Mass spectrometry

Dr.s. H. Burungale

1. Mass spectrometry Dr. S. H. Burungale

2. Components of Mass Spectrometer Inlet system Ion source Ion source Mass analyzer Ion collection or detector Data display

3. ION SOURCE Liquids and solids are first converted in to gases from the gaseous sample, ions are produced in a Box like enclosure called Ion Source. Function • Produces ion without mass discrimination of the sample. • Accelerates ions into the mass analyzer. Classification of ion sources Gas Phase Sources. • Electron Impact Ionization (EI). • Chemical Ionization (CI). • Field Ionizations (FI).

4. Desorption Sources. • Field Desorption (FD). • Electrospray Ionization (ESI). • Matrix assisted desorption/ Ionisation (MALDI). • Plasma desorption (PD). • Fast Atom Bombardment (FAB). • Thermospray Ionization (TS). • Secondary Ion Mass Spectrometry (SIMS).

5. It is the most widely used and highly developed method. It is also known as Electron bombardment or Electron Ionization. Electron impact ionization source consists of a ionizing chamber which is maintained at a pressure of 0.005 torr and temperature of 200 ± 0.25 degrees. Electron gun is located perpendicular to chamber. Electrons are emitted from a glowing filament (tungsten or rhenium) by thermionic emission and accelerated by a potential of 70 V applied between the filament and anode. These electrons are drawn in the ionization chamber through positively charged slits. The number of electrons is controlled by filament temperature and energy . The sample is brought to a temperature high enough to produce molecular vapors.

6. is an ionization method in which energetic electrons interact with solid or gas phase atoms or molecules to produce ions

7. The gaseous Neutral molecules then pass through the molecular leaks and enter the ionization chamber. The gaseous sample and the electrons collide at right angles in the chamber and ions are formed by exchange of energy during these collisions between electron beam and sample molecules. M Analyte molecule e- Electrons M.+ Molecular ions The positive ions formed in the chamber are drawn out by a small potential difference (usually 5eV) between the large repeller plate (positively charged) and first accelerating plate (negatively charged).

8. Strong electrostatic field (400 – 4000 V) applied between the first and second accelerating plates accelerates the ions according to their masses (m1, m2, m3 etc) to their final velocities. The ions emerge from the final accelerating slit as a collimated ribbon of ions. The energy and velocity of ions are given by :- zV = ½ (m1v1) = ½ (m2v2) = ½ (m3v3) z = charge of the ion V = accelerating potential v = velocity of ion

9. Advantages • Gives molecular mass and also the fragmentation pattern of the sample. • Extensive fragmentation and consequent large number of peaks gives structural information. • Gives reproducible mass spectra. • Can be used as GC/MS interface. Disadvantages • Sample must be thermally stable and volatile. • A small amount of sample is ionized (1 in 1000 molecules). • Unstable molecular ion fragments are formed so readily that are absent from mass spectrum.

10. Chemical Ionization In chemical ionization the ionization of the analyte is achieved by interaction of it’s molecules with ions of a reagent gas in the chamber or source. Chemical ionization is carried out in an instrument similar to electron impact ion source with some modifications such as:- • Addition of a vacuum pump. • Narrowing of exit slit to mass analyzer to maintain reagent gas pressure of about 1 torr in the ionization chamber. • Providing a gas inlet.

11. It is a two part process. Step-I Reagent gas is ionized by Electron Impact ionization in the source. The primary ions of reagent gas react with additional gas to produce stabilized reagent ions. step-II Reagent ions interact with sample molecules to form molecular ions. In this technique the sample is diluted with a large excess of reagent gas. Gasses commonly used as reagent are low molecular weight compounds such as Methane, tertiary Isobutane , Ammonia, Nitrous oxide, oxygen and hydrogen etc. Depending upon the type of ions formed CI is categorized as:- • Positive Chemical Ionization. • Negative Chemical Ionization.

