Paper No. : 06 Atomic Spectroscopy
Module : 30 Introduction to Mass Spectrometry
Principal Investigator: Dr. Nutan Kaushik, Senior Fellow
The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry,
Jamia Hamdard University, New Delhi
Paper Coordinator: Dr. Mymoona Akhtar, Associate professor, Dept. of Pharm.
Chemistry, Jamia Hamdard, New Delhi.
Content Writer: Dr. S.K.Raza, Former Director,Institute of Pesticide formulation Technology, Gurugram
Content Reviwer: Dr. Nutan Kaushik, Senior Fellow , The Energy and Resouurces Institute (TERI), New Delhi
Introduction to Mass Spectrometry
1. Aim of the Module
To introduce mass spectrometry, one of the most sought after analytical technique.
To give an account of the historical background of the technique along with the timeline of the events.
To describe the various components of mass spectrometer.
To discuss the basic principle of mass spectrometry.
To give an account of the Terms and Definitions being used in this and subsequent modules related to mass spectrometric techniques.
2. Objectives of the Module
At the end of this module one should be able to understand:
a. What is mass spectrometry
b. The historical background of the technique along with the timeline of the events.
c. Mass spectrometry instrumentation d. The basic principle of mass spectrometry.
e. The Terms and Definitions related to mass spectrometric techniques.
3. Introduction
Mass spectrometry is one of the most sensitive analytical techniques used for the structure determination of chemicals. It can be used to identify unknown compounds, quantify known materials, studying molecular structure, probing the fundamental principles of chemistry and elucidate the chemical properties of molecules. The most significant part is that it is all accomplished with very small quantities.
Description of Module
Subject Name Analytical Chemistry / Instrumentation Paper Name Atomic Spectroscopy
Module Name/Title Introduction to Mass Spectrometry
Module Id 30
Pre-requisites Objectives Keywords
Mass spectrometry is a sophisticated "weighing machine" for determining the molecular masses of the molecules. The technique is based upon the motion of a charged particle, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z)of the ion affects this motion. Since the charge of an electron is known, the mass to charge ratio is a measurement of an ion's mass. The mass spectrometry research focuses on the formation of gas phase ions, the chemistry of ions, and applications of mass spectrometry. The term mass spectrometry is used in preference to mass spectroscopy which is a departure from usual UV, IR or NMR spectroscopy. It is because this technique involves quantitative mass measurement and draws analogy from gravimetry or volumetry.
4. Historical Background
Mass spectrometry evolved from physical and chemical studies regarding the nature of matter. The studies conducted on the gas discharge during mid-19th century led to the discovery of anode and cathode rays, which were identified as positive ions and electrons.
The improvements in capabilities for the separation of these positive ions led to the discovery of stable isotopes of the elements. The first such discovery was with the element neon, which was shown by mass spectrometry to have at least two stable isotopes: 20Ne (neon with 10 protons and 10 neutrons) and 22Ne (neon with 10 protons and 12 neutrons). Manhattan Project used mass spectrometry for the separation of isotopes of uranium necessary to create the atomic bomb.
Timeline:
Year Event
1898 Wein showed deflection of +ve ions by electric and magnetic fields 1898 J. J. Thomson measures the mass-to-charge ratio of electrons.
1901 Walter Kaufmann uses a mass spectrometer to measure the relativistic mass increase of electrons.
1905 J. J. Thomson begins his study of positive rays.
1913 Thomson was able to separate particles of different mass-to-charge ratios.
1919 Francis Aston constructs the first velocity focusing mass spectrograph with mass resolving power of 130.
1934 Josef Mattauch and Richard Herzog develop the double-focusing mass spectrograph.
1937 Aston constructs a mass spectrograph with resolving power of 2000.
1943 Westinghouse markets its mass spectrometer and proclaims it to be "A New Electronic Method for fast, accurate gas analysis".
1946 William Stephens presents the concept of a time-of-flight mass spectrometer.
1954 A. J. C. Nicholson proposes a hydrogen transfer reaction that will come to be known as the McLafferty rearrangement.
