Gas chromatography mass spectrometry

a brief view of gcms

1. Gas chromatography-mass spectrometry S.Bhagya M.pharmacy Dept of Pharmacology

2. CONTENTS INTRODUCTION - HYPHENATED TECHNIQUES - GCMS BASIC PRINCIPLES -GAS CHROMATOGRAPHY -MASS SPCTROMETRY -GCMS INSTRUMENTATION APPLICATIONS

3. INTRODUCTION HYPHENATED TECHNIQUE: A hyphenated technique is combination (or) coupling of two different analytical techniques with the help of proper interface. Mainly chromatographic techniques are combined with spectroscopic techniques. In the chromatography, the pure or nearly pure fractions of chemical components in a mixture was separated and spectroscopy produces selective information for identification using standards or library spectra. “The coupling of the separation technique and an on-line spectroscopic detection technology will lead to a hyphenated technique.” The term “hyphenation” was first adapted by Hirschfeld in 1980.

4. Advantages of hyphenated techniques: Shorted analysis time, Fast and accurate Reduction of contamination due to its closed system Higher degree of automation Higher sample throughput Both separation and quantification at same time Better reproducibility. TYPES OF DIFFERENT HYPHENATED TECHNIQUES: Gas chromatography-Mass spectrometry (GC-MS) Gas chromatography-Infra red spectroscopy Gas chromatography-Infra red spectroscopy-Mass spectroscopy Gas chromatography-Thin layer chromatography Liquid chromatography-Mass spectrometry Capillary electrophoresis-Mass spectrometry.

5. 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. The use of a mass spectrometer as the detector in Gas chromatography was developed during 1950s by ROLAND GOHIKE and Mc LAFFERTY. GC= separation; MS= Identification.

6. GCMS Gas chromatography–mass spectrometry (GC MS) is an analytical method that combines the features of gas-chromatography and mass spectrometry to identify different substances within a test sample. Gas chromatography is a technique capable of separating, detecting and partially characterizing the organic compounds particularly when present in small quantity. Mass spectroscopy provides some definite structural information from in small quantity. The separation and identification of the components of complex natural and synthetic mixture are achieved more quickly than any other technique with less sample. Compounds that are adequately volatile, small, and stable in high temperature in GC conditions can easily be analyzed by GC-MS.

7. Ga s C h r o m a tog r aph y / M as s S p ec t r om e t r y (G C -MS) Gas Chromatography Mass spectrometry Gas chromatography-Mass spectrometry. Separates mixture of chemicals so each can be identified individually. Identifies (de t ects) che m ic a ls based on their m olecul a r weight or m ass. A Chemical Analysis Technique combining two instruments to provide for powerful separation and identification capabilities.

8. PRINCIPLE G A S C H R OM A T OGRAPH Y PRINCIPLES: May be Gas Liquid [ G L C] or Gas Solid C h ro m atography [ G S C] but G L C i s preferred. GLC works by part i t i on but G S C works by adso r pt i on. In G LC the subs t ance to be stud i ed first conve r ted to gas which works as the m obi l e phase. M A S S SPEC T R OS C O PY PRINCIPLES: Io n F or m ation Io n Det e ction & Separation.

9. ION F OR M A TION : T e c h n iqu e s used to form i o n s a r e: Elec t ron Io n i z a t ion C h e m i c al Io n i z a t ion Field Ionization De s orption Ioniz a t i on Se c o n dary Ion Ma s s S p e c tros c o p y [SIMS] Plasma Desorption [PD] Field Desorption [FD] F as t Atom B o m bardm e nt [ F AB] Mat r ix Assoc i a t ed Las e r De s or p t i on Io n i z a t ion [MAL D I] Thermo spray Ionization. Electron spray Io n i z a t ion.

10. Ion sources: The ion source is the part of the mass spectrometer that ionizes the material under analysis (analyte). The ions are then transported by magnetic or electric fields to the mass analyzer. Molecular ions are formed when energy of the electron beam reaches 10-15 eV. Fragmentation of the ion reaches only at higher bombardment energies at 70 eV.

11. Ionizing agents in mass spectroscopy

12. Ionization methods in mass spectroscopy

13. Electron impact I n t h e E l e c t ron I m pa ct ( E I ) p r o c e s s , e le c t r o n s a re e mi tt e d f r o m a he a t e d fi l am e n t ( u s u ally m a d e of t u n g s t e n o r r h en ium ) a n d a re a c c el e r a t e d a c ro ss t h e s o u r ce b y u sing an appropriate potential (5-100V) to a c h i e ve t h e r equi r e d e le c t r o n ene r g y ( s u f f i c i e n t to io n i ze the molecule).

14. Chemical Ionization A reactive gas like methane is introduced along with the sample into the ionization chamber. When the reactive gas bombards with the electron beam it undergoes ionization to produce ions which further react with neutral molecules to form chemically reactive species that interact with the sample molecules to produce positive ions.

15. Field Ionization The analyte molecules exposed to a very high electrostatic field of about 10 -10V/cm, which is about 1000 times higher than that of a typical MALDI ion source. Thin metal wire is used as anode. The anode and cathode is placed very closely to the ionization chamber. When the sample is introduced between the electrodes with of high potential the sample ionizes and produce electrons.

