Gas Chromatography Introduction, theory, instrumentation, - PowerPoint Presentation

1. Gas ChromatographyIntroduction, theory, instrumentation, derivatization,Temperature programming, advantages, disadvantages and applications1Dr. Nisha Sharma, Associate Professor, Pharmacy, C.S.J.M. University

2. INTRODUCTION: GAS CHROMATOGRAPHY Origin of gas chromatography: 1905, W. Ramsey Separated mixture of gases and vapors Used solid adsorbent: activated charcoal. Gas used as mobile phase: introduced in 1952 by James and Martin. The technique was based on a suggestion made 11 years earlier by Martin and Synge on partition chromatography Martin and Synge were presented the Nobel Prize in chemistry in 1952. Used to analyse volatile substances 2

3. Partition takes place b/w gas & solid or gas & liq.Nature of stationary phase– Fixed stat. phase-solid mat. like granular silica/alumina/C.---GSCFixed phase. Non vol. liq. Held as thin layer on solid support- (diatomacious earth or keisulguhr)--- GLCGSC- limited application. Difficult to reproduce surface areas, excessive retention of active gases on solid surfaces which reduce available area, tailing of elution peaks.GLC- Most imp. Widely used.Principle: liq. Partition chrom. Mobile phase in gas liq. Chrom. is gas rather than liquid.3

4. Theory: Retention timeTime of emergence of peak max of a component after injection. Sum of times the components spends in mobile phase (tM) & in stationary phase . Adjusted Retention time t’R: time the component spends in stationary phase.t’R=tR-tMtM – measuring time to elute an unretained subs. Eg. Air or methane.4

5. 5

6. TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS 6

7. 7Retention Times

8. Filters/TrapsAirHydrogenGas Carrier ColumnINSTRUMENTATION: Gas Chromatographgas systeminletcolumndetectordata system8Data systemSyringe/SamplerInletsDetectorsRegulatorsHRESETBasic Instrumentation:1. Tank: of carrier gas 2. Injection port of sample 3. Column 4. Detector

9. Schematic Diagram of Gas Chromatography9

10. Carrier gas:He, H, N, Choice of gas-type of detector. Additional regulating valves-for good control of pressure in inlet of column. Gas- inert, available at low cost, should be suitable for detector & type of sample analysed, available in high purity, should not cause risk of fire or explosion hazard.H- dangerous to use, better T.C., Low density, but may react with unsaturated compds & create a fire or explosive hazard.He- 2nd best but explosive, gen used, good T.C. Inert, Low density, great flow rates.N- inexpensive but low sensitivityAir- used only when O in air is useful to the detector or separation.Ex. H or He gives highest sensitivity with TCD because of difference in TC between organic mol. & H/He is greater than other gases10

11. Gas cylinderHigh pressure gas cylinder (gas in compressed) – carrier gas reservoir.Pressure regulator- To ↓ & control gas flow.Soap bubble meter- To reproduce the rate of carrier gas. Soap film is formed in path of gas when a rubber bulb containing aq sol of soap or detergent if squeezed. Time req for soap film to move b/w 2 graduations on burette is measured & converted to flow rate.11

12. Sample IntroductionColumn inlet-sample port injector. Solute-chrom-Vapor state. Inj port is heated to temp-rapid vaporization, but no thermal degradation of soluteConstruction of port- Heavy mass, maint at ↑Temp. Sample should be intro immediately into column.Liq/gas sample- inj by syringe-0.1-100µL. Rapid inj. into gas stream.Liq- injected- near- as solutions with syringe-0.5-10 µL. Solid- dissolved in suitable solvent-injected as solution.Injection of samples which can’t be vaporised at operating temp. are avoided. B’cos compds not move appreciably in liq or solid form may clog the port & damage the column.12

13. 13G.C INJECTION SYRINGE:* To rapidly vaporize the sample.* Slow vaporization ↑ses band broadening, by ↑sing the sample “plug”.* Injection port temperature mostly held 50°C higherthan BP of least volatile compd.