12. Application of very strong electric field induces emission of electrons. Sample molecules in vapour phase is brought between two closely spaced electrodes in the presence of high electric field (107 - 108 V/cm) it experiences electrostatic force. If the metal surface (anode) has proper geometry (a sharp tip, cluster of tips or a thin wire ) and is under vacuum (10-6 torr ) this force is sufficient to remove electrons from the sample molecule without imparting much excess energy. The electric field is produced by applying high voltage ( 20 KV ) to these specially formed emitters ( made up of thin tungsten wire ).

13. In order to achieve high potential gradients necessary to effect ionization, the anode is activated by growing carbon microneedles or whiskers. These whiskers are 10 micro meters in length and greater than 1μm in diameters. These whiskers are capable of removing valence electrons from the organic molecules by quantum mechanical tunneling mechanism. As concentration of sample molecules is high at the anode ion-molecule reactions often occur which results in formation of protonated species (M+H)+. Thus both M+ and (M+H) + is observed in FI spectrum .

14. These cations are accelerated out of the source and their mass is analyzed by analyzer . Advantages • As fragmentation is less, abundance of molecular ions (M+) is enhanced, hence this method is useful for relative molecular mass and empirical formula determination. Disadvantages • Not suitable for thermally unstable and non volatile samples. • Sensitivity is les than EI ion source. • No structural information is produced as very little fragmentation occurs

15. DESORPTION SOURCES Field desorption In field desorption method a multitipped emitter (made up of tungsten wire with carbon or silicon whiskers grown on its surface). The electrode is mounted on a probe that can be removed from the sample compartment and coated with the solution of the sample. The sample solution is deposited on the tip of the emitter whiskers either by dipping the emitter into analyte solution or by using a microsyringe . The probe is then reinserted into the sample compartment which is similar to CI or EI unit. Then the sample is ionized by applying a high voltage to the emitter. In some cases it is necessary to heat the emitter by passing a current through the wire to evaporate the sample.

16. FI sample is heated in a vacuum so as to volatize it on to an ionization surfaces It is uesed for volatile and thermally stable compounds FD The sample is placed directly on to the surface before ionization FD is used for nonvolatile compounds or thermally labile substances Comparison of FD and FI

17. ELECTRON SPARY IONIZATION ESI is a most popular ionization technique. • It is a soft ionisation technique. • The electrospray is created by putting a high voltage on a flow of liquid at atmospheric pressure. The charged liquid droplets are produced by atomisation or nebulization . • The created spray is directed in the vacuum system of the mass spectrometer, where these droplets are evaporated by heat in high-vacuum region. • So, the ions are ejected from the droplets and accelerated into the mass analyser by voltages. For larger molecules, the ions may contain multiple charges, allowing the detection of very large molecules on analysers that have limited mass to charge (m/Z)) ratio ranges. • It is mostly used with liquid chromatography (LC). Electrospray ionization (ESI)

18. Advantage:- • Can analyse large biomolecule upto 150-200 Kda • This technique is more sensitive and can accurately measure the sample both in term of quality and quantity. • Suitable for polar compounds. Disadvantage- • Not good for complex mixture • The apparatus is also very difficult to clean • The multiple charges that are attached to the molecular ions can make for confusing spectral data Continued..

19. Advantages • Large (M+H)+ ion identifies molecular weight (M) • Soft ionization technique cause simple fragmentation • Require low energy Disadvantages • Simple fragmentation gives little structural information • Sample must be thermally volatile and stable • Ion source can easily contaminated. Continued .