16. Fast Atom Bombardment Argon gas is ionized by hot filament and focused beam that bombards the sample. Beam impinges the sample a series of molecular reactions occur and analyze in mass spectroscopy analyzer. Example: Insulin, Amino glycosides.

17. Matrix Assisted Laser Desorption/Ionization MALDI is an LIMS method of vaporizing and ionizing the sample molecules are dispersed in solid matrix such as Nicotinic acid. A UV laser pulse ablates the matrix which carries some of the large molecules into the gas phase in an ionized form so they can be extracted into the Mass Spectrometer.

18. Plasma Desorption This technique was first developed by Macfarlane and Torgerson in 1976. It was first used for the ionization of thermo labile and non volatile molecules including peptides, proteins, nucleotides etc. In this technique, californium252 is used to ionize the sample. The sample is dissolved in a suitable solvent and deposited on a thick nickel foil. The sample foil is then positioned closed to californium 252 source. When fission occurs two fragments having kinetic energy up to 200MeV are released. These fragments are travel in opposite directions one moves towards the sample and other moves towards fission fragment detector.

19. The fragment penetrating into the sample imparts sufficient energy to the sample and thereby causes volatilization and ionization in sample. The resulting samples get desorbed from the surface of the sample into the gas phase. The gaseous ions are then detected and recorded.

20. Electron spray Ionization ESI consist of very fine needle and series of skimmers. A Sample solution is sprayed into source chamber to form droplets. When droplets carry the charge exit the capillary end, as the solvent evaporates , the droplets disappear leaving highly charged analyte molecules.

21. ION S E P ARATION AND DET E CTION: Mass analyzer also called Ion separator After ionization , the gaseous ions enter the mass analyzer where they are separated according to their m/e ratio. It plays a important role in the instrument’s accuracy and mass range. Ty p es of ins t ru m ents used are- Di r e c t Focus i ng T yp e Sing l e Focus i ng Dou ble Focusing Quadrapole Ma s s An aly zer Magnetic sector Mass Analyzer Fourier Transfer Ion cyclotron Resonance T ime Of Fli g ht Ma s s Anal y zer.

22. Quadrapole Mass Analyzer In a Quadrapole m a s s analyzer, a s e t o f four rods a r e a rr a n g e d p a r al le l t o the direction. Here a DC current and radio frequency RF is applied to generate oscillating electrostatic field in between the rods. Based on this only m/z is been determined and stable oscillation takes place. And ion travels in Quadrapole axis with cork screw type of trajectory.

23. Magnetic sector Mass Analyzer In magnetic sector analyzers ions are accelerated through a flight tube, where the ions are separated by charge to mass ratios. As moving charges enter a magnetic field, the charge is deflected to a circular motion of a unique radius in a direction perpendicular to the applied magnetic field. Basically the ions have certain m/e value will have unique path radius, if similar ions passed through magnetic field they will be deflected in same degree.

24. Ion trap Mass Analyzer T he ion t r a p m a ss analyzer operate s by s i m i l a r p r inciple s w h e r e it consists of circular ring electrode Plus two end caps that form a chamber. Here AC or DC power along RF potential is applied between the cups and the ring electrode. There the ions entering into the chamber are trapped by electromagnetic fields and they oscillates in concentric trajectories. Th is p r o c e ss is calle d re s onant eje c t i on.

25. ION DETECTORS Detectors plays important role in identifying charged ions. Different types of detectors used are: - Electron Multiplier Detector - Photo Multiplier tube Detector - Micro channel plate Detector.

26. Electron Multiplier detector

27. This is used when positive and negative ions need to be detected on same instrument. An electron multiplier is a vacuum-tube structure that multiplies incident charges. In a process called secondary emission, a single electron when bombarded on secondary-emissive material, induce emission of roughly 1 to 3 electrons. If an electric potential is applied between this metal plate and yet another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons. This can be repeated a number of times, resulting in a large shower of electrons all collected by a metal anode, all having been triggered by just one.

28. Photomultiplier detector

29. These are typically constructed with an evacuated glass housing containing a photocathode, several dynodes, and an anode. Incident photons strike the photocathode material, which is usually a thin vapor-deposited conducting layer on the inside of the entry window of the device. Electrons are ejected from the surface as a consequence of the photoelectric effect. These electrons are directed by the focusing electrode toward the electron multiplier, where electrons are multiplied by the process of secondary emission. The electron multiplier consists of a number of electrodes called dynodes . Each dynode is held at a more positive potential, by ≈100 Volts, than the preceding one. A primary electron leaves the photocathode with the energy of the incoming photon. This last stage is called the anode. This large number of electrons reaching the anode results in a sharp current pulse that is easily detectable.

30. Micro-channel plate (MCP) It is a planar component used for detection of single particles (electrons, ions and neutrons ) and low intensity impinging radiation (ultraviolet radiation and X-rays). It is closely related to an electron multiplier, as both intensify single particles or photons by the multiplication of electrons via secondary emission. However, because a micro channel plate detector has many separate channels, it can additionally provide spatial resolution.

