed gas cylinder. Sometimes H2 or another gas is used in GC. Now, H 2 is not used as fire hazard is associated with it. Choice of gas is dependent upon type of detector. H e is widely used. 9 CHIRAG SOLANKI

Pressure regulator: :

Pressure regulator: Inlet pressure from 10 – 50psi which leads to flowrate of 25 – 150 ml/min with packed column and 1- 25 ml/min for OTC. Flowrate can be established by rotameter at column but soap-bubble meter is accurate which is located at end of column. Now, electronic flowmeter that are computer controlled to maintain flowrate are used. 10 CHIRAG SOLANKI

Sample injector system:

Sample injector system Column efficiency required that samples be of suitable size and introduced as a plug of vapor. Slow injection of oversized sample cause band broadening and poor resolution. Injector changes sample to gas and reproducibly introduces resulting gaseous sample into column. Types of injector system are dependent upon physical state of sample. 11 CHIRAG SOLANKI

For gaseous sample :

For gaseous sample Rotary Valve is preferred and that is same as in HPLC but here sample loop is larger. In position 1 carrier gas flows directly into column and valve is in that position, the sample is flushed and filled with gaseous sample. Upon switching value to inject position 2, carrier gas sweeps through sample loop and flushes sample into column. 12 CHIRAG SOLANKI

For liquid sample :

For liquid sample A gas syringe is used. Sample is injected with 1-.5- or 10-µL graduated syringe through septum into heated flash evaporation injector. The sample is rapidly vaporized because injector’s temperature is maintained above B.P. of highest boiling component in sample. Carrier gas flushes vaporized sample from injector and into column. Typical liquid sample volume used with packed column varies from 0.1 – 5µL and injector temperature is held at least 10  C above the column temperature. 13 CHIRAG SOLANKI

For solid sample :

For solid sample A solid sample is more difficult to convert to gas. Sometimes it can be chemically converted to volatile derivative. E.g. Fatty acid is converted to volatile methyl ester with BF3 or BCl3 in methanolic solution In some cases pyrolysis equipment is attached to GC and used to vaporize solid sample In pyrolysis apparatus sample is placed and heated sufficiently (1000  C) to vaporize the sample or to form volatile sample decomposition product and volume sample swept into GC with carrier gas 14 CHIRAG SOLANKI

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Pyrolysis gas chromatography (PGC) The sample is heated in 2 steps. After sample is placed into injector port, it is initially heated at 270  C. At that temperature all volatile components are flash-vaporized to flush into column with carrier gas and chromatogram is recorded of volatile components. After chromatogram is recorded temperature of injection port is increased to 1000  C and program of pyrolyzed product is recorded. 15 CHIRAG SOLANKI

HEAD SPACE GAS CHROMATOGRAPY :

HEAD SPACE GAS CHROMATOGRAPY U sed to assay solid or liquid solution that contain both volatile and non volatile components. 16 CHIRAG SOLANKI

Columns:

Columns 2 types of columns : Packed columns Open tubular columns 17 CHIRAG SOLANKI

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18 Packed columns: Columns which contain particles of stationary phase packed into metallic or glass tube. Column diameter: 2 – 10 mm Column length: 1 – 4 m Stationary phase: Either small particles of solid adsorbent (GSC). or liquid supported on small particles of solid (GLC). CHIRAG SOLANKI

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19 Open tubular column / Capillary column: Columns which contains liquids stationary phase coated on inner wall of glass or fused silica column. Because pressure drop through OTC is less than that of packed column, use of OTC are longer than packed. Column length:10 – 100m Column diameter: 0.2 – 0.5mm Diameter: 10µm CHIRAG SOLANKI

Packed Column:

Packed Column Characteristic of solid stationary phase Provide inert surface on which liquid phase can be coated. Provide appropriate physical structure for stationary phase. It must be thermally stable at column temperature. It consists of uniformly sized particles. Most common solid supports are derived from diatomaceous earths. Diatomaceous earth primarily consists of hydrated silica groups. Chromosorb P is primarily used as support for separation of samples containing non polar components. 20 CHIRAG SOLANKI