1959 Researchers at Dow Chemical interface a gas chromatograph to a mass spectrometer.
1966 F. H. Field and M. S. B. Munson develop chemical ionization.
1968 Malcolm Dole develops electrospray ionization.
1969 H. D. Beckey develops field desorption.
1974 Comisarow and Marshall develop Fourier Transform Ion Cyclotron Resonance mass spectrometry.
1976 Ronald MacFarlane and co-workers develop plasma desorption mass spectrometry.
1984 John Bennett Fenn and co-workers use electrospray to ionize biomolecules.
1985 Franz Hillenkamp, Michael Karas and co-workers describe and coin the term matrix-assisted laser desorption ionization (MALDI).
1989 Wolfgang Paul receives the Nobel Prize in Physics "for the development of the ion trap technique".
1999 Alexander Makarov presents the Orbitrap mass spectrometer.[29]
5. Basic Principles of Mass Spectrometry
In mass spectrometer, a molecule is bombarded with fast moving, high energy (70 eV) electrons resulting in the generation of multiple ions from the sample under investigation.
These ions are then separated according to their mass-to-charge ratio (m/z), and the ions are recorded as per their relative abundances.
Fast-moving electrons knock off an electron from the neutral molecule giving rise to a cation- radical known as “molecular ion”. It may be emphasized that the electrons do not strike the molecule in the classical sense but pass through the molecule or very close to the sample molecule. Although the majority of the organic molecules have their first ionization potential in the range of 8 – 15 eV, an electron beam of 25 – 70 eV is used in most of the mass spectrometers. The amount of energy used if far higher than required to break any C – X bond (X = any other element or Carbon itself) homolytically or heterolytically. The use of electron beam with such an excessive energy can be explained with the help of energy efficiency diagram for the production of positive ions (as shown below). The ion efficiency diagram is a plot of energies of electrons against the number of cation-radicals produced.
The point marked as ‘a’ in the diagram corresponds to the ionization energy of the compound.
It is apparent from the ionization energy diagram that the ionization is maximum around 35 eV but there is a tendency for sharp reversal of energy. A plateau like situation is obtained after 50 eV which remains steady over a large range of increased energy. Thus 70 eV may be regarded as the optimum energy which is to be associated with an electron beam.
A – B : → A – B +• + 2e Organic Molecule Molecular ion
The resultant cation-radical (molecular ion) is loaded with excess of energy and therefore, tends to decompose giving rise to different charged and neutral (radical/molecule) species.
The resultant charged species are called fragment ions. Fragmentation may involve simple bond cleavages which may be homolytic or heterolytic.
A – B +. → A• + B +:
Homolytic Cleavage A – B +. → A+ + B•
Heterolytic Claevage
It is worth mentioning here that mass spectrometer records only the charged species such as AB+, A+, A+• Neutral species such as radicals or neutral molecules are not recorded by a mass spectrometer.
Fragmentation pattern is of diagnostic value for a particular class of compounds and it has been observed that one class of compounds usually varies from another in their fragmentation behavior.
6. Mass Spectrometer
A mass spectrometer comprises of three major components:
a) Ion Source: For producing gaseous ions from the substance being studied.
b) Analyzer: It resolves the ions into their characteristic mass components according to their mass-to-charge ratio.
c) Detector: It detects the ions and records the relative abundances of each of the resolved ions.
In addition to the above, a sample introduction system is used to admit the samples to the ion source while maintaining the high vacuum requirements (~10-6 to 10-8 mm of mercury). A new generation mass spectrometer is always accompanied with a state of the art data system which controls the instrument, acquire and manipulate data, and compare spectra to reference libraries.
Figure: Components of a Mass Spectrometer
A mass spectrometer is expected to perform the following processes:
i) Produce ions from the sample in the ionization source.
ii) Separate these ions according to their mass-to-charge ratio in the mass analyzer.
iii) Fragment the selected ions and analyze the fragments in a second analyzer.
iv) Detect the ions emerging from the last analyzer and measure their abundance with the detector that converts the ions into electrical signals.
v) Process the signals from the detector that are transmitted to the computer and control the instrument using feedback.
6.1 Inlet Devices
An inlet device is required to inject the sample into the ion source of the mass spectrometer.
