Mass Ionisa Techni MKR 3 6 11

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1 Ionization Techniques In mass Spectroscopy

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2 Ionization Techniques In Mass Spectroscopy Mass Spectroscopy Mass spectroscopy is a technique in which the sample is converted into rapidly moving positive ions which are then separated and characterized. Mass spectroscopy deals with the study of the charged molecules and fragment ions produced from a sample exposed to ionizing conditions, and also of the relative intensity spectrum which results from the correlation of the ions with their mass to charge ratio (Mass Number), and designated as m/e.

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3 Mass Spectrometer It is an instrument used which is used to obtain the mass spectra of a sample. Functions Creates gaseous ion fragments from the sample. Sorts these ions according to mass. Measures the relative abundance of ion fragments . Mass Spectrum Mass spectrum is a record of relative number of different kinds of ions and is characteristic of every compound.

components of mass spectrometer includes :

4 components of mass spectrometer includes Ion Source Mass Analyzer Ion collection System or Ion detector. Data Handling System Vacuum System Inlet System

Instrumentation of Mass Spectrometer:

5 Instrumentation of Mass Spectrometer Ion sources Inorganic Mass spectrometry Organic Mass spectrometry Thermal/surface ionisation Electron ionisation/bombardment Radio frequency spark source Glow discharge source Inductively coupled plasma source Secondary ion source Electron impact ionization (EI) Chemical ionization (CI) Field ionization (FI) Field desorption (FD) Fast atom bombardment (FAB) Plasma desorption (PD) Laser desorption (LD) Electrospray ionization (ESI) Matrix assisted laser desorption/ionization (MALDI)

Instrumentation of Mass Spectrometer:

6 Instrumentation of Mass Spectrometer

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7 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).

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8 Desorption Sources. Field Desorption (FD) Electrospray Ionization (ESI). Matrix Assisted Laser Desorption/Ionisation (MALDI). Plasma Desorption (PD). Fast Atom Bombardment (FAB). Thermospray Ionization (TS). Secondary Ion Mass Spectrometry (SIMS). ION SOURCE

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9 Ionisation Volatile Thermal Size Amount Examples EI Yes Stable Small 1-2mg Organics CI Yes Stable Small 1-2mg Organics FI Yes Stable Small 1-2mg Organics FAB No Labile Medium 50 μ g-1mg Polar/ionic organics,peptides,biomolecules,organometallics FD No Labile Medium 1-2mg Non-polar organics,organometallics, porphyrins, MALDI No Labile Large 250fmol- 500pmol Peptides, Proteins, DNA/RNA, polymers, dendrimers ESI No Labile Large 1-300pmol/ μ L Polar/ionic organics,peptides,biomolecules,organometallics, proteins, polymers

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10 Gas Phase Ionization Methods Electron Impact Ionization

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12 Gas Phase Ionization Methods Electron Impact Ionization 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.

Gas Phase Ionization Methods:

13 Gas Phase Ionization Methods These electrons are drawn in the ionization chamber through positively charged slits. The number of electrons is controlled by filament temperature and energy of energy is controlled by filament potential. The sample is brought to a temperature high enough to produce molecular vapors. The gaseous neutral molecules then pass through the molecular leaks and enter the ionization chamber. Electron Impact Ionization Gas Phase Ionization Methods Electron Impact Ionization

Gas Phase Ionization Methods:

14 Gas Phase Ionization Methods 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. Electron Impact Ionization M Analyte molecule e - Electrons M + Molecular ions

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15 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). Strong electrostatic field (400 – 4000 V) applied between the first and second accelerating plates accelerates the ions according to their masses (m 1 , m 2 , m 3 etc) to their final velocities. Gas Phase Ionization Methods Electron Impact Ionization

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16 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 (m 1 v 1 ) = ½ (m 2 v 2 ) = ½ (m 3 v 3 ) z = charge of the ion V = accelerating potential v = velocity of ion

Gas Phase Ionization Methods:

17 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 . Gas Phase Ionization Methods Electron Impact Ionization

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18 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.