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Other derivatives are chromosorb W, chromosorb A and G. Other substances used as solid support are powdered Teflon, Graphitized carbon black, Cadmium chloride, volcanic scoria, Activated carbon, Organic polymer, Vermiculite and glass beads. Keiselguhr and embacel are widely used. If solid support is more polar than desired, it can be deactivated by coating with DMCS- Dimethyl Chloro Silane or by rinsing support with solution of strong acid HCl . 21 CHIRAG SOLANKI

Liquid stationary phase:

22 Liquid stationary phase Characteristics: It must be nonvolatile at column temperature. Sample component must be slightly soluble in it in order to be separated. It should be pure compound if possible. Polar samples are retained longer in stationary polar liquid phases than are non polar components and separation is atleast partially based on polarity of sample components. With nonpolar stationary liquid phase, components are separated according to their relative solubilities in liquid and gaseous. In short, polar substances are better separated on polar stationary phase and non polar substances on nonpolar stationary phase CHIRAG SOLANKI

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23 3 types according to their polarity. Highly polar : e.g. polyester Used for polar samples: alcohol, acids, amines. Medium polarity : e.g. containing –CN, -OH, -CO Used for medium polar samples: ethers, ketones, aldehydes . Non polar : Hydrocarbon based e.g. poly dialkyl siloxanes. Used for non polar samples: hydrocarbons. CHIRAG SOLANKI

Liquid Crystalline Stationary Phase:

24 Liquid Crystalline Stationary Phase Allows separation of geometric isomers. Lyotropic liquid crystals and thermotropic liquid crystals Ideal properties: thermally stable, have low vapor pressure, capable of adhering to solid support. Separation is based upon geometric shape of components. Length width ratio of component molecules Polar interaction between component and stationary phase. E.g. o, m, p isomers of xylene . CHIRAG SOLANKI

Open Tubular Column:

Open Tubular Column Support Coated OTC . Solid support: Diatomaceous earth, silica gel, graphite, metal oxides and silicates. Due to support internal surface area increases so sample handling ability increases. Wall Coated OTC. Thickness of film should be ≤ 1 µm to prevent bleeding. Bleeding can be prevented by – crosslinking and chemical bonding. 25 CHIRAG SOLANKI

Cross linking:

26 Cross linking Reacting vinyl linkage of stationary phase with decomposed peroxides . Irradiating stationary phase with gamma radiation from source of Co 60 e.g. IFSOTC ( Irradicated fused silica OTC) Normal film thickness: 0.2 – 0.5 µm For analytical studies: 0.1µm and 0.25µm CHIRAG SOLANKI

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27 Chemical bonding Longer lifetime. Glass or fused silica column are used. S.P. in bonded phase is chemically bonded to Silicon atoms on inner surface of column . Other types of OTC Porous Layer OTC (PLOT) Whisker Walled OTC (WWOT) CHIRAG SOLANKI

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28 Glass Columns Less expensive. Contains small amount of metallic ions act as active sites that increases retention of polar components. If column wall is thick, leaching occurs leading to brittle glass. Fused Silica Columns High purity glass with SiO 2 . More expensive. Not having metallic impurities. Resolution and efficiency is higher. Lack brittleness. Easy to handle . CHIRAG SOLANKI

Advantage of OTC over Packed:

29 Advantage of OTC over Packed Smaller diameter – less band broadening due to no. of different paths is minimized. Heat transfer to column with small diameter is superior. Coating in WCOT and bonded phase columns is more uniform. Produces better resolution. CHIRAG SOLANKI

DETECTORS:

DETECTORS 1.Thermal conductivity detector (Katherometer or TCD) 2.Flame ionization detector (FID) 3.Flame photometric detector (FPD) 4.Electron capture detector (ECD) 5. Thermionic detector 6. Photo ionization detector 30 CHIRAG SOLANKI

Ideal requirements :

