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Mass Spectrometry
Submitted to :
Rani Mansuri
Submitted by:
Surbhi
M.Pharma 1st sem
Contents• Definition
• Principle
• Instrumentation
• Components of MS
• Ion Source & Types of ionization
• Ionizing Agents
• Ionization Methods :
 Electron Impact Ionization (EI)
 Chemical Impact Ionization (CI)
 Field Ionization (FI)
 Fast Atom Bombardment (FAB)
 Matrix Assisted Laser Desorption /Ionization (MALDI)
 Atmospheric Pressure Chemical Ionisation (APCI)
 Electro Spray Ionization (ESI)
 Atmospheric Pressure Photo Ionization (APPI)
 Comparing APPI with ESI and APCI
 Other Ionization methods : FD, PD, LD.
• Mass Analysers :
 Quadrupole Mass Analyzer
 Time of flight
• Mass Spectrometric Analysis
• Applications of Mass Spectrometry
Definition
• Mass Spectrometry (MS) is a technique for the analysis of a
substance in which the molecule is subjected to
bombardment by high-energy electrons or atoms to cause
ionization and fragmentation to give a series of ions in the
gas phase that constitutes the fragmentation pattern
observed by using a mass spectrometer.
• It is a process, which used to identify chemicals in a
substance by their mass and charge.
• Mass spectrometers are instruments that measure mass
and charge of molecules.
• A mass spectrometer also can determine how much of a
compound is present in a mixture, also known as mass
spectroscopy.
Principle of Mass Spectrometry (MS)
• A mass spectrometer generates multiple ions from the sample
under investigation; it then separates them according to their
specific mass-to-charge ratio (m/z), and then records the
relative abundance of each ion type.
Mass Spectrum
Instrumentation
• The instrument used in MS is called mass
spectrometer.
• It produces a mass spectrum that plots the mass-to-
charge (m/z) ratio of compounds in a mixture.
• Mass spectrometry is used in many different fields and
is applied to pure samples as well as complex mixtures.
• Mass Spectrometry (MS) is used for both qualitative
and quantitative chemical analysis.
• It may be used to identify the elements and isotopes
of sample, to determine the masses of molecules, and
as a tool to help identify chemical structures.
• It can measure sample purity and molar mass.
Components of MS
Inlet system
Ion source
Mass analyzer
Ion detectors
Vaccum system
• 1.Solid
• 2.Liquid
• 3.Gas
Inlet system
• ESI,EI,FAB, CI,FI,FD, MALDI,PD, TIIonization
• Quadrupole, TOF, Ion trap, FT-ICRMass Analyser
• Photo multiplier, Electron
multiplier, Faraday cup, Micro
channel plate
Detector
Ion source and Ionization Types
• Molecular ions are formed when energy of the
electron beam reaches to 10-15 eV.
• Fragmentation of the ion reaches only at
higher bombardment energies at 70 eV.
Ionization Types:
•gases and vapour1.Gas Phase
Ionization
•liquid and solid2. Desorption
Technique
1.Gas Phase Ionization (gases and vapour)
• Samples are ionized outside the ion source.
• This technique include:
1.Electron
impact
ionization (EIS)
2.Chemical
ionization.(CI)
3.Field
ionization.(FI)
2.Desorption Technique(liquid and solid)
• Samples are ionised inside the ion source.
• This technique include:
1. Field
desorption.(FD)
2. Fast atom
bombardment.(FAB)
3.Laser
desorption.(LD)
Ionizing Agents
Ionization Method
Electron impact ionization
• A beam of electrons passes through a gas-phase sample and
collides with neutral analyte molecules (M) to produce a
positively charged ion or a fragment ion.
• Generally electrons with energies of 70 eV are used to form a
fragment ions.
• The positive ions are collected in focusing plates and passed
to mass analyzer.
Chemical Impact Ionization
• Chemical Impact Ionization between
interactions of sample with large amount of
reagent gas.