14. Columns: Heart of InstrumentGlass/metal, 4-8 mm(D)- few cm-hundred mts.(L)Mounted in const. temp. oven, basic process takes place. Size: 6ft L, 1/4” I.D., Cu, SS tubing, U shaped. Coiled to spiral, saves space.Tubing- packed with inert, pulverized solid material of large surface area. (Diatomacious earth or firebrick)Solid is used as mechanical support to liq.Before packing solid mat. is impregnated with desired liq. Which serves as real stationary phase. Liq.- stable, Non vol. at temp. of column. 14

15. Types of ColumnsIn GLC-2 typesCapillary columns- form capillary tubingBore coated with a thin film of liq. Phase 0.166” or less Dia., upto 200ft. L, ↓ sample capacity. Film of liq. is deposited on inner wall of tubing.Packed Columns: SS/Cu, tube packed with solid substrate- GSC- or a liq coating on inert solid (GLC), D of tube: 0.25”, L- 5-50 ft. Tubes-folded/coiled 15

16. PackedCapillary (Open Tubular) Columnsglass, metal (stainless), Cu, Teflonmetal, plastic, glass (FRAGILE), More recently: Fused Silica - robust, flexiblefew meters in length, L- 5-50 ftUpto 200ft. Lfew mm in diameter (i.d.), 0.25”. Int. col. Dia. Should be atleast 8 times D of support particles.Small diameter (typically <500 μm), 1/6” or less Support Materials:small particles, uniform (spherical) shape, porous, inert. 100-300 μm diameterSupport methods: Support-Coated OT (SCOT)Wall-Coated OT (WCOT)diatomaceous earth, polymeric materials16

17. Stationary Phase MaterialsGas-Liquid Chromatography (GLC)Characteristics:– appropriate chemical nature (“like dissolves like”)– low volatility– thermal stability– chemical inertnessTypical coatings (< 1 to several μm thickness):– Polyethylene Glycol (PEG, Carbowax) -(O-CH2-CH2-)n– Poly(dialkyl)silane–Vary R groups to vary retention characteristics17

18. Most Common Stationary Phases181. For Separation of mixture of polar compoundsCarbowax 20M (polyethylene glycol)2. For Separation of mixtures of non-polar compoundsOV101 or SE-30 (polymer of methylsilicone)3. For Methylester of fatty acidsDEGS (diethylene glycol succinate)

19. Lifetime of column is limited by:– adhesion of liquid coating to stationary phase (bleeding)– irreversible adsorption of contaminants to columnTo Minimize the bleeding– Cross-linking: on-column reaction– “Bonding”: Utilize surface chemistry of column (or packing)Minimizing AdsorptionGas-solid Chromatography (GSC):– Typically porous solid adsorbed to walls (OT) or a porous solid supportmolecular sievesPorous polymers (beads or coatings)19

20. 20Gas Chromatography Packed Column 1/8” x 6’Gas Chromatography Capillary Column 250 μm x 10mPacked column longitudinal cross section

21. ColumnsMost popular-capillary columns- WCOT column- fused-silica wall-coated (FSWC) open tubular column. Walls of fused-silica columns- made of purified silica containing minimal metal oxides. Much thinner than glass columns, Dia 0.1 mm & lengths as long as 100 m. To protect the column, a polyimide coating applied to outside of the tubing and bent into coils to fit inside the thermostatted oven of gas chromatography unit.FSWC columns commercially available, have increased chemical inertness, greater column efficiency and smaller sampling size requirements. It is possible to achieve up to 400,000 theoretical plates with a 100 m WCOT column, the world record for the largest number of theoretical plates is over 2 million plates for 1.3 km section of column.

22. 22

23. PACKED Columns Packed columns are made of a glass or a metal tubing which is densely packed with a solid support like diatomaceous earth. Due to the difficulty of packing the tubing uniformly, these types of columns have a larger diameter than open tubular columns and have a limited range of length.As a result, packed columns can only achieve about 50% of the efficiency of a comparable WCOT column.Furthermore, the diatomaceous earth packing is deactivated over time due to the semi-permanent adsorption of impurities within the column. In contrast, FSWC open tubular columns are manufactured to be virtually free of these adsorption problems.

24. 24Blood Alcohol Content

25. Depending on the type of sample, some GC columns are better than the others. Ex. FSWC column Designed specially for blood alcohol analysis. It produces fast run times with baseline resolution of key components in under 3 minutes. It displays enhanced resolutions of ethanol and acetone peaks, this helps in determining the BAC levels. Known as Zebron-BAC & made with polyimide coating on outside & inner layer is made of fused silica & inner diameter 0.18 mm to 0.25 mm. Ex. Of Zebron GC column – known Zebron-inferno. Its outer layer is coated with a special type of polyimide that is designed to withstand high temperatures. It contains extra layer inside. It can withstand up to 430°C & designed to provide true boiling point separation of hydrocarbons distillation methods. Moreover, it is also used for acidic and basic samples.