Gas Phase Ionization Methods:

19 Gas Phase Ionization Methods 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. Chemical Ionization

Gas Phase Ionization Methods:

20 Gas Phase Ionization Methods Chemical Ionization

Gas Phase Ionization Methods:

21 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. Gas Phase Ionization Methods Chemical Ionization

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22 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, iso-butane, ammonia, nitrous oxide, oxygen and hydrogen etc. Depending upon the type of ions formed CI is categorized as:- Positive Chemical Ionization. Negative Chemical Ionization.

     A possible mechanism for ionization in CI occurs as follows :

23 A possible mechanism for ionization in CI occurs as follows Reagent (R) + e- → R + + 2 e- R + + RH → RH + + R RH + + Analyte (A) → AH + + R

Gas Phase Ionization Methods:

24 1. Positive Chemical Ionization In this technique positive ions of the sample are produced. In positive chemical ionization gasses such as Methane, Ammonia, Isobutane etc are used For example:- Ammonia is used as reagent gas. First ammonia radical cations are generated by electron impact and this react with neutral ammonia to form ammonium cation (reactive species of ammonia in CI). NH 3 NH 3 .+ + 2 e - NH 3 .+ NH 4 + + NH 2 e - Gas Phase Ionization Methods Chemical Ionization

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25 NH 4 + reacts with the sample molecules by proton transfer or adduct formation to produce sample ions M + NH 4 + [M + H] + + NH 3 Proton transfer M + NH 4 + [M + NH4] + adduct formation

Gas Phase Ionization Methods:

26 When Methane is used as Reagent gas. Methane is ionized by electron impact: CH 4 + e - CH 4 + + 2e - Primary ions react with additional reagent gas molecules to produce stabilized reagent ions: CH 4 + + CH 4 CH 5 + + CH 3 CH 3 + + CH 4 C 2 H 5 + + H 2 The reagent ions then react with the sample molecules to ionize the sample molecules: CH 5 + + MH CH 4 + MH 2 + (Proton transfer) CH 3 + + MH CH 4 + M + (hydride abstraction) CH 4 + + MH CH 4 + MH + (Charge transfer ) Gas Phase Ionization Methods Chemical Ionization

Gas Phase Ionization Methods:

27 Negative chemical ionization is counterpart of Positive chemical ionization. Negative ions of the sample are formed; oxygen and hydrogen are used as reagent gasses. This method is used for ionization of highly electronegative samples. The negative ions are formed by following reactions :- Resonance electron capture M + e - M - Dissociative electron capture RCl + e - R + Cl - H 2 O + e - H + OH - 2. Negative Chemical Ionization Gas Phase Ionization Methods Chemical Ionization

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28 The ion molecule reaction occurring between negative ion formed in the chamber source and the sample molecule include:- Charge transfer. Hydride transfer. Anion- Molecule adduct formation.

Gas Phase Ionization Methods:

29 Advantages: Used for high molecular weight compounds. Used for samples which undergo rapid fragmentation in EI. Limitations: Not suitable for thermally unstable and non-volatile samples. Relative less sensitive then EI ionization. Samples must be diluted with large excess of reagent gas to prevent primary interaction between the electrons and sample molecules Gas Phase Ionization Methods Chemical Ionization

Gas Phase Ionization Methods:

30 Atmospheric Pressure Chemical Ionization (APCI) APCI produces ions using a reagent gas generated from solvent vapour. The solvent - a mixture of methanol, acetonitrile and water at 0.5 ml/min - is supplied to the APCI probe by a pump (either from HPLC or LC). Liquid spray is produced by passing co-axial nebuliser gas (nitrogen). The solvent spray is vaporized by heating. Once formed, the vapour emanates from a corona pin held at 3 kV. Gas Phase Ionization Methods Chemical Ionization