• Commonly used reagent gases include
methane, ammonia, isobutane.
• Oxygen and hydrogen are used in Negative ion
chemical ionization in MS.
• The vaporised sample is introduced into the
mass spectrometer with an excess of a
reagent gas (methane) at pressure of about 1
torr.
• The excess carrier gas is ionized by electron
impact to the primary ions CH4
.+ and CH3
+.
• These may react with the excess methane to
give secondary ions
Field Ionization
• In this method the molecule pass through
sharp metal anode carrying an electric field of
10
10
vm-1 .
• Electrons are analysed in primary focusing
cathode slit.
• ADV :- abundance of molecular ions.
• DISADV :- lower resolution.
Fast Atom Bombardment (FAB)
• Argon gas ionised by hot filament and focused beam that bombards the
sample.
• Beam impinges the sample, a series of molecular reactions occur and
analyse in MS analyser.
• Ex:-Insulin, Amino glycosides, Phospholipids.
Matrix Assisted Laser Desorption
/Ionization (MALDI)
• MALDI is a LIMS method of vaporizing and ionizing and
sample molecules are dispersed in a 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 a mass spectrometer.
APCI
• Atmospheric pressure chemical ionisation (APCI) is an
analogous ionisation method to chemical ionisation(CI).
• Corona discharge is used to ionize the analyte in the
atmospheric pressure region.
Electro Spray Ionization (ESI)
• The ESI source consists of a very fine needle and a series of
skimmers.
• A sample solution is sprayed into the source chamber to form
droplets.
• When droplets carry charge exit the capillary end, as the
solvent evaporates, the droplets disappear leaving highly
charged analyte molecules.
Atmospheric Pressure
Photoionization (APPI)
• APPI mechanism: The Photoionization mechanism is
simplified under vacuum conditions: photon absorption by
the analyte molecule, leading to electron ejection, forming a
molecular radical cation M.+ .
• APPI provides excellent coverage of a wide range of
compounds for a wide range of LC–MS conditions.
• RF discharge Kr lamps used for APPI provide high photon
radiance.
Comparing APPI with ESI and APCI
• All three sources do well on polar, basic-type molecules, such as
drugs.
• However, less polar compounds, such as steroids and especially
PAHs, are ionized poorly by either ESI or APCI in comparison to the
relatively efficient APPI. APCI has an advantage in ionizing less polar
compounds than ESI, but is less effective than APPI at low-polar to
nonpolar molecule ionization.
• ESI is the best choice for polar compounds such as drugs and is by
far the best choice for larger molecules, such as peptides and
proteins. For other compounds, APCI and APPI should be the
preferred choice, with an advantage toward APPI in many important
respects, except at very high flow rates (for example, 1 mL/min) and
for small molecules such as smaller alkanes (smaller than hexane)
and halocarbons.
Other Ionization Methods
Field Desorption
• Useful for nonvolatile and thermo-labile compounds.
• Sample is applied to field ion emitter and the solvent allowed
to evaporate.
• Evaporated sample that leads to chemical ionization or EIS. •
• Example:- Nucleotides & Quaternary ammonium compounds.
PLASMA DESORPTION
• Sample is coated with a high energetic fragment
Californium 252.
• This fission fragment desorbs positive, negative, and
neutral molecules.
• 252cf generates 1012 power at 10,000k, this may ionize
the target molecule.
LASER DESORPTION
• This method involves the interaction of laser beam
with sample to produce both vaporization and
ionization.
• The vaporized sample passed to mass spectrometers
for analysis.
• Appl:-used for elemental analysis.
Mass Analyzers
• An ion, after leaving ion source, the ions are separated according to their
m/e ratio.
• Mass Analyzer In this area, the ions are accelerated by both electrostatic
and magnetically.
