1. 1

2. Gas Chromatography Mass Spectrometry (GC-MS) Presented by :Ayesha Abdul Ghafoor Student ID : MS (I) (Analytical Chemistry section) Session : 2011-13 2

3. GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC- MS) Latest Advances in Chemistry 3

4. GC-MS Introduction History Instrumentation Gas Chromatograph Interface Mass Spectrometer Data System GC-MS operation Analysis of Results Calibration of Instrument Applications Limitations Good Practise of GC-MS Conclusion Refrences 4

5. Hyphenated Techniques term hyphenated techniques introduced by Hirschfeld “It refers to an on-line combination of a chromatographic sepration technique with a sensitive and element-specific Spectroscopic detector”. + = Hybrid Techniques Chromatography Spectroscopy 5

6. Hyphenated Techniques Hybrid techniques exploit both qualitative and quantitavie advantages. Examples of Hyphenated techniques are  LC-FTIR  LC-NMR  ICP-ES  GC-MS 6

7. Gas Chromatography It seprates components of sample Interface Combines both techniques by removing pressure incompatibility problem between GC and MS Mass spectrometry Ionise eluted componet and seprate, identify it according to its mass to charge ratio GC-MS GC-MS is an integrated composite analysis Instrument Combining GC which is excellent in its ability for separation with mass spectrometry ideal in identification and elucidate structure of separated component. 7

8. Introduction Gas chromatography-mass spectroscopy (GC-MS) is a hyphenated analytical technique exquisitely sensitive but also specific and reliable GC can separate volatile and semi-volatile compounds with great resolution, but it cannot identify them. MS provide detailed structural information on most compounds such that they can be exactly identified, but can’t readily separate them. 8

9. Continued............ Therefore, marriage of both instruments have been propsed shortly after the development of GC in the mid-1950s. we obtain both qualitative and quantitative information of our sample in a single run within the same instrument Today computerized GC/MS instruments are widely used in environmental monitoring,in the regulation of agriculture and food safety, and in the discovery and production of medicine. 9

10. Historical Background of GC-MS Roland Gohlke and Fred McLafferty introduce use of MS as detecot of GC in 1950s Miniaturized computers has helped in the simplification of instrument In 1968, the Finnigan Instrument Corporation delivered the first quadrupole GC/MS By the 2000s computerized GC/MS instruments using quadrupole technology had become essential In 2005 GC tandem MS/MS have been intoduced 10

11. Principle of GC-MS The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium). The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions and detected according to their mass to charge m/z ratio 11

12. Instrumental Layout GC-MS comprise following major blocks 1. the gas chromatograph 2. Interface 3. the mass spectrometer 4. A data system is necessary to handle results obtained during a sample run 12

13. GC-MS Instrument Fig 1:The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right. 13

14. GAS CHROMATOGRAPHY Gas chromatography leads to Separation of volatile organic compounds Separation occurs as a result of unique equilibrium established between the solutes and the stationary phase (the GC column) An inert carrier gas carries the solutes through the column 14

15. 1.Gas Chromatograph Basic Components: Carrier Gas Gas Controls The Injector The Column Two Groups: Packed Column Capillary Column The Oven The Detector (Mass Spectrometer) 15

16. Carrier Gas/Mobile Phase Gas Requirements : Inert Column requirements Detectors Purity Better than 99.995% Better than 99.9995% for Mass Spec. Cost and Availability 16

17. Injector Packed column injectors Split Injection Splitless Injection Programmed Split/Splitless Injector Programmed On-Column Injector 17

18. Fig 1.1:Programmed Split/Splitless Injector Septum purge Carrier gas in Split vent Split point Capillary column Liner Cooling fins Heater block Cooling fan 18

19. Columns There are two kinds of columns used i.e. Packed or capillary columns The gas chromatograph GCMS utilizes a capillary column which most widely used columns for GC-MS are those in which the stationary phase has been chemically bonded to the fused silica DB-5 is a common trade name. 19

20. FIG 1.2 :PACKED AND CAPILLARY COLUMN COMPARISON Fig1.3 :( a) Uninstalled Capillary Columns (b)Installed capillary Columns 20