The inlet device is selected based on the type of sample and the sample matrix. The ionization techniques in mass spectrometers are designed for gas phase molecules and hence the inlet must transfer the analyte into the ion source as a gas phase molecule. A variety of inlets are available for volatile and thermally stable analytes. Gases and samples with high vapor pressure are introduced directly into the ion source of the mass spectrometer. Liquids and solid analytes are heated to increase the vapor pressure for analysis. The thermally labile analytes which decompose at high temperature or those which do not have a sufficient vapor pressure are directly ionized from the condensed phase. These direct ionization techniques
require special instrumentation and are more difficult to use. However, they greatly extend the range of compounds that may be analyzed by mass spectrometry. A modern mass spectrometer is generally equipped with more than one inlet device. The commonly used inlet devices are:
i) Direct Inlet device ii) Gas Chromatography iii) Liquid Chromatography
Direct Inlet Device: Direct inlet Device is the most commonly used inlet device for pure compounds. The analyte is introduced directly into the ion source of the mass spectrometer through a needle valve. This inlet works well for gases, liquids, or solids with a high vapor pressure. Samples with low vapor pressure are heated to increase the vapor pressure. This inlet works for only limited stable compounds which can be analysed at modest temperatures.
samples.
Gas Chromatography (GC): The most commonly used inlet device in modern mass spectrometry used for introducing volatile analytes is the Gas chromatography. This device has the capability of separating complex mixtures by gas chromatography which is subsequently identified by mass spectrometry. A GC couple to a MS is used to identify and quantify the individual components in a complex mixture. The coupling of these two techniques has been a challenging task but has since been successfully resolved with technological advancements. The major concern in coupling of GC to MS was the entry of large amount of GC carrier gas that enters the mass spectrometer which will breach the vacuum present in the ion source of the mass spectrometer. Several interfaces have been developed and used to cut off the carrier gas flow and connect these two instruments. The invention of capillary columns has played a vital role in the direct coupling of a GC to the mass spectrometer.
Liquid Chromatography: The introduction of non-volatile and thermally labile analytes was quite a difficult task by using GC-MS. The coupling of High Performance Liquid Chromatography (HPLC) to MS revolutionized the mass spectrometry technique and made the analysis of non-volatile, thermo-labile and polar compounds possible by using LC-MS.
Like the coupling of GC to MS, the coupling of LC to MS was still more difficult job due to the higher vapour pressure associated with the liquids being used as mobile phase in HPLC.
A number of interfaces have since been developed and used for the coupling of LC to MS.
These inlets have undergone considerable development and are now fairly routine. The development of Electrospray Ionization and its other modified form namely Atmospheric Pressure Chemical Ionization (APCI) has revolutionized the analysis of not only volatile and thermolabile compounds but also large biomolecules due to the formation of multiply charged ions.
6.2 Ion Source
A wide variety of ionization methods are available for the ionization of the molecule depending upon the nature of sample. All these sources ionize the samples in gas phase with a positive or negative charge. They differ in the energies imparted to the target molecule.
Some of the most commonly used ionization techniques are as follows.
• Electron Ionization (EI)
• Chemical Ionization (CI)
• Fast Atom Bombardment (FAB)
• Electrospray Ionization (ESI)
• Atmospheric Pressure Chemical Ionization (APCI)
• Matrix Assisted Laser Desorption Ionization (MALDI) These ion sources will be discussed in details in the next module.
6.3 Analyzers
The ions formed in the ion source are accelerated through a voltage towards the source slit.
The slit reduces the spread of divergent ions and forms the object plane of the analyser.
Some of the commonly used analyzers are listed below.
Sector analyser (magnetic/electrostatic)
Quadrupole analyser
Ion trap analysers
Time of flight analyser
Ion cyclotron resonance analyser
A detailed discussion about each of the above listed analyzers will follow in Module 3.
6.4 Detectors
The ions formed in the ion source, separated in analysers finally reach the detector of the mass spectrometer. These ions are detected based on their charge or momentum. A faraday cup is used to collect ions and measure the current for large signals. Photographic plates were used to measure the ion abundance at each mass to charge ratio in older instruments.
Modern detectors use amplified ion signal using a collector similar to a photomultiplier tube.
These detectors include: electron multipliers, channeltrons and multichannel plates. The signal gain is controlled by changing the high voltage applied to the detector. A detector is selected based on its its speed, dynamic range, gain, and geometry. Some detectors are sensitive enough to detect single ions.
6.5 Vacuum System
High vacuum is one of the primary requirements of a mass spectrometer. It reduces the chance of ions colliding with other Molecules in the mass analyzer and hence avoiding any collision which may cause the ions to react, neutralize, scatter, or fragment. This vacuum thus reduces the chances of interference with the mass spectrum. In order to minimize collisions, the experiments are conducted under high vacuum conditions, typically 10-2to 10-5Pa (10-5to 10-7torr) depending upon the geometry of the instrument. This high vacuum is achieved by using two pumping stages. The first stage is a mechanical pump providing rough vacuum down to 0.1 Pa (10-3torr) while the second stage uses diffusion pumps or turbo-molecular pumps to provide high vacuum.