Gas Phase Ionization Methods:

31 The sample ions are then accelerated out of the atmospheric pressure source and into the mass analyzer by application of a small voltage (typically 20-70 V) to the skimmer cone. The pressure differential between source and analyzer regions is maintained by the presence of an area of intermediate vacuum. Little energy is transferred to the sample molecule during ionization as a result fragmentation is minimal. During acceleration of the sample ions through the hot solvent vapour collisional activation and subsequent fragmentation may occur. Gas Phase Ionization Methods Atmospheric Pressure Chemical Ionization

Gas Phase Ionization Methods:

32 The electric field is sufficiently strong to ionize solvent vapour by either removal (positive ion mode) or donation (negative ion mode) of an electron. Ion/molecule reactions then result in the formation of a reactive species. Ex:- with Methanol: Positive Ion AP-CI Negative Ion AP-CI Gas Phase Ionization Methods Atmospheric Pressure Chemical Ionization

Gas Phase Ionization Methods:

33 Acid-base reaction then takes place between the sample and reagent gas, resulting in protonation (positive ion mode) or deprotonation (negative ion mode) of the sample molecule (M). Positive In AP-CI Negative Ion AP-CI Gas Phase Ionization Methods Atmospheric Pressure Chemical Ionization

Gas Phase Ionization Methods:

34 Gas Phase Ionization Methods Atmospheric Pressure Chemical Ionization

Gas Phase Ionization Methods:

35 Applications of APCI APCI is suitable for the analysis of organic compounds with medium - high polarity. Since positive ionization is dependent on protonation, molecules containing basic functional groups such as amino, amide esters, aldehyde/ketone and hydroxyl can be analyzed. Negative ionization depends upon deprotonation, molecules containing acidic functional groups such as Carboxylate, Phenol and Imide are analyzed by this method. Can be used as LC/MS interface Gas Phase Ionization Methods

Gas Phase Ionization Methods:

36 Field Ionization FI is used to produce ions from volatile compounds that do not give molecular ions by EI. It produces molecular ions with little or no fragmentation. Gas Phase Ionization Methods

Gas Phase Ionization Methods:

37 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 (10 7 - 10 8 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. Gas Phase Ionization Methods Field Ionization

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38 The electric field is produced by applying high voltage ( 20 KV ) to these specially formed emitters ( made up of thin tungsten wire ). In order to achieve high potential gradients necessary to effect ionization, the anode is activated by growing carbon micro-needles or whiskers.

Gas Phase Ionization Methods:

39 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. Gas Phase Ionization Methods Field Ionization

Gas Phase Ionization Methods:

40 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. Gas Phase Ionization Methods Field Ionization

Gas Phase Ionization Methods:

41 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 Disadvantages : Gas Phase Ionization Methods Field Ionization

Desorption Sources:

42 In field desorption method a multitipped emitter (made up of tungsten wire with carbon or silicon whiskers grown on its surface) is used. 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 micro-syringe. 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. Field desorption Desorption Sources

Desorption Sources:

43 Ionization takes place by quantum mechanical tunneling mechanism which involves transfer of electron from the sample molecule to the anode (emitter). This results in formation of positive ions which are radical ions (M + ) and cations attached species such as (M+Na) + . (M+Na) + are produced during desorption by attachment of trace alkali metal ions present in analyte. Advantages Works well for small organic molecules, low molecular weight polymers and petrochemical fractions. Desorption Sources

Desorption Sources:

44 Sensitive to alkali metal contamination. Sample must be soluble in a solvent. Not suitable for thermally unstable and non volatile samples. Structural information is not obtained as very little fragmentation occurs. Disadvantages Desorption Sources

Extrel’s Thermal Desorption Systems:

45 Extrel’s Thermal Desorption Systems

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46 Thermal Desorption Systems (TDS) , (complete with mass spectrometer, vacuum system and pumps, mounting rack, sample heater with control and pressure measurement), provide high sensitivity temperature programmed desorption analysis with precise temperature control. TDS Systems can be configured for a number of different sample sizes (up to 300 mm), temperature ranges and mass ranges.