21. INTERFACE The pressure incompatibility problem between GC and MS was solved by Inserting an Interface. Interface join GC with MS. There are many interfaces like jet,Electrospray, thermospray, direct electrical ionization, moving wire or belt interface. Commercially available interface are: 1. Jet Interface 2. Direct Interface 21

22. 1.Jet Interface device takes advantage of the differences in diffusibility between the carrier gas and the organic compound. These jet separators work well at the higher carrier gas flow rates (10 to 40 mL/min) is sprayed through a small nozzle, indicated into a partially evacuated chamber (about 10 –2 torr). The carrier gas is almost always a small molecule with a high diffusion coefficient, whereas the organic molecules have much lower diffusion coefficients. 22

23. JET SEPARATORS ARE MADE FROM GLASS BY DRAWING DOWN A GLASS CAPILLARY, SEALING IT INTO A VACUUM ENVELOPE, AND CUTTING OUT THE MIDDLE SPACING Fig:1.4: Jet Interface 23

24. 2.Direct Capillary Infusion Interface Most GC-MS interfacing is now done by simply inserting the capillary column directly into the ion source. Using a column that is 25 to 30 m long by 220 to 250 μm inner diameter gives an ion source pressure of 10 –6 to 10 –5 torr This gives a helium or hydrogen GC carrier gas velocity of 25 to 35 cm/sec or a flow of about 1 to 2 mL/min. Pumping Speed of Mass spectrometer should be high 24

25. Fig1.5 :Direct Capillary Column Interface Capillary Column Interface 25

26. 3.Mass spectrometer “Mass spectrometry is a technique used for measuring the molecular weight and determining the molecular formula of an organic compound” In general a mass spectrometer consists of an ion source, High-vacuum system a mass-selective analyzer, and an ion collector 26

27. Ion Source Electron Impact Ioniser In an electron-impact mass spectrometer (EI-MS), a molecule is vaporized and ionized by bombardment with a beam of high-energy electrons. The energy of the electrons is ~ 1600 kcal (or 70eV). The electron beam ionizes the molecule by causing it to eject an electron. 27

28. Electron Impact Ionization => 28

29. Chemical Impact Ionizer CI begins with ionization of methane, ammonia or another gas, creating a radical cation (e.g. CH 4 + or NH 3 + ). sample molecule M will produce MH + molecular ions. positively charged species will be detected. CI Used for determination of molecular ion while EI for detailed structure Information. hence the two methods are complementary. 29

30. Chemical Ionisation First - electron ionization of CH 4 : – CH 4 + e -  CH 4 + + 2e - Fragmentation forms CH 3 +, CH 2 +, CH + Second - ion-molecule reactions create stable reagent ions: – CH 4 + + CH 4  CH 3 + CH 5 + – CH 3 + + CH 4  H 2 + C 2 H 5 + CH 5 + and C 2 H 5 + are the dominant methane CI reagent ions 30

31. Mass Analyzers Mass analyzers scan or select ions over a particular m/z range. contribute to the accuracy, range and sensitivity of an instrument. common types of mass analyzers are quadrupole, magnetic sector, time-of-flight, Fourier transform-ion cyclotron resonance (FT-ICR). 31

32. Quadrupole Analyzer Quadrupoles are four precisely parallel rods with a direct current (DC) voltage and a superimposed radio-frequency (RF) potential. The field on the quadrupoles determines which ions are allowed to reach the detector. Quadrupoles thus function as a mass filter. the B 1.6 32

33. Time-of-flight The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector. If the particles all have the same charge, the kinetic energies will be identical, and their velocities will depend only on their masses. Lighter ions will reach the detector first. 33

34. 34

35. Data System GCMS system purchased with a powerful (but small) computer acting as a data system. Data System of GC-MS used to identify and measure the concentration of one or more analytes in a complex mixture. Quantitation can be based on peak areas  mass chromatograms  from selected ion monitoring(SIM). 35