6.6 Data System
A strong data system is a prominent component of all modern mass spectrometers. The revolutionary developments during the last decade or so in information technology has led to extraordinarily efficient data systems which can take care of the mass spectrometric operations very smooth. It has evolved from photographic plates and strip chart recorders to
data systems that control the instrument, acquire hundreds of spectra in a minute and search tens of thousands of reference spectra to identify the unknowns.
7. Terms and Definitions
Adduct Ion : An ion formed by the interaction of an ion with one or more atoms or molecules to form an ion containing all the constituent atoms of the precursor ion as well as the additional atoms from the associated atoms or molecules.
α-Cleavage : A homolytic cleavage where the bond fission occurs between at the atom adjacent to the atom at the apparent charge site and an atom removed from the apparent charge site by two bonds.
Base Peak : The peak in a mass spectrum that has the greatest intensity. This term may be applied to the spectra of pure substances or mixtures.
β-Cleavage : A homolytic cleavage where the bond fission occurs between at an atom removed from the apparent charge site atom by two bonds and an atom adjacent to that atom and removed from the apparent charge site by three bonds.
Collisional Excitation : The reaction of an ion with a neutral species in which all or part of the translational energy of the collision is converted into internal energy of the ion.
Collision Gas : An inert gas used for collisional excitation and ion/molecule reactions.
Collision-Induced Dissociation : The dissociation of an ion after collisional excitation. The term collisional activated dissociation is not recommended.
Dalton : A non-SI unit of mass (symbol Da) that is equal to the unified atomic mass unit:
1.660 538 86x 10- 27 kg.
Diagnostic Ion : A product ion whose formation reveals structural or compositional information of its precursor. For instance, the phenyl cation in an electron ionization mass spectrum is a diagnostic ion for benzene and derivatives.
Electron Energy : The magnitude of the electron charge multiplied by the potential difference through which electrons are accelerated in order to effect electron ionization.
Fragmentation : the dissociation of energetically unstable molecular ions formed from the passing the molecules in the ionization chamber. The fragment ions thus formed are useful in determining the structure of the molecule.
Fragment Ion : A product ion that results from the dissociation of a precursor ion.
Hard Ionization : The formation of gas-phase ions accompanied by extensive fragmentation.
Heterolytic Cleavage : Fragmentation of a molecule or ion in which both electrons forming the single bond that is broken remain on one of the atoms that were originally bonded. This term is synonymous with heterolysis.
Homolytic Cleavage : In general, fragmentation of an ion or molecule in which the electrons forming the single bond that is broken are shared between the two atoms that were originally bonded. For an odd electron ion, fragmentation results from one of a pair of electrons that form a bond between two atoms moving to form a pair with the odd electron on the atom at the apparent charge site. Fragmentation results in the formation of an even electron ion and a radical. This reaction involves the movement of a single electron and is represented by a single-barbed arrow synonymous with homolysis.
Hybrid Mass Spectrometer : A mass spectrometer that combines m/z analyzers of different types to perform tandem mass spectrometry.
Ion : An atomic, molecular or radical species with an unbalanced electrical charge. The corresponding neutral species need not be stable.
Ion Desolvation : The removal of solvent molecules clustered around a gas-phase ion by means of heating and/or collisions with gas molecules.
Ionization Efficiency : The ratio of the number of ions formed to the number of molecules consumed in the ion source.
Ion/Molecule Reaction : The reaction of an ion with a molecule. The term ion-molecule reaction is not recommended because the hyphen suggests a single species that is that is both an ion and a molecule.
Ion Source : The region in a mass spectrometer where ions are produced.
Mass Resolution : The smallest mass difference � m between two equal magnitude peaks so that the valley between them is a specified fraction of the peak height
Mass Spectral Library : A collection of mass spectra of different compounds, usually expressed as arrays of signal intensity vs. the m/z value rounded off to the integral mass number. In some cases the library may consist of monoisotopic mass spectra.
Mass Spectrum : A plot of the relative abundances of ions forming a beam or other collection as a function of the their m/z values.
McLafferty Rearrangement : A dissociation reaction triggered by transfer of