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47 Specifications and Advantages of Extrel’s TDS/TDA Systems include: High Precision Proportional Integral Derivative (PID) Controller for Temperature Ramp High Speed, Multi-Component Trend Analysis Mass Ranges of 1-60, 1-300, 1-500, 1-1000 amu Partial Pressure Detection limits down to 10-16 Torr Temperature Gradient across the wafer > 4% Up to 500oC Maximum Temperature

Desorption Sources:

48 Electro-spray ionization The method generates ions from solution of a sample by creating fine spray of charged droplets. A solution of sample is pumped through a fine, charged stainless steel capillary needle at a rate of few microlitres/minute. The needle is maintained at a high electric field (several kilovolts) with respect to cylindrical electrode. Desorption Sources

Electro-spray ionization:

49 Electro-spray ionization

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Desorption Sources:

52 The liquid pushes itself out of the capillary as a mist or aerosol of fine charged droplets. Set of aerosol droplets is produced by a process involving formation of Taylor cone and a jet from the tip of this cone. These charged droplets are then passed through desolvating capillary where the solvent is evaporated in the vacuum and attachment of charge to the analyte molecules takes place. Desorption Sources

Desorption Sources:

53 Desolvating capillary uses warm nitrogen as nebulizing gas. The desolvating capillary is maintained under high pressure. As the droplets evaporate the analyte molecules comes closer together. These molecules become unstable as the similarly charged molecules comes closer together and the droplets explode once again. This is referred as Coulombic fission . The process repeats itself until the analyte is free from solvent and is lone ion. The ion then moves to the mass analyzer . Desorption Sources

Desorption Sources:

54 In this method quassimolecular ions are produced by addition of a proton (hydrogen ion) to give (M+H) + or other cations such as sodium ion (M+Na) + or removal of hydrogen ion (M-H). Multiply charged ions are often observed and these ions are even electron species indicating that electrons have neither been added nor removed. Desorption Sources

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Advantages:

59 Advantages Used for analysis of high molecular weight biomolecules such as polypeptides, proteins, oligonucleotides and synthetic polymers. Can be used along with LC and capillary electrophoresis. softest ionization technique. Can be combined with a number of methods Useful for large molecules M + , M 2+

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60 practical mass range of up to 70,000 Da good sensitivity with femtomole to low picomole sensitivity typical softest ionization method, capable of generating noncovalent complexes in the gas phase easily adaptable to liquid chromatography easily adaptable to tandem mass analyzers such as ion traps and triple quadrupole instruments multiple charging allows for analysis of high mass ions with a relatively low m/z range instrument no matrix interference

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61 Disadvantages Multiply charged ions are confusing and needs careful interpretation. Sensitive to contaminants such as alkali metals or basic compounds. Not suitable for low polarity compounds.

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62 the presence of salts and ion-pairing agents like TFA can reduce sensitivity complex mixtures can reduce sensitivity simultaneous mixture analysis can be poor multiple charging can be confusing especially in mixture analysis sample purity is important carryover from sample to sample

Desorption Sources:

63 In this method ionization is carried out by bombarding a laser beam on the sample dissolved in a matrix solution. Desorption Sources Matrix Assisted Laser Desorption/Ionization (MALDI)

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JMS-S3000 SpiralTOF™ Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometer :

65 JMS-S3000 SpiralTOF™ Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometer

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66 JEOL offers an original target plate. The target plate has 384 MTP sample spots and one calibration spot per 4 sample spots. A unique ID is assigned to each target plate. The system automatically identifies a target plate when it is loaded. The ID is saved with the data acquired.