Ideal requirements Adequate sensitivity Good stability and reproducibility Short response time Similarity in response towards all solute compounds Linear response should be obtained Temperature range from R.T.-400 ºC should be feasible Highly reliable and easily used Should not cause destruction of sample 31 CHIRAG SOLANKI

Thermal conductivity Detector (TCD):

Thermal conductivity Detector (TCD) Principle Themal conductivity of gas. TCD operates on principle that ability of a gas to dissipate heat (TC) varies with composition of the gas. Here actual detecting element is heated wire filament. Temperature of wire is proportional to electrical resistance through wire. The relative resistance of the two heated filaments monitored with Whetstone Bridge. Thermisters can also be used instead of filaments. 32 CHIRAG SOLANKI

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Factor affecting detectors performance :

Factor affecting detectors performance Types of detecting elements used. Nature and flow rate of carrier gas. Effect of filament current. Temperature. Presence of o 2 or air 34 CHIRAG SOLANKI

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Applications TCD is a universal detector . It detects most compounds including hydrocarbon and inorganic salt. Has unique capacity to detect water and can detect water level below that detectable by Karl Fischer titration. For determination of water in ketonic solvents and other solvents contaminated with water. Advantages Non destructive . So sample can be reused. TCD is a rugged detector which can take a good deal of maltreatment and continue to function well. 35 CHIRAG SOLANKI

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Flame ionization detector Principle :-Ionization of sample by air-hydrogen flame 36 CHIRAG SOLANKI

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Factors affecting performance Flow rate should be in the range of 20-30ml/min. Air flow should be approximate 10 times that of water. e.g. H 2 flow rate-25-30ml/m Air flow-300ml/min Minimum detectable quantity -10 -10 -10 -11 37 CHIRAG SOLANKI

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Advantages 1000 times more sensitive than TCD Less noise level Easy to use and strong enough for operations Disadvantages Sample is destroyed It does not respond to most inorganic compound like N 2, O 2 , CO 2 and water. And it response to organic compound decrease as the no. of O 2 , N 2 , S and X in compound increase. 38 CHIRAG SOLANKI

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Applications CS2 and water used as solvents for sample introduction . GLC-FID used for Analysis of belladonna alkaloids. Chlorphereramine maleate , phenylpropanol amine HCL in cold tablet, capsules and ampoules. Also for biological drug analysis like determination of chlorpropamide , tolbutamide and codeine in plasma. 39 CHIRAG SOLANKI

Flame photometric detector:

Flame photometric detector Principle: FPD uses a hydrogen-air flame as does FID, but rather that measure ionization, FPD uses photomultiplier due to measure radiation emitted in flame by sample component. FPD can be made selective for compounds which contain a single element which emitted radiation at the particular wavelength. E.g. S is 394 nm and P is 526 nm So appropriate optical filters are placed between the flame and the PM tube to isolate this wavelength. N2 is used as a carrier gas. N2/O2are used in ratio 4:1. Air velocity-200ml/min. It is selective detector for P & S containing compound . 40 CHIRAG SOLANKI

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Pesticides residue analysis To determine cannabinol and Δ9-tetrahydrocannabiniol in blood after conversion to diethyl PO-4 ester. Determine S/P containing drugs like penicillins , cephalosporins , phenothiazines and sulphonamides . Analysis of environmental samples such as those containing pollutant S compounds. Applications 42 CHIRAG SOLANKI

Electron Capture Detector:

Principle:- Capturing of electrons by the component present in the compound, which are produced during the collision between β particle and carrier gas. That will results in decrease in current in external circuit. Electron Capture Detector 43 CHIRAG SOLANKI

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Factors affecting performance Non pulsed operation Pulsed operation 2. Carrier gas Pulsed flow- Argon +10-15 %CH4 Non pulsed flow- N2 is used The detector has been described as a halogen detector because it’s response to molecules containing halogen is relatively large due to it’s electro negativity. 44 CHIRAG SOLANKI