31. Vacuum system All mass spectrometers need vacuum to allow ions to reach the detector without colliding with other gaseous molecules or Atoms.

32. PRINCIPLE O F W ORKI N G OF GC-MS A ND INTER F A CES PRINCIPLE OF GCMS 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. Then ions are detected by Mass spectrometer. T h e tim e el a p s ed bet w e e n injection and el u sio n is call e d “ r etention time” (t R ) .

33. Samples: Nature: Samples should be organics, must be volatile or semi-volatile thermally stable. State: Organic compounds must be in solution for injection into the gas chromatograph. The solvent must be volatile and organic (for example, hexane or dichloromethane) Amount: Depending on the ionization method, analytical sensitivities of 1 to 100 pg per component are routine. Preparation: Sample preparation can range from simply dissolving some of the sample in a suitable solvent to extensive. Clean up procedures using various forms of liquid chromatography.

34. INTERFACES OF GC-MS 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 SEPARATOR PERMSELECTIVE MEMBERANE MOLECULAR EFFUSION DIRECT INTRODUCTION.

35. Commercially available interfaces are: 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). In these separators, the GC flow is introduced into an evacuated chamber through a restricted capillary. Light particle dispersed away.

36. Direct Interface Most GC-MS interfacing is now done by simply inserting the capillary column directly into the ion source. 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. In this method capillary column is directly inserted into MS ionization chamber.

37. Perm selective membrane interface: It is made of a silicone-rubber membrane that transmits organic non-polar molecules and acts as a barrier for (non-organic) carrier gases.

38. Molecular effusion /Watson- Biemann interface: It is based on the molecular filtering of the gas effluent by means of a porous glass fritted tube.

39. INSTRUMENTATION GAS CHROMATOGRAPHY Gas Chromatography – “It is a process of separating components from the given crude drug by using a gaseous mobile phase.” It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

40. INSTRUMENTATION PARTS OF GC : Carrier Gas Sample Injection System Oven Separation Column Detectors Amplification Recorder.

41. MASS SPECTROSCOPY The mass spectroscopy is an technique in which, the compound under investigation is bombarded with beam of electrons which produce an ionic molecule or ionic fragments of the original species. The mass spectrometer is an instrument in which the substance in gaseous or vapor state is bombarded with a beam of electrons, to form a positively charged ions (cations) which are further sorted according to their mass to charge ratio to record their masses and their abundances.

42. INSTRUME N T ATIO N P ART S OF MS : Sample Handling System Ionizat ion Cha mber Ion Separator or Mass Analyzer Ion Collecto r , Det ector and Rea d Out System V acuum System.

43. INSTRUMENTATION OF GCMS Parts of GCMS: Pneu m at i c cont r ols In j ec t or Oven Colu m n In t erface Ion Source Mass Analyzer Detector V acuum System Control Elec t ron i c s

44. C h r o m at o g ram generated by Gas Chromatography W h i le t he i n s t r u m e n t r un , t he computer generated a graph from signal called chromatogram. X-axis show the Rt. Y-axis show the intensity of the signal.

45. Spectrum generated by Mass Spectrometry Th e computer record a graph for each scan called spectrum The mass spectrum is essentially a fingerprint for the molecule and can be used to identify the compound.

46. I n t e r p ret a t i o n o f R e s u l t s Through GC a CHROMATOGRAM is obtained. Through MS a SPECTRUM is obtained. GC-MS gives a 3D graph which has both chromatogram and spectrum to each separated components in the chromatogram.

47. APPLICATIONS Elucidation of organic, inorganic, and biological molecules. Impurity profiling of pharmaceuticals. Identification of components in Thin layer and Paper chromatography. Identification of drugs of abuse & metabolites of drugs of abuse in blood, urine and saliva. Testing of the presence of the drugs in blood in race horses and in Olympic athletic (in forensic GC-MS). Analysis of aerosol particles. Determination of pesticide residues in food. Polymer characterization. Drug monitoring and toxicological studies.

48. Detection of lipophilic compounds in diverse plant tissues. Analysis of biologically important aromatic amines. Application of human Dosimetry. Identification of volatile components. For the determination of pyrethoid residues in vegetable samples. Environmental and forensic applications. Toxicity assessment. GC-M S is increasi n gl y use d for detectio n o f illegal narcotic s marijua n a , cocain e , opioids , oxycodein e an d oxymorphine. Petrochemica l an d hydrocarbon s analysis. Food, beverages and perfume analysis.

49. Advantages: High sensitivity Excellent detection limits. Typically low ppb to high ppt High selectivity Identification is based on two parameters not one (retention time and mass spectrum must match standard) selects analyte of interest with very high confidence. Typical analysis takes from 1/2 hour to approx. 1 hour analysis can contain upwards of 80 and more pollutants. It can provides sensitive response to most analytes. Good Accuracy and Precision. Disadvantages: Higher capital cost. Higher maintenance (time, expertise and money) For optimum results, it requires analyst of knowledge in both chromatography and mass spectrometry.