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Desorption Sources:

68 MALDI matrix – A nonvolatile solid material facilitates the desorption and ionization process by absorbing the laser radiation. As a result, both the matrix and any sample embedded in the matrix are vaporized. The matrix also serves to minimize sample damage from laser radiation by absorbing most of the incident energy. Absorb the laser energy. Prevent analyte agglomeration. Protect analyte from being destroyed by direct laser beam. Desorption Sources

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69 Matrix consists of a crystallized molecules of which the most commonly used are :- 3,5 – dimethoxy – 4 – hydroxy cinnamic acid (sinapinic acid). α – cyano – 4 – cinnamic acid (α – cyano or α – matrix). 2,5 – dihydroxy benzoic acid (DHB)

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70 UV MALDI Matrix List Compound Abbreviation Mass (Da) Solvent Wavelength (nm) Applications 2,5-dihydroxy benzoic acid DHB 154 ACN, water, methanol, acetone, chloroform 337, 355, 266 peptides, nucleotides, oligonucleotides, oligosaccharides 3,5-dimethoxy-4-hydroxycinnamic acid sinapic acid; sinapinic acid; SA 224 ACN, water, acetone, chloroform 337, 355, 266 peptides, proteins, lipids 4-hydroxy-3-methoxycinnamic acid ferulic acid 194 ACN, water, propanol 337, 355, 266 proteins α-cyano-4-hydroxycinnamic acid CHCA 189 ACN, water, ethanol, acetone 337, 355 peptides, lipids, nucleotides Picolinic Acid PA 123 Ethanol 266 oligonucleotides 3-Hydroxypicolinic acid HPA 139 Ethanol 337, 355 oligonucleotides Some of the more commonly used matrices are

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71 Solution of the matrix is made in a mixture of highly purified water and another organic compound (acetonitrile or ethanol). Triofluoro acetic acid (TFA) is also added. If sinapinic acid is used as a matrix the solution is prepared by adding: 20 mg/ml of sinapinic acid. Water: acetonitrile: TFA (50:50:0.1).

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73 Matrix solution is then mixed with the analyte to be investigated. The organic compound acetonitrile dissolves hydrophobic proteins present in the sample while water dissolves hydrophilic proteins. The solution is then spotted in a air tight chamber on the tip of the sample probe.

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74 With a vacuum pump the air is removed and vacuum is created which leads to evaporation of the solvent leaving behind a layer of recrystalized matrix containing analyte molecules. Laser Wavelength (nm) Reference Nitrogen Laser 337 (Tanaka 1988) CO 2 10600 (Overberg 1991)

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75 Laser desorption methods involves interaction of pulsed laser beam with the sample to produce both vaporization and ionization. Laser beam is usually of different wavelengths from far U.V to far IR depending upon the sample to be analyzed. Requirements Laser wavelength must be at absorption wavelength of the molecule. In order to avoid decomposition absorbed energy must be quickly dispersed in the molecules . Desorption Sources Laser desorption

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76 The solid mixture is then exposed to pulsed laser beam. The matrix absorbs the laser energy and transfers some of this energy to the analyte molecules which results in the sublimation of sample molecules as ions or the matrix after absorbing the laser energy gets ionized and transfer part of this charge to the sample molecules and ionize it. Nitrogen or carbon lasers are most commonly used.

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77 The ions produced in this process are quassimolecular ions that are ionized by addition of proton (M+H) + or a cation such as sodium (M+Na) + or removal of a proton (M-H) - . It generally produces singly charged ions in some cases doubly charged ions such as (M+2H) 2+ are also observed. Desorption Sources

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78 The chamber consists of two electrodes and the ions are produced between the electrodes. When the polymers form cations the cathode is placed right behind the sample and anode in front of the sample. The cations get attracted towards the negatively charged anode. This acceleration is used to move the ion to the detector. When the polymer forms anions the electrodes are interchanged

