Ionizaion Techniques - Mass Spectroscopy

Various Ionization Techniques used In Mass Spectroscopy
Presented to Ritu Mam
Presented By Pema Chodon
1 – M.Pharma
Dept. Of Pharmaceutics
Al – Ameen College Of Pharmacy Bangalore
Re-Edited by
Suraj C.
1st M.Pharm
AACP
Ionization Techniques In Mass Spectroscopy
Mass Spectrometer
ION SOURCE
• Since the mass analyzer utilizes only gaseous ions i.e., starting point of mass spectrometric analysis is formation of gaseous analyte ions.
• Non –Volatile solids are first converted in to gases and from the gaseous sample the 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 Source:
On the basis of the nature of the substance and the method by which ions are generated the ion sources are classified as
Ion Source
Mass Analyzer
Ion collection System
Data Handling System
Vacuum System
Inlet System
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Gas Phase Sources
 Electron Impact Ionization (EI)
 Chemical Ionization (CI)
 Field Ionizations (FI)
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)
Gas Phase Ionization Methods
1. Electron Impact Ionization
 INTRODUCTION
• Electron impact (EI) is the classical ionization method in mass spectrometry.
• It is the most widely used and highly developed method.
• It is also known as Electron bombardment or Electron Ionization.
 CONSTRUCTION & WORKING:
• 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 of energy is controlled by filament potential.
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• 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 (which is maintained at a pressure of 0.005 torr and a temperature of 200 ± 0.250 C).
 MECHANSIM:
• 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
• In this example, 20eV is transferred to a molecule following it’s collision with a 70eV electron.
• Since the ionization energy of most of the organic molecules is 15eV an electron is expelled to produce a radical cation with 5eV excess energy.
• 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) is 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)
where: z = charge of the ion
V = accelerating potential
v = velocity of ion
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 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.
 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.
NOTE: 70eV, the de Broglie wavelength of an electron matches with the length of typical bonds in organic molecules (0.14 nm) and energy transferred to organic molecules is maximized at this wavelength.
2. Chemical Ionization
 INTRODUCTION
• 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.
• It is a two part process.
• In the first step
 A reagent gas is ionized by Electron Impact ionization in the source.
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 The primary ions of reagent gas react with additional gas to produce stabilized reagent ions.
• In the second step, the reagent ions interact with sample molecules to form molecular ions.
• In this technique the sample is diluted with a large excess of reagent gas so that the probability of ionizing collisions between sample molecules and the electrons is negligibly small and primary ions are formed entirely from interaction with reagent gas ions.
• Gases commonly used as reagent are low molecular weight compounds such as Methane, tertiary Isobutane, Ammonia, Nitrous oxide, oxygen and hydrogen etc.
 TYPES OF CI:
• Depending upon the type of ions formed CI is categorized as:-
1. Positive Chemical Ionization
2. Negative Chemical Ionization
1. Positive Chemical Ionization
 In this technique positive ions of the sample are produced.
 In positive chemical ionization, gases 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 CI).
NH3 NH3.+ + 2 e-
NH3.+ NH4+ + NH2
 NH4+ reacts with the sample molecules by proton transfer or Adduct formation to produce sample ions.
M + NH4+ [M + H]+ + NH3 Proton transfer
M + NH4+ [M + NH4]+ Adduct formation
 When Methane is used as Reagent gas. Methane is ionized by electron impact:
CH4 + e- CH4+ + 2e-
 Primary ions react with additional reagent gas molecules to produce stabilized reagent ions:
CH4+ + CH4 CH5+ + CH3
CH3 + CH4 C2H5+ + H2
 The reagent ions then react with the sample molecules to ionize the sample molecules:
CH5+ + MH CH4 + MH2+ (Proton transfer)
CH3+ + MH CH4 + M+ (Anhydride abstraction)
CH4+ + MH CH4 + MH+ (Charge transfer)
e-
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2. Negative Chemical Ionization
 Negative chemical ionization is counterpart of Positive chemical ionization.
 In this technique, 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 :-
A. Resonance electron capture
M + e- M-
B. Dissociative electron capture
RCl + e- R + Cl-
H2O + e- H + OH-
 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.
 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.
3. Atmospheric Pressure Chemical Ionization
 INTRODUCTION:
• It is a variant of chemical ionization and is carried out using an ion source similar to ESI.
• 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 vaporised by a heating.
• Once formed, the vapour is emanating from a corona pin held at 3 kV.
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• 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.
• For example, with Methanol:
Positive In AP-CI
Negative Ion AP-CI
• 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
• 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.
• During the ionization process itself, little energy is transferred to the sample molecule, and fragmentation is minimal.
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• Upon acceleration of the sample ions through the hot solvent vapour, however, collisional activation and subsequent fragmentation is common.
 APPLICATIONS
 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 are analyzed by this method.
 Can be used as LC/MS interface.
4. Field Ionization
 INTRODUCTION:
• 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.
• Application of very strong electric field induces emission of electrons.
• In this technique, sample molecules in vapour phase is brought between two closely spaced electrodes in the presence of high electric field (107 - 108V/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).
• In order to achieve high potential gradients necessary to effect ionization, the anode is activated by growing carbon microneedles or whiskers.
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• 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.
• 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.
5. Field desorption
 INTRODUCTION:
• In field desorption method, a multitipped emitter (made up of tungsten wire with carbon or silicon whiskers grown on its surface) similar to that used in FI is used.
 CONSTRUCTION & WORKING
• 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
 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.
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NOTE: In some cases it is necessary to heat the emitter by passing a current through the wire to evaporate the sample.
• Ionization takes place by quantum mechanical tunneling mechanism, which involves transfer of ions 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.
 DISADVANTAGES
 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.
6. Electrospray ionization
 INTRODUCTION:
• Electrospray ionization is a technique used in mass spectrometry to produce ions from macromolecules such as proteins, polypeptides and oligonucleotides having molecular weights of 10,000 Da or more.
• 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. 9