• Types:-
Magnetic sector mass analysers
Double focussing analysers
Quadrupole mass analyzers
Time of Flight analysers(TOF)
Ion trap analyser
Ion cyclotron analyser
Quadrupole Mass Analyzer
• The quadrupole consists of two pairs of parallel rods with
applied DC and RF voltages.
• Ions are scanned by varying the DC/Rf quadrupole voltages.
Time Of Flight
• TOF analyzer – ions are accelerated through a flight tube and
the time of flight to the detector is measured.
• Typical flight times are 1 to 50μs.
Mass Spectrometric Analysis
The first step in the mass spectrometric analysis of
compounds is the production of gas phase ions of the
compound, basically by electron ionization.
This molecular ion undergoes fragmentation.
Each primary product ion derived from the molecular ion, in
turn, undergoes fragmentation, and so on.
The ions are separated in the mass spectrometer according to
their mass-to-charge ratio.
Detected in proportion to their abundance.
A mass spectrum of the molecule is thus produced.
It displays the result in the form of a plot of ion abundance
versus mass-to-charge ratio.
Ions provide information concerning the nature and the
structure of their precursor molecule.
In the spectrum of a pure compound, the molecular ion, if
present, appears at the highest value of m/z and gives the
molecular mass of the compound.
How Mass Spectrometry Works?
• The three main parts of a mass spectrometer are the
ion source, the mass analyzer, and the detector.
• Ionization – The initial sample may be a solid, liquid, or
gas. The sample is vaporized into a gas and then
ionized by the ion source, usually by losing an electron
to become a cation. The ionization chamber is kept in a
vacuum so the ions that are produced can progress
through the instrument without running into
molecules from air. Ionization is from electrons that are
produced by heating up a metal coil until it releases
electrons.
• Acceleration – In the mass analyzer, the ions are then
accelerated through a potential difference and focused
into a beam. The purpose of acceleration is to give all
species the same kinetic energy, like starting a race
with all runners on the same line.
• Deflection – The ion beam passes through a magnetic
field which bends the charged stream. Lighter
components or components with more ionic charge
will deflect in the field more than heavier or less
charged components.
• Detection – A detector counts the number of ions at
different deflections. The data is plotted as a graph or
spectrum of different masses. Detectors work by
recording the induced charge or current caused by an
ion striking a surface or passing by.
Applications of Mass
Spectrometry
1. Sequencing of different proteins
• Mass Spectrometry (MS) has emerged as an
indispensable method for the characterization
and sequencing of different proteins.
• It has gained popularity for protein analysis due
to its ability to tackle the intricacies underlying
the proteome.
• The three fundamental applications of the
technique in proteomics are determining protein
interactions, categorizing protein expression, and
locating sites of protein modification.
2. To determine the molecular weight
• Mass Spectrometry (MS) represents a powerful
technique with a myriad of different applications
in biology, chemistry, and physics, but also in
clinical medicine and even space exploration.
• It is used to determine the molecular weight of
compounds by separating molecular ions on the
basis of their mass and charge.
• Various types of mass spectrometry with high
specificity are being increasingly utilized as tools
in the clinical laboratory, but also in different
research settings.
3. Space-related applications
• There are also space-related applications of
mass spectrometry, two important ones being
observing air quality of manned space mission
and examining the composition of planetary
atmospheres.
• The success of its use in planetary missions
can be attributed to space vacuum that can be
used to decrease resource demands.
4. Drug discovery
i. Mass Spectrometry has also played a pivotal
role in many phases of drug discovery.
ii. Drug reaction optimization, structural analysis
of library products, and the evaluation of library
compound quality are just few examples of how
this technique can be employed.
iii. It is also becoming increasingly used in geologic
research for petroleum composition
measurement and carbon dating.
iv. The technique can also be used to test water
quality or potential food contamination.
v. Elucidation of the structure of the organic and
biological molecules.
vi. Determination of molecular mass of peptides,
proteins, and Oligonucleotides.
vii. Monitoring gases in patients breath during
surgery.
viii.Identification of drugs abuse and metabolites of
drugs of abuse in blood, urine, and saliva.
ix. Analyses of aerosol particles.
x. Determination of pesticides residues in food.