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79 Atmospheric pressure-matrix assisted laser desorption AP-MALDI is a variant of MALDI which is carried out at atmospheric pressure (760 torr). AP-MALDI is performed using an instrument similar to ESI source with spray replaced by a sample probe or MALDI target. Main difference MALDI and AP- MALDI is the pressure at which ions are produced. In MALDI ions are produced at 10 mtorr while in AP- MALDI ions are formed at (760 torr) atmospheric pressure as a result AP- MALDI provides better and faster cooling which makes it softer ionization technique than MALDI . Desorption Sources

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80 Ionization is carried out by two techniques :- Microprobe techniques Laser beam is focused to a very small spot on the back side of a thin metal foil that holds a thin film of sample. Ions emerge out on the front side from a small cratered hole in the foil.

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81 Bulk analysis techniques The laser beam produces micro-plasma that consists of neutral fragments with elementary and fragment ions. The ions produced are largely protonated and deprotonated species that have a unit charge.

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82 Advantages Used for larger biomolecules such as proteins and carbohydrates. It has since become a widespread analytical tool for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids). The efficient and directed energy transfer during a matrix-assisted laser-induced desorption event provides high ion yields of the intact analyte, and allows for the measurement of compounds with sub-picomole sensitivity. In addition, the utility of MALDI for the analysis of heterogeneous samples makes it very attractive for the mass analysis of complex biological samples such as proteolytic digests.

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83 practical mass range of up to 300,000 Da. Species of much greater mass have been observed using a high current detector; typical sensitivity on the order of low femtomole to low picomole. Attomole sensitivity is possible; soft ionization with little to no fragmentation observed; tolerance of salts in millimolar concentrations; suitable for the analysis of complex mixtures.

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84 Disadvantage Laser pulse lasts only for a few micro seconds, suitable mass analyzers are limited to time-of-flight and Fourier Transform spectrometers. Molecules of molecular weight less than 1000 Da for biopolymers and 10000 Da for synthetic polymers cannot be studied as they get decomposed.

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85 matrix background, which can be a problem for compounds below a mass of 700 Da. This background interferences is highly dependent on the matrix material; possibility of photo-degradation by laser desorption/ionization; acidic matrix used in MALDI my cause degradation on some compounds.

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86 Applications Used in proteomics. Estimation of DNA, RNA and oligosaccharides. Used in analysis of lipids, phosphopeptides and synthetic polymers. widespread analytical tool for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).

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88 Plasma desorption produces molecular ions from the samples coated on a thin foil when a highly energetic fission fragments from the Californium -252 “blast through” from the opposite side of the foil. The fission of Californium -252 nucleus is highly exothermic and the energy released is carried away by a wide range of fission fragments which are heavy atomic ion pairs. Ion pair fission fragments depart in opposite directions. Each fission of this radio active nucleus gives rise to two fragments traveling in opposite directions (because necessity of momentum conversation). Desorption Sources Plasma desorption

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89 A typical pair of fission fragments is 142 Ba 18+ and 106 Tc 22+ , with kinetic energies roughly 79 and 104 MeV respectively. When such a high energy fission fragments passes through the sample foil, extremely rapid localized heating occurs, producing a temperature in the range of 10000 K. Consequently, the molecules in this plasma zone are desorbed, with the production of both positive and negative ions. These ions are then accelerated out of the source in to the analyzer system. Desorption Sources

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90 It is an ionization technique in which the analyte and non-volatile liquid matrix mixture is bombarded by a high energy beam of inert gas such as Argon or Xenon. Desorption Sources Fast Atom Bombardment

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92 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. Desorption Sources Fast Atom Bombardment

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93 m -nitrobenzyl alcohol (NBA) glycerol

Fast Atom Bombardment:

94 Fast Atom Bombardment 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.

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95 During this passage high energy cation obtain electrons from Xenon atoms to become high energy atoms (Xe). The lower energy ions are removed by electrostatic deflector. (Xe) ++ Xe .+ + Xe (Xe) .+ + Xe (Xe) + Xe .+ The analyte is dissolved in the liquid matrix such as glycerol and applied as a thin layer on the sample probe shaft. Desorption Sources Fast Atom Bombardment

Fast Atom Bombardment:

96 Fast Atom Bombardment The mixture is bombarded with the high energy beam of Xenon atoms. Xenon ionizes the glycerol molecules to give glycerol ions. These ions react with the surrounding glycerol molecules to produce (G+H) + as reactant ions. The sample molecules then undergo proton transfer or hydride transfer or ion-pair interaction with reactant ions to give quassimolecular or psuedomolecular ions such as (M+H) + , (M-H) - or (M+G+H) + .

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97 Advantages Used for ionization of polar high molecular weight samples. Provides rapid heating of samples and reduces sample fragmentation. Rapid ionization. Desorption Sources These ions are then extracted from slit lens system designed to collect ions and directed to mass analyzer. Fast Atom Bombardment

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98 Disadvantages Difficult to distinguish between low molecular weight compounds. Compounds must be soluble in liquid matrix. Not good for multiply charged compounds

Desorption Sources:

99 Desorption Sources SIMS is a measurement technique that is being used for the compositional analysis of small samples. In a SIMS instrument (or " ion microprobe ") a high energy primary ion beam is directed at an area of the sample whose composition is to be determined . Secondary ion mass spectrometry (SIMS)

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100 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. Desorption Sources Secondary ion mass spectrometry

Secondary ion mass spectrometry:

101 Secondary ion mass spectrometry Argon or Cesium gas is ionized by heating the filament to produce mono-energetic 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.

Secondary ion mass spectrometry:

102 Secondary ion mass spectrometry

Secondary ion mass spectrometry:

103 Secondary ion mass spectrometry

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104 The interaction of the primary ions with the sample surface ("sputtering") has three major effects: (1) It leads to a mixing of the upper layers of the sample, resulting in an amorphization of the surface; (2) atoms from the primary ion beam are implanted in the sample and (3) some secondary particles (atoms and small molecules) are ejected from sample. Among the ejected particles , Charged particles of one polarity ("secondary ions") can then be extracted from the sputtering area with the help of an electrical field between the sample and an extraction lens.

Secondary ion mass spectrometry:

105 Secondary ion mass spectrometry These accelerated secondary ions constitute a secondary ion beam which is then led into a mass spectrometer. There, the secondary ions are sorted by mass (and energy) and finally counted in an ion detector The count rates of different secondary ion species give information about the composition of the sample in the sputtered area. Since the size of the sputtered area depends only on the primary ion beam diameter, which typically is in the order of micro-meter, a SIMS analysis has a relatively high lateral resolution.

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106 This results in the formation of secondary sample ions by charge transfer interaction between the sample molecules and the primary gas ions 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. Desorption Sources Secondary ion mass spectrometry

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107 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 evaporated. As the sample evaporates it undergoes ionization. Desorption Sources

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108 The probability of ionization is predictable and is a function of work function :- Ionization potential of the sample = E1 Work function of the filament material = Φ Filament temperature = T This can be summarized as follows n + /n 0 = exp[z(Φ – E1)/KT] z = electronic charge K = Boltzmann’s constant n + = Number of ions formed n O = Number of neutral species Desorption Sources

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109 Principles of Instrumental analysis. Fifth Edition by Douglas. A. Skoog, F. James Holler and Timothy A. Nieman. Page No. 499 – 511. Instrumental Methods Of Analysis. Seventh Edition by Willard Meritt. Page No. 468 – 74. http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Lecture/oxmain_lectureCI.html http://www.astbury.leeds.ac.uk (A.E. Ashcroft's MS web pages and tutorial) "http://en.wikipedia.org/wiki/Atmospheric_pressure_chemical_ionization References

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110 Thankyou