• The needle is maintained at a high electric field (several kilovolts) with respect to cylindrical electrode.
• The liquid pushes itself out of the capillary as a mist or aerosol of fine charged droplets.
• In the 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.
• Desolvating capillary uses warm nitrogen as nebulising 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.
NOTE: In electrospray process, the ions observed are quassimolecular ions that are ionized 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).
NOTE: Furthermore, multiple charged ions are often observed and these ions are even electron species indicating that electrons have neither been added nor removed.
 ADVANTAGES
 Most important techniques for analysis of high molecular weight biomolecules such as polypeptides, proteins, oligonucleotides and synthetic polymers.
 Can be used along with LC and capillary electrophoresis.
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7. Matrix Assisted Laser Desorption (MALDI)
 INTRODUCTION:
• Matrix assisted laser desorption is a technique in mass spectrometry for ionization of biomolecules (polymers such as proteins, polypeptides and sugars) and synthetic polymers that are more fragile and form fragments when ionized by conventional methods.
• It is most similar to ESI in both softness and ions produced.
A) Matrix
 Matrix is used in MALDI to
 Absorb the laser energy.
 Prevent analyte agglomeration.
 Protect analyte from being destroyed by direct laser beam.
 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)
 Preparation of Amtrix:
a) Solution of the matrix is made in a mixture of highly purified water and another organic compound (acetonitrile or ethanol).
b) Triofluoro acetic acid (TFA) is also added.
c) 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)
d) Matrix solution is then mixed with the analyte to be investigated.
NOTE: The organic compound acetonitrile dissolves hydrophobic proteins present in the sample while water dissolves hydrophilic proteins.
e) The solution is then spotted in a air tight chamber on the tip of the sample probe.
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f) 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.
 Some of the more commonly used matrices are:
B) Laser
 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
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
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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.
 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.
 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.
i) 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.
Lasers Used for MALDI
Laser
Wavelength(nm)
Reference
Nitrogen Laser
337
(Tanaka 1988)
CO2
10600
(Overberg 1991)
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• 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 .
 APPLICATIONS
 Used in proteomics
 Estimation of DNA, RNA and oligosaccharides.
 Used in analysis of lipids, phosphopeptides and synthetic polymers.
8. Plasma desorption
 INTRODUCTION
• 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).
• A typical pair of fission fragments is 142Ba18+ and 106TC22+, 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 10000K.
• 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.
9. Laser desorption
 INTRODUCTION:
• 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.
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 In order to avoid decomposition absorbed energy must be quickly dispersed in the molecules.
IONIZATION TECHNIQUE:
• 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.
 Bulk analysis techniques
 The technique uses a less focused beam and larger samples.
 The laser beam produces microplasma that consists of neutral fragments with elementary and fragment ions.
 The ions produced are largely protonated and deprotonated species that have a unit charge.
 ADVANTAGES
 Used for larger biomolecules such as proteins and carbohydrates.
 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 9000 Da for synthetic polymers cannot be studied as they get decomposed.
10. Fast Atom Bombardment
 INTRODUCTION:
• 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.
• 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.
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• 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.
• 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.+
 MATRIX PREPARATION:
• The analyte is dissolved in the liquid matrix such as glycerol and applied as a thin layer on the sample probe shaft.
• 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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• These ions are then extracted from slit lens system designed to collect ions and directed to mass analyzer.
 ADVANTAGES
 Used for ionization of polar high molecular weight samples.
 Provides rapid heating of samples and reduces sample fragmentation.
 Rapid ionization.
 DISADVANTAGES
 Difficult to distinguish between low molecular weight compounds.
 Compounds must be soluble in liquid matrix.
 Not good for multiply charged compounds.
11. Secondary ion mass spectrometry
 INTRODUCTION:
• 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.
• 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.
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12. 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.
• The probability of ionization is predictable and is a function of work function of :-
 Ionization potential of the sample E1
 Work function of the filament material Φ
 Filament temperature T
• This can be summarized as follows
n+/n0 = exp[z(Φ – E1)/KT]
Where,z = electronic charge
K = Boltzmann’s constant
n+ = Number of ions formed
n0 = Number of neutral species
REFERENCES
1. Principles of Instrumental analysis. Fifth Edition by Douglas. A. Skoog, F. James Holler and Timothy A. Nieman. Page No. 499 – 511.
2 Instrumental Methods Of Analysis. Seventh Edition by Willard Meritt. Page No. 468 – 74.
3 http://www.chem.ox.ac.uk/spectroscopy/mass-spec/Lecture/oxmain_lectureCI.html
4 http://www.astbury.leeds.ac.uk (A.E. Ashcroft's MS web pages and tutorial)
5 "http://en.wikipedia.org/wiki/Atmospheric_pressure_chemical_ionization
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Ionizaion Techniques - Mass Spectroscopy

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