References
• Instrumental methods of chemical analysis by
Willard
• Organic spectroscopy by William Kemp
• Spectroscopic identification of organic
compounds by Silverstein
• Instrumental analysis by Skoog
• Wikipedia
Mass spectrometry and ionization techniques

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Mass spectrometry and ionization techniques

  • 1. Mass Spectrometry Submitted to : Rani Mansuri Submitted by: Surbhi M.Pharma 1st sem
  • 2. Contents• Definition • Principle • Instrumentation • Components of MS • Ion Source & Types of ionization • Ionizing Agents • Ionization Methods :  Electron Impact Ionization (EI)  Chemical Impact Ionization (CI)  Field Ionization (FI)  Fast Atom Bombardment (FAB)  Matrix Assisted Laser Desorption /Ionization (MALDI)  Atmospheric Pressure Chemical Ionisation (APCI)  Electro Spray Ionization (ESI)  Atmospheric Pressure Photo Ionization (APPI)  Comparing APPI with ESI and APCI  Other Ionization methods : FD, PD, LD. • Mass Analysers :  Quadrupole Mass Analyzer  Time of flight • Mass Spectrometric Analysis • Applications of Mass Spectrometry
  • 3. Definition • Mass Spectrometry (MS) is a technique for the analysis of a substance in which the molecule is subjected to bombardment by high-energy electrons or atoms to cause ionization and fragmentation to give a series of ions in the gas phase that constitutes the fragmentation pattern observed by using a mass spectrometer. • It is a process, which used to identify chemicals in a substance by their mass and charge. • Mass spectrometers are instruments that measure mass and charge of molecules. • A mass spectrometer also can determine how much of a compound is present in a mixture, also known as mass spectroscopy.
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  • 7. Principle of Mass Spectrometry (MS) • A mass spectrometer generates multiple ions from the sample under investigation; it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type.
  • 9. Instrumentation • The instrument used in MS is called mass spectrometer. • It produces a mass spectrum that plots the mass-to- charge (m/z) ratio of compounds in a mixture. • Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures. • Mass Spectrometry (MS) is used for both qualitative and quantitative chemical analysis. • It may be used to identify the elements and isotopes of sample, to determine the masses of molecules, and as a tool to help identify chemical structures. • It can measure sample purity and molar mass.
  • 10. Components of MS Inlet system Ion source Mass analyzer Ion detectors Vaccum system
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  • 12. • 1.Solid • 2.Liquid • 3.Gas Inlet system • ESI,EI,FAB, CI,FI,FD, MALDI,PD, TIIonization • Quadrupole, TOF, Ion trap, FT-ICRMass Analyser • Photo multiplier, Electron multiplier, Faraday cup, Micro channel plate Detector
  • 13. Ion source and Ionization Types • Molecular ions are formed when energy of the electron beam reaches to 10-15 eV. • Fragmentation of the ion reaches only at higher bombardment energies at 70 eV. Ionization Types: •gases and vapour1.Gas Phase Ionization •liquid and solid2. Desorption Technique
  • 14. 1.Gas Phase Ionization (gases and vapour) • Samples are ionized outside the ion source. • This technique include: 1.Electron impact ionization (EIS) 2.Chemical ionization.(CI) 3.Field ionization.(FI)
  • 15. 2.Desorption Technique(liquid and solid) • Samples are ionised inside the ion source. • This technique include: 1. Field desorption.(FD) 2. Fast atom bombardment.(FAB) 3.Laser desorption.(LD)
  • 18. Electron impact ionization • A beam of electrons passes through a gas-phase sample and collides with neutral analyte molecules (M) to produce a positively charged ion or a fragment ion. • Generally electrons with energies of 70 eV are used to form a fragment ions. • The positive ions are collected in focusing plates and passed to mass analyzer.