36. SELECTED ION MONITORING With the selected ion monitoring technique, the mass spectrometer is not scanned over all masses; instead, the instrument jumps from one selected mass to another. the mass spectrometer spends much more time at a given mass Difference b/w SELECTED ION MONITORING the responses from only a few preselected masses are recorded MASS CHROMATOGRAM all of the masses are scanned; thus, no preselection is required 36

37. Fig 1.7 :Block Diagram of GC-MS 37

38. Sampling  State compounds must be in solution form The solvent must be volatile and organic  Amount  1 to 100 pg per component are routine.  Preparation 38

39. GC-MS Operation 1.START-UP PROCEDURE Turn on the computer, monitor, data transfer module and the printer. Remove the MS detector from STAND-BY and engage the pumping unit and heater. Turn on the gauge controller - depress the power and degas buttons simultaneously. You are now ready to make a run. 39

40. 2.Operating Conditions: of GC-MS Column Temp. Profile Initial temp. = 110°C, 4 min. hold Ramp at 15°C/min to 140°C Hold at 140°C for 2 min Sample: 1000 ppm acetic acid in water 40

41. 3.Working of GC-MS Vaporized Sample introduced into GC inlet swept onto the column by He carrier gas & separated on column. Sample components eluted from column moved to the MS (He removed). The computer drives the MS, records the data Identification based on it's mass spectrum A large library of known mass spectra is stored on the computer and can be searched for identification 41

42. Fig1.8 : Schematic of a Gas Chromatography-Mass Spectrometry (GC-MS) Instrument interface 42

43. Analysis Time In addition to sample preparation time, the instrumental analysis time usually is fixed by the duration of the gas chromatographic run, typically between 20 and 100 min. Data analysis can take another 1 to 20 hr (or more) depending on the level of detail necessary. 43

44. Analytical Information Obtained from GC-MS There are three ways of examining GC-MS data. First, the analyst can go through the gas chromatogram The second approach is to look at each mass spectrum in turn, in essence stacking up the mass spectra one behind the other and examining them individually. 44

45. Continued.......... The third approach is to look at the intensity of one particular mass as a function of time i.e mass chromatogram. This third approach makes use of the three- dimensional nature of GC-MS data. Two of these dimensions are the mass versus intensity of the normal mass spectrum; the third dimension is the GC retention plot of the intensity of one selected mass as a function of time is called a mass chromatogram 45

46. Fig1.9 : Three dimensional view of Mass Chromatogram 46

47. Fig 1.10 :GC trace of a three component mixture.The mass spectrometer gives a spectrum for each component 47

48. Definition of Terms Molecular ionThe ion obtained by the loss of an electron from the molecule Base peakThe most intense peak in the MS, assigned 100% intensity Radical cation+ve charged species with an odd number of electrons Fragment ionsLighter cations formed by the decomposition of the molecular ion. Isotope abundance Peak These often correspond to stable carbocations. “A” Element—an element that is monoisotopic “A + 1” an element with an isotope that is 1 amu above that of the most abundant isotope 48

49. Consider the mass spectrum of CH 4 below: Fig1.11 : Mass Spectrum of Methane (CH 4 ) 49

50. list of some of problems and their Solutions in GC-MS Analysis contaminations from solvents,glass ware ;solved by high-quality solvents, the latter by heating the glassware to 450 °C after solvent and acid washing sample decompose before or after workup can be identified by spike recovery GC column or GC-MS interface is not working properly can be find out using a mixture of standard compounds of varying polarities and acidities. either the mass spectrometer itself or the data system may not be working properly can be determined these problems is to run an overall mass spectrometer performance standard. The one recommended (mandated in many cases) by the EPA is decafluorotriphenylphosphine. 50

51. Quality Assurance of GC-MS results First, the mass spectra of the unknown compound and of the authentic compound must agree over the entire mass range Second, the GC retention times of the unknown compound and of the authentic compound must agree within about ±1 to 2 sec. Third, a compound cannot be considered fully identified in a mixture unless two other questions are addressed: Is the identification plausible? Why is it present in a given sample? 51

52. Applications of GC-MS Petrochemical and hydrocarbons analysis Geochemical research Forensic (arson, explosives, drugs, unknowns) Environmental analysis Pesticide analysis, food safety and quality Pharmaceutical and drug analysis Clinical toxicology Food and fragrance 52