20. Matrix Assisted Laser Desorption Ionization (MALDI) MALDI is a method of ionization in which the sample is bombarded with a laser. • The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp , Michael Karas and their colleagues. • In 1987, Koichi Tanaka and his co-workers used ultra fine metal plus liquid matrix method that contain 30 nm cobalt particles in glycerol with a 337 nm nitrogen laser for ionization. Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the 34,472 Da protein carboxypeptidase -A. • Tanaka received Nobel Prize in Chemistry in 2002 for demonstrating the proper combination of laser wavelength and matrix for the ionization of protein . Matrix Assisted Laser Desorption Ionization (MALDI)

21. The sample is typically mixed with a matrix that absorbs the laser radiation and transfer a proton to the sample. • Some small mass samples can be ionized without matrix, but this is typically called laser desorption. • Mostly forms singly charged ions and mostly performed on specially built time-of-flight instruments. • The matrix consists of crystallized molecules. • Most commonly used compounds sinapinic acid, α-cyano-4- hydroxycinnamic acid (alpha- cyano or alpha-matrix) and 2,5- dihydroxybenzoic acid(DHB). Continued..

22. Advantage:  Mostly used to analyse biomolecules  High sensitivity  Simple structure  Easy operation & maintenance Disadvantage:  Delayed extraction  Different calibration for different mass range & experimental condition (laser, matrix) Continued..

23. This technique is used for ionization of polar high molecular weight compounds such as polypeptides. Commonly used matrices include :- Glycerol Monothioglycerol Carbowax 2,4 – dipentyl phenol 3 – nitrobenzyl alcohol (3 – NBA) These solvents easily dissolve organic compounds and do not evaporate in vacuum. The bombarding beam consists of Xenon or Argon atoms of high translational energy. This beam is produced by first ionizing the Xenon (or Argon atoms with electrons to give Xenon radical cations. Xe + e- = Xe .+ +2e- The radical cations are then accelerated to 6 – 10 KeV to give radical cations of high translational energy ( Xe )++, which are then passed through a chamber containing Xenon atoms at a pressure of 10-5 torr .

24. Secondary ion mass spectrometry Secondary ion mass spectrometry is nearly identical to FAB except the primary ionizing beam is an ion beam rather than a neutral atom beam. The Cesium or Argon ions are most commonly used. The source consists of a cylindrical grid and a vertically placed ion gun or filament. Argon or Cesium gas is ionized by heating the filament to produce monoenergetic noble gas ions. The ion gun can produce an ion beam of diameter ranging from 0.1mm to 1mm. The ions are accelerated to a potential of 300 to 3000 eV . This ion beam is then bombarded on to the surface of the sample. This results in the formation of secondary sample ions by charge transfer interaction between the sample molecules and the primary gas ions.

25. The ions formed in the cylindrical grid are then extracted from one end and focused on the target or mass analyzer by an electrostatic lens system. Advantages • Higher sensitivity . • Selection of Beam diameter permits for rapid transition from a small. • surface analysis with a small beam to a large surface area. Thermal ionization or Surface ionization Thermal surface ionization source is useful for inorganic solid materials. Samples are coated on a tungsten ribbon filament and then the filament is heated until the sample is evaporates. As the sample evaporates it undergoes ionization

26. MASS ANALYZER Mass analyzer is also called ion separator . Mass analyzer is the Heart of the mass spectrometer that takes ionized masses and separates them based on charge to mass ratios.  Mass analyzer must posses the following characteristics. I . It should have a high resolution. II . It must have a high rate of transmission of ions

27. TYPES OF MASS ANALYZERS  Quadrupole mass analyzer  Time of flight analyzer (TOF)  Magnetic sector mass analyzer Single focusing  Double focusing  Quadrupole ion trap mass analyzer Ion cyclotron resonances

28. It consists of 4 voltage carrying rods.  The ions are pass from one end to another end  During this apply the radiofrequency and voltage complex oscillations will takes place.  Here the single positive charge ions shows the stable oscillation and the remaining the shows the unstable oscillations Mass scanning is carried out by varying each of the rf and voltage frequencies ratios keeping their ratios constant. ◦ Quadrupole ion storage (ion trap) ◦It store the unsorted ions temporarily, they released to the detector by scanning the electric field.