26. DETECTORS Flame Ionization Detector (Nanogram - ng)↑ Temp. of H2 flame (H2+O2+N2) ionizes compds eluted from column into flame. Ions collected on collector electrode and recorded on recorder due to electric current. Potential of 400 V across 2 electrode- lowers the resistance b/w electrodes & causes current to flow.Flame jet & collector position is 0.5-1 cm above the tip of flame. Forms twin electrode 26I=V/R

27. 27

28. Current arises from the ions & free e-, generate in pure H2/air.When ionisable material from column effluent enters flame & burned, current rises abruptly. Current flows thru external resistor, sensed by voltage drop-amplified-sends output device – recorder/ microprocessor.FID –enclosed in chimney-heated sufficiently-to avoid conditions of H2O droplets-from combustion process.FID-responds- no. of –CH2- grps. Introduced into flame.Eg. Response of equimolar amt. of butane is twice to ethane.No responses from fully oxidized C- carbonyl,-COOH gp. Ether grp.Response from C attached to –OH, -NH grp. is ↓If desired CO, CO2--- converted to CH4 by redn. with H over Ni cat.--- Measured 28

29. ExhaustChimneyIgniterHydrogen InletColumn EffluentPolarizing ElectrodeCollector Electrode29Flame Ionization Detector Column effluent is passed through a H2-Air flame – Produces ions and electronsCharged particles are accelerated by voltage applied between jet and collector– results in current Number of ions depends on number of reduced (methylene) carbons in molecule– one molecule of ethane gives twice the signal of one molecule of methane– less sensitive for non-hydrocarbon groups– insensitive to H2O, CO2, SO2 and other noncombustiblesHigh sensitivity, low noise, destructive

30. Thermal Conductivity detector30 A heated filament is cooled by the flow of carrier gas .Metal blockReferencesampleFilamentCarrier gassRLeads to W.S. bridgeCarrier gas outkatharometer

31. Thermal Conductivity Detector31Principle: Electrical power is converted to heat in a resistant filament and the temperature climbs until heat power loss from the filament equals the electrical power input.The filament may loose heat by radiation to a cooler surface and by conduction to the molecules coming into contact with it.The ability of a colliding molecule to carry off heat depends on its thermal conductivity. Hydrogen and helium have high thermal conductivity and therefore will be more efficient at “cooling” a heated filament than other gases will.

32. The detector contains two filaments: one exposed only to carrier gas, while the other is exposed to the carrier gas for sample analysis.When the gas for the sample analysis is only carrier gas , the two filaments can be balanced. Instead of a direct measurement of filament temperature, the filament resistance, which is a function of temperature, is measured.Thermal Conductivity Detector32

33. Thermal Conductivity Detector Measures the changes of thermal conductivity due to the sample (mg). Sample can be recovered.  Heated filament is placed on the emerging gas stream. Amount of heat lost from filament by conduction to the detector walls depends on the thermal conductivity of gas phase With in the cavity of metal block- coiled filament- Tunsten metal, Tungsten rhenium alloy, tungsten sheathed with gold. Filament is heated to constant temp. but less than dull red, regulated by DC current Loss from filament to metal block is constant, when only carrier gas flows Thermal conductivity of H & He is 6-10 times greater than organic compounds 33

34. Thermal Conductivity Detector Presence of small amount of organic material results in relatively large decrease in TC of column effluent. Filament retains max. heat & Temp. rises & electrical resistance increases Standard detector: 4 identical filaments, mounted in 1 brass block Filaments form arms of a wheatstone bridge Through one pair of filament column effluent is passed & 2nd pair is places in gas stream near sample injection port Any imbalance b/w pair of filaments are recorded 34

35. Thermal Conductivity Detector When analyte comes, Filament Temp. rises When compound elutes, T.C. of gas mixture of carrier gas & compound gas is decreased Filament in sample column becomes hotter than other control columnResistance increases and imbalance b/w control and sample filament resistance is measuredAbility of colliding molecules to carry off heat depends upon its TC.H & He have high TC & more efficiently cools in heated filament conc. sensing detector35

36. Thermal Conductivity Detector Responds to all compounds, simple construction, Good & Adequate sensitivity to many compounds Good linear range of signal Simple construction Signal quite stable provided carrier gas flow rate, block temperature, and filament power are controlledThe TCD is a nondestructive, concentration sensing detector.36

37. Thermal Conductivity Basics37When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal. A heated filament is cooled by the flow of carrier gas .FlowFlowMetal blockReferencesampleFilamentCarrier gassRLeads to W.S. bridgeCarrier gas out