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  • 20. Chemical Impact Ionization • Chemical Impact Ionization between interactions of sample with large amount of reagent gas. • Commonly used reagent gases include methane, ammonia, isobutane. • Oxygen and hydrogen are used in Negative ion chemical ionization in MS.
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  • 22. • The vaporised sample is introduced into the mass spectrometer with an excess of a reagent gas (methane) at pressure of about 1 torr. • The excess carrier gas is ionized by electron impact to the primary ions CH4 .+ and CH3 +. • These may react with the excess methane to give secondary ions
  • 23. Field Ionization • In this method the molecule pass through sharp metal anode carrying an electric field of 10 10 vm-1 . • Electrons are analysed in primary focusing cathode slit. • ADV :- abundance of molecular ions. • DISADV :- lower resolution.
  • 24. Fast Atom Bombardment (FAB) • Argon gas ionised by hot filament and focused beam that bombards the sample. • Beam impinges the sample, a series of molecular reactions occur and analyse in MS analyser. • Ex:-Insulin, Amino glycosides, Phospholipids.
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  • 26. Matrix Assisted Laser Desorption /Ionization (MALDI) • MALDI is a LIMS method of vaporizing and ionizing and sample molecules are dispersed in a 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 a mass spectrometer.
  • 27. APCI • Atmospheric pressure chemical ionisation (APCI) is an analogous ionisation method to chemical ionisation(CI). • Corona discharge is used to ionize the analyte in the atmospheric pressure region.
  • 28. Electro Spray Ionization (ESI) • The ESI source consists of a very fine needle and a series of skimmers. • A sample solution is sprayed into the source chamber to form droplets. • When droplets carry charge exit the capillary end, as the solvent evaporates, the droplets disappear leaving highly charged analyte molecules.
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  • 30. Atmospheric Pressure Photoionization (APPI) • APPI mechanism: The Photoionization mechanism is simplified under vacuum conditions: photon absorption by the analyte molecule, leading to electron ejection, forming a molecular radical cation M.+ . • APPI provides excellent coverage of a wide range of compounds for a wide range of LC–MS conditions. • RF discharge Kr lamps used for APPI provide high photon radiance.
  • 31. Comparing APPI with ESI and APCI • All three sources do well on polar, basic-type molecules, such as drugs. • However, less polar compounds, such as steroids and especially PAHs, are ionized poorly by either ESI or APCI in comparison to the relatively efficient APPI. APCI has an advantage in ionizing less polar compounds than ESI, but is less effective than APPI at low-polar to nonpolar molecule ionization. • ESI is the best choice for polar compounds such as drugs and is by far the best choice for larger molecules, such as peptides and proteins. For other compounds, APCI and APPI should be the preferred choice, with an advantage toward APPI in many important respects, except at very high flow rates (for example, 1 mL/min) and for small molecules such as smaller alkanes (smaller than hexane) and halocarbons.
  • 32. Other Ionization Methods Field Desorption • Useful for nonvolatile and thermo-labile compounds. • Sample is applied to field ion emitter and the solvent allowed to evaporate. • Evaporated sample that leads to chemical ionization or EIS. • • Example:- Nucleotides & Quaternary ammonium compounds.
  • 33. PLASMA DESORPTION • Sample is coated with a high energetic fragment Californium 252. • This fission fragment desorbs positive, negative, and neutral molecules. • 252cf generates 1012 power at 10,000k, this may ionize the target molecule. LASER DESORPTION • This method involves the interaction of laser beam with sample to produce both vaporization and ionization. • The vaporized sample passed to mass spectrometers for analysis. • Appl:-used for elemental analysis.