29. Advantage:  Classical mass spectra.  Good reproducibility.  Relatively small and low-cost systems.  Limitations :  Limited resolution.  Peak heights variable as a function of mass.  Not well suited for pulsed ionization methods . Application : In GC/MS and LC/MS systems. Triple quadrupole MS/MS systems. Bimolecular detection.

30. 2.TIME OF FLIGHT MASS ANALYZER (TOF) In this type of analyzer the sorting of ions is done in absence of magnetic field. The ions produced are acquiring different velocities depending on their masses Here the particles reach the detector in the order of the increasing order of their masses  Here electron multiplier detector is used. The resolution power of this is 500-600

31. It has horse shoe shaped glass tube which is evacuated, consists of sample inlet, electron bombarding source, accelerating plates on one end,& collector slit at other end. At curvature of tube there is provision to apply electric/magnetic field Sample in the form vapour is allowed through inlet and bombarded with electron beam at 70eV. It knocks off one electron from every molecule then they become + vely charged ion. As these molecules become + ve charged, they are accelerated by accelerating plates and travel in straight path.

32. By application of electric or magnetic field they travel in curved path & molecular ions are separated according to their masses and collected. Different fragments fall on detector then mass spectrum is recorded.

33. It is used differentiate the small mass differences of the fragment.  These provides the high resolution  To achieve better focusing, energy has to be reduced before ions are allowed to enter the magnetic field and increase resolving power can be obtained two mass analyzers in series.  In a double-focusing mass analyzer beam is first passes radial Electrostatic field. Advantages:  Classical mass spectra.  Very high reproducibility.  High resolution.  High sensitivity.  High dynamic range. 

34. Limitations:  Not well-suited for pulsed ionization methods (e.g. MALDI).  Usually larger and higher cost than other mass analyzers. Applications:  All organic MS analysis methods .  Accurate mass measurement.  Isotope ratio measurements.

35. 4.QUADRUPOLE ION TRAP MASS ANALYZERS There are two principal ion trapped mass analyzers Three- dimensional quadrupole ion traps (" dynamic“ traps), and ion cyclotron resonance mass spectrometers ("static" traps).  Both Operate by storing ions by using DC and RF electric fields.

36. Advantage :  The highest recorded mass resolution of all mass spectrometers.  Powerful capabilities for ion chemistry and MS/MS experiments.  Well-suited for use with pulsed ionization methods such as MALDI.  Non-destructive ion détection ; ion remesurèrent .  Stable mass calibration in superconducting magnet FTICR systems.

37. Limitations:  Limited dynamic range.  Strict low-pressure requirements mandate an external source for most analytical applications . Subject to space charge effects and ion molecule reactions. Applications:  Ion chemistry.  High-resolution electrospray experiments for high-mass analytes .  Laser desorption for materials and surface characterization.

38. DETECTORS Electron multiplier.  Faraday cups .  Micro-channel plate detectors.

39. Continuous dynode electron multiplier .  An electron multiplier (continuous dynode electron multiplier) is a vacuum-tube structure that multiplies incident charges.  In a process called secondary emission, a single electron can, 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 . 

40.   A Faraday cup is a metal (conductive) cup designed to catch charged particles in vacuum .  The resulting current can be measured and used to determine the number of ions or electrons hitting the cup.  When a beam or packet of Ions hits the metal it gains a small net charge while the ions are neutralized.  The metal can then be discharged to measure a small current equivalent to the number of impinging ions.  By measuring the electrical current (the number of electrons flowing through the circuit per second) in the metal part of the circuit the number of charges being carried by the ions in the vacuum part of the circuit can be determined

41.  It is a planar component used for detection of particles (electrons or ions) and 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

42. Mass spectrometry has both qualitative and quantitative uses. 1.Structure elucidation 2.Detection of impurities 3.Quantitative analysis 4.Drug metabolism studies 5.Clinical , toxicological and forensic applications 6.GC-MS-MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.

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