38. Electron Capture Detector38ECD detects ions in exiting from the gas chromatographic column by the anode electrode.3H or 63Ni emits  particles.Ionization : N2 (Nitrogen carrier gas) +  (e) = N2+ + 2e These N2+ establish a “base line”X (F, Cl and Br) containing sample +  (e) → X-Ion recombination : X- + N2+ = X + N2The “base line” will decrease and this decrease constitutes the signal.Insecticides, pesticides, vinyl chloride, and fluorocarbons

39. Electron Capture Detector (ECD):Carrier gas (and analyte) passes over β-emitter, resulting in ionization and e- productionThis Produces current between electrodesIn the presence of other compounds (especially halogens, etc.) electrons are captured, causing decrease in currentMost commonly used for halogenated organics (insecticides, etc.)As an analyte flows through Ni-63 source, electron capture is possible by electron-withdrawing species: A + e- => A-Current decreases as a result of e- capture by analyte. This is one of the few instances in which a signal is produced by a decrease in detectable phenomenon.39

40. Electron capture detectorColumn effluent is passed b/w 2 electrodesOne electrode has radioisotope on its surface that emits ↑ energy electrons (β particles as it decays)These electrons are bombarded with carrier gas N2 Results in formation of +ve ions, radicals, thermal electronsProcess is very rapid < 0.1µ sec.When Voltage is applied→ e- capture cell → collect the thermal e- → produce –ve ions of large massesRate of combination b/w –ve & +ve ions is greater than thermal e- & +ve ions ↓se in detector current due to removal of thermal e- in presence of e- capturing compounds → measured40

41. Electron capture detectorRadio active sources: tritium adsorbed in titanium or scandium & Nickel 63 as foil → placed on interior of cathode chamberNi-63: 400° CLinearity of ECD- cell design, composition of carrier gas, method of applying P.D. to collect thermal e-ECDs - high selectivity, sensitivity towards certain organic species with electronegative functional groups. However, the detector has a limited signal range & is potentially dangerous owing to its radioactivity.In addition, the signal-to-noise ratio is limited by radioactive decay & presence of O2 within the detector.41

42. ECD42cathodeelectode

43. Mass Spectrometry DetectorsMost powerful of all detectors. Scans the masses continuously throughout the separation. When sample exits column, it passes through transfer line into the inlet of detector. The sample is then ionized & fragmented by electron-impact ion source. During this process, the sample is bombarded by energetic electrons which ionize the molecule by causing them to lose an electron due to electrostatic repulsion. Further bombardment causes the ions to fragment. 43

44. Mass Spectrometry DetectorsThe ions are then passed into a mass analyzer where the ions are sorted according to their m/z value, or mass-to-charge ratio. Most ions are only singly charged.The Chromatogram will point out the retention times and the mass spectrometer will use the peaks to determine what kind of molecules exist in mixture. mass spectrum of water with the absorption peaks at the appropriate m/z ratios.44100%2010Water molecule in which H is removed by fragmentation

45. Mass Spectrometry DetectorsOne of the most common types of mass analyzer in GC/MS is the quadrupole ion-trap analyzer, which allows gaseous anions or cations to be held for long periods of time by electric and magnetic fields. A simple quadrupole ion-trap consists of a hollow ring electrode with two grounded end-cap electrodes as seen in figure. Ions are allowed into the cavity through a grid in the upper end cap. A variable radio-frequency is applied to the ring electrode and ions with an appropriate m/z value orbit around the cavity. As the radio-frequency is increased linearly, ions of a stable m/z value are ejected by mass-selective ejection in order of mass.Ions that are too heavy or too light are destabilized and their charge is neutralized upon collision with the ring electrode wall.45

46. Mass Spectrometry DetectorsEmitted ions then strike an electron multiplier which converts the detected ions into an electrical signal. This electrical signal is then picked up by the computer through various programs. As an end result, a chromatogram is produced representing the m/z ratio versus the abundance of the sample.GC/MS units are advantageous because they allow for the immediate determination of the mass of the analyte and can be used to identify the components of incomplete separations.They are rugged, easy to use and can analyze the sample almost as quickly as it is eluted. The disadvantages of mass spectrometry detectors are the tendency for samples to thermally degrade before detection and the end result of obliterating all the sample by fragmentation.46

47. 47GC/MS system.Arrangement of the poles in Quadrupole and Ion Trap Mass spectrometers

48. Other Detectors:Atomic Emission detectorMicrowave induced plasma, grating monochromator, diode array detectorThermionic DetectorPhoto ionization detectorGC Chemiluminiscence detectors48

49. DERIVATIZATIONProcess of modifying chemically a compd. to generate a new one so as to enable the analysis Chemical structure remains same, only sp. fn. grp. of reacting compd. is modified to changes in chemical & physical prop. for easy analysis.Why derivatization? For compds. that cannot be directly analyzed due to improper stability & volatility, imparts volatility to non volatile compds.To improve chromatographic behaviour/ detection 49