  • 34. Mass Analyzers • An ion, after leaving ion source, the ions are separated according to their m/e ratio. • Mass Analyzer In this area, the ions are accelerated by both electrostatic and magnetically. • Types:- Magnetic sector mass analysers Double focussing analysers Quadrupole mass analyzers Time of Flight analysers(TOF) Ion trap analyser Ion cyclotron analyser
  • 35. Quadrupole Mass Analyzer • The quadrupole consists of two pairs of parallel rods with applied DC and RF voltages. • Ions are scanned by varying the DC/Rf quadrupole voltages.
  • 36. Time Of Flight • TOF analyzer – ions are accelerated through a flight tube and the time of flight to the detector is measured. • Typical flight times are 1 to 50μs.
  • 37. Mass Spectrometric Analysis The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. The ions are separated in the mass spectrometer according to their mass-to-charge ratio. Detected in proportion to their abundance. A mass spectrum of the molecule is thus produced.
  • 38. It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio. Ions provide information concerning the nature and the structure of their precursor molecule. In the spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z and gives the molecular mass of the compound.
  • 39. How Mass Spectrometry Works? • The three main parts of a mass spectrometer are the ion source, the mass analyzer, and the detector. • Ionization – The initial sample may be a solid, liquid, or gas. The sample is vaporized into a gas and then ionized by the ion source, usually by losing an electron to become a cation. The ionization chamber is kept in a vacuum so the ions that are produced can progress through the instrument without running into molecules from air. Ionization is from electrons that are produced by heating up a metal coil until it releases electrons.
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  • 41. • Acceleration – In the mass analyzer, the ions are then accelerated through a potential difference and focused into a beam. The purpose of acceleration is to give all species the same kinetic energy, like starting a race with all runners on the same line. • Deflection – The ion beam passes through a magnetic field which bends the charged stream. Lighter components or components with more ionic charge will deflect in the field more than heavier or less charged components. • Detection – A detector counts the number of ions at different deflections. The data is plotted as a graph or spectrum of different masses. Detectors work by recording the induced charge or current caused by an ion striking a surface or passing by.
  • 43. 1. Sequencing of different proteins • Mass Spectrometry (MS) has emerged as an indispensable method for the characterization and sequencing of different proteins. • It has gained popularity for protein analysis due to its ability to tackle the intricacies underlying the proteome. • The three fundamental applications of the technique in proteomics are determining protein interactions, categorizing protein expression, and locating sites of protein modification.
  • 44. 2. To determine the molecular weight • Mass Spectrometry (MS) represents a powerful technique with a myriad of different applications in biology, chemistry, and physics, but also in clinical medicine and even space exploration. • It is used to determine the molecular weight of compounds by separating molecular ions on the basis of their mass and charge. • Various types of mass spectrometry with high specificity are being increasingly utilized as tools in the clinical laboratory, but also in different research settings.
  • 45. 3. Space-related applications • There are also space-related applications of mass spectrometry, two important ones being observing air quality of manned space mission and examining the composition of planetary atmospheres. • The success of its use in planetary missions can be attributed to space vacuum that can be used to decrease resource demands.
  • 46. 4. Drug discovery i. Mass Spectrometry has also played a pivotal role in many phases of drug discovery. ii. Drug reaction optimization, structural analysis of library products, and the evaluation of library compound quality are just few examples of how this technique can be employed. iii. It is also becoming increasingly used in geologic research for petroleum composition measurement and carbon dating. iv. The technique can also be used to test water quality or potential food contamination.
  • 47. v. Elucidation of the structure of the organic and biological molecules. vi. Determination of molecular mass of peptides, proteins, and Oligonucleotides. vii. Monitoring gases in patients breath during surgery. viii.Identification of drugs abuse and metabolites of drugs of abuse in blood, urine, and saliva. ix. Analyses of aerosol particles. x. Determination of pesticides residues in food.
  • 48. References • Instrumental methods of chemical analysis by Willard • Organic spectroscopy by William Kemp • Spectroscopic identification of organic compounds by Silverstein • Instrumental analysis by Skoog • Wikipedia