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Applications Used as X and P detector Determination of insecticides in environment and biological system. Determination of organometallic compound. Selective determination of submicro levels of drug level in biological specimen like blood, urine and tissues. Compound which do not have high electron affinities are derivatized usually with polyhalogenated group to confer electron capturing properties on molecules. E.g . trifluroacetyl , pentafluropropionyl and heptaflurobutyryl . 45 CHIRAG SOLANKI

Thermoionic detector:

Principle and operation:- Thermal ionization of an alkali metal salt occurs by the flame. This is done by suspending a salt-coated wire in the flame or placing a cylinder filled with salt as a compressed disc of salt on the top of the flame jet. Flame containing alkali metal atoms elicits an enhanced response for organic compound containing N 2 , P, S or halogen. Salt tends to reduce the response to carbon relative to ordinary FID. Thermoionic detector 46 CHIRAG SOLANKI

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Mechanism Gas-phase reaction- where alkali metal is present in the flame Solid phase reaction- where active intermediates are formed which then reacts with surface of salt to form volatile compound of the alkali metals. Photovapourization - which is based on theory that photons emitted by the flame are absorbed by the alkali metal salt, causing it to evaporate. 48 CHIRAG SOLANKI

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Factors affecting performance 1.Alkali metal used - Rubidium is mostly preferred. -Al oxide , Na 2 SO 4 , LiCl , RbCl , NaCl , CeCl 2 provides response for P containing compound. 2.Bead surface temperature - Adjust the bead temperature for optimum signal to noise ratio. -Typical range-700-900 0 C Gas flow rate -For H 2 - 3.6 ml/min Air- 200 ml/min Carrier gas- 10-100 ml/min 49 CHIRAG SOLANKI

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Applications Analysis of N2 drugs and their metabolites. Used for analysis of pesticides. Analysis of lidocaine in plasma and anticonvulsant in blood . Disadvantage Less stable Gradual depletion of alkali metal salt and a concomitant change in noise and sensitivity. 50 CHIRAG SOLANKI

Photoionization detector:

Principle and operation It induces ionization via photons emitted by an UV lamp. Detectors uses a photon energy of 10-2 eV emitted as a L yman alpha line. Compound with ionization potential of less than 10-2 eV will absorb the UV radiation and be converted into the positive ions. An electron field is set up in the detector by 2 charged electrodes, the cathode becoming the collector electrode for the ions. Photoionization detector 51 CHIRAG SOLANKI

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Applications It is considered as “Universal detector”. Compounds with ionization potential less than 10-2 eV are detected by the PID. It’s response is proportional to no. of H.C. (for H.C. of greater than 5 carbons ) Advantage It is non destructive so that it can be used in series with other detectors. Disadvantage Dis -advantage Because of ionization potential limitation compound like H2O, SO2, saturated H.C. smaller than hexane, CHCl3, CHCl2 & acetonitrile are not detected. 52 CHIRAG SOLANKI

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CHIRAG SOLANKI 53 Type of Detector Applicable Samples Detection Limit Mass Spectrometer (MS) Tunable for any sample .25 to 100 pg Flame Ionization (FID) Hydrocarbons 1 pg/s Thermal Conductivity (TCD) Universal 500 pg/ml Electron-Capture (ECD) Halogenated hydrocarbons 5 p g/s Atomic Emission (AED) Element-selective 1 pg Chemiluminescence (CS) Oxidizing reagent Dark current of PMT Photoionization (PID) Vapor and gaseous Compounds .002 to .02 µg/L

References:

References Braun Robert D., I ntroduction to Instrumental Analysis, chapter no.26, first edition, year2006, pharma book syndicate, page no. 890 - 928. Christian Gary D., Analytical Chemistry, chapter no.17, fifth edition, year2001, library of congress cataloging in publication data, page no. 535 – 536. Munson James W., Pharmaceutical Analysis part A, chapter no.1, year2001, Ashish arts, page no. 4 – 54. 54 CHIRAG SOLANKI

Thank You:

55 Thank You CHIRAG SOLANKI