50. DERIVATZATION↑ses detectability ex. Steroids↑ses stability- thermo-sentitivity↑ses sensitivity to detector: ECD- halogenated acyl grp. when introduced – allows detection↑ses volatility- ex. sugars;- eliminates polar grps- OH, NH, SH↓ces adsorption of polar samples on active surfaces of column walls & solid supportDerivatization agent must be- stable, Deriv Ag & its products- must not be detectable, must be separable, Analyte – reactive with DA, Non toxic, 50

51. DERIVATIZATION: Types Silylation: easily volatilizes sample, silyl derivatives- more volatile, thermal stable, replaces active H with TMS (tri methyl silyl gr)Silylation- SN2 grps- better the leaving grp- better silylation Alkylation: reduces mol. Polarity, replaces active H with alkyl grp. They modify acidic H containing compd.-carboxylic acids, phenols They make- esters, ethers, alkyl amines, alkyl amides, mech - nucleophilic displacementEx: BCl3 in MeOH, BF3 in MeOH, MeOH in acid51

52. DERIVATZATIONAcylation: reduces polarity of amino, -OH, thiol grps. Adds halogenated fnc. for ECDTargets are high polar, multifunctional compds., carbohydrates, amino acidsIt converts- active H to esters, thioesters, amidesFormed with acyl halide, anhydride, acyl amideCarried out in pyridine, THF capable of accepting acid by productUsed for increasing retention timesEx. Acetic anhydride, TFAA Anhydride52

53. Effects of Derivitization531. Time consumption2. Side reaction3. Loss of sample

54. Temperature Programming54

55. Temperature programmingComplete & efficient separation T high enough to evaporate sample componentsEffects partition coefficient of analytes↑ T ↓ retention vice versaSample with components close similar B.P.- Isothermal separation- short time at a single Temp.- obtained55

56. Temperature programmingBut with compounds having wide B.P. – efficient sep.- not possible in short timeGo for Temp-prog.- allows efficient & complete separation of both components with early & late elution in less time.Initial Temp: nearly below the B.P. of lowest boiling componentFinal Temp.: near to B.P. of highest boiling of component in sampleTemp. rate: linear/multi linear etc56

57. Linear Temp. programmimg57Isothermal vs Temperature Gradient

58. Chromatographic behaviour of solutesDescribed in no. of waysRetention Vol. VR , Retention time: tRPartition coefficient K’By varying stationary mobile phase combinations & operating parameters, degrees of retention can be varied from total retention to free migration 58

59. 59

60. 60

61. Column Efficiency61

62. 62

63. Plate Ht. Plate No.63

64. 64

65. GLC ADVANTAGES 1.Very good separation2.Time (analysis is short)3.Small sample is needed - ml4.Good detection system available5. Quantitatively analysis 6. Universal detectors for organic 7. Carrier gases can’t be detected65

66. GLC DISADVANTAGES Less application- fixed Gas analysisLow column efficiencySample must be volatileNot good for thermolabileCarrier gas must be pureDoes not respond to common inorganic compMobile phase impurities can’t be detected66

67. DISADVANTAGES OF GAS CHROMATOGRAPHY Material has to be volatilized at 250°C without decomposition.67

68. ApplicationsAs a tool for doing separations- complex organic, metal organic, & Biochemical systemsFor providing means for completion of an analysisQuanlitative identification: carried by using tR, VR , quantitative information- by using peak ht. or area68

69. ApplicationsIn petroleum Industry- for analysis of crude petroleum products, gasoline, waxes, LPG, S, N compounds & unsaturation etc. In food industry: For color, flavour, detectors based on ECD. To determine residual solvents in spices, oleoresins, for pesticides in foods.In Biochemical & clinical fields: Blood gases, estrogens, vanilimandelic acid, Hydroxy cortico steroids etc. determined & analysed in medicine69

70. ApplicationsIn cosmetic & perfumery: To determine composition of various cosmetics, quality of ingredients,Determination of styrene monomer, vinyl toulene, latex toulene di-isocyanate in paint, varnish, lacquer etc.In plastic Industry: To identify plastics, determination of esters in acrylic co-polymers, styrene monomers in styrene plastics, vinyl acetate in its co-polymers etc.70

71. ApplicationsFor analysis of coal tar productsFor analysis of Alcoholic beveragesFor analysis of FertilizersFor determination of water in butane, gas, creams, emulsions, ointments, pastes etc. Pesticides in water, aquatic herbicide. 71