53. Applications of GC-MS Criminal forensics GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. accelerant is significant evidence in a fire investigation because it suggests that the fire was set intentionally. 53

54. Law enforcement GC-MS is increasingly used for detection of illegal narcotics marijuana, cocaine, opioids Clinicians oxycodone and oxymorphone Piperazines are not detectable by typical immunoassay testing, but they may be detectable via GC-MS Sports anti-doping analysis 54

55. Astrochemistry Several GC-MS have left earth. Two were brought to Mars by the Viking program. Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS. 55

56. Petrochemical and hydrocarbons analysis PONA is an acronym for Paraffins, Olefins, Naphthenes and Aromatics. Environmental monitoring and cleanup GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. 56

57. Medicine Inborn error of metabolism are now detectable by newborn screening tests, especially the testing using gas chromatography–mass spectrometry. possible to test a newborn for over 100 genetic metabolic disorders by a urine test at birth based on GC-MS 57

58. Security Thermo Detection (formerly Thermedic explosive detection systems have become a part of all US airports. These systems run on a host of technologies, many of them based on GC-MS. 58

59. Food, beverage and perfume analysis Foods and beverages contain numerous aromatic compounds identification and in Foodpairing 59

60. Cost The major factor influencing the cost  ionization methods available on the instrument and the  mass range of the mass spectrometer. only electron impact ionization and have a mass range of 20 to 700 cost about $50,000. Those capable of both CI and EI and with mass ranges of 20 to 2000 cost about $200,000. Operating costs In most laboratories are about 5% of the instrument cost per year. 60

61. Limitation Only compounds with vapor pressures exceeding about 10 –10 torr can be analyzed by gas chromatography-mass spectrometry (GC-MS). Determining positional substitution on aromatic rings is often difficult. Certain isomeric compounds cannot be distinguished by mass spectrometry (for example, naphthalene versus azulene), but they can often be separated chromatographically. 61

62. Good Practise Always wear clean, lint-free, nylon gloves when handling parts which will come in contact with the sample stream. Before login, please check the standard spectrum to make sure the machine is in good condition for the day. to avoid possible carryover from previous sample, run a blank. Don’t overload the machine with too concentrated sample  Concentration >0.001mg/mL may carryover. If there is a problem, please notify respective company for help 62

63. Required Level of Training and Maintenance The required level of training and expertise varies as a function of the level of data interpretation and instrument maintenance For interpretation of the data, some chemistry training is needed, particularly organic chemistry Refreshable courses specific in mass spectrometry through 1- to 2-week offered through professional societies (such as the American Chemical Society or the American Society for Mass Spectrometry). 63

64. Conclusions Gass Chromatography mass spectrometry is 64

65. References McLafferty, F. W.Hertel, R. H. and Villwock, R. D. (1974), "Probability based matching of mass spectra. Rapid identification of specific compounds in mixtures". Organic Mass Spectrometry 9 (7): 690– 702 Amirav, A.Gordin, A. Poliak, M. Alon, T. and Fialkov, A. B. (2008), "Gas Chromatography Mass Spectrometry with Supersonic Molecular Beams". Journal of Mass Spectrometry 43: 141–163. R. A. Hites and K. Biemann (1968), Analytical Chemistry, 40,1217–21. McMaster, C.McMaster, Marvin C. (1998). GC/MS: a practical user's guide. New York: Wiley. 65

66. References R. S. Gohlke (1959), Analytical Chemistry, 31 535–41. J. T. Watson and K. Biemann (1965), Analytical Chemistry, 37, 844–51. R. Ryhage (1964), Analytical Chemistry, 36, 759–64. T. E. Jensen and others (1982), Analytical Chemistry, 54, 2388–90. Giannelli, Paul C. and Imwinkelried, Edward J. (1999). Drug Identification: Gas Chromatography. In Scientific Evidence 2, pp. 362. R. A. Hites and K. Biemann, Analytical Chemistry, 42 (1970), 855–60. 66

67. THANK YOU HAVE A NICE DAY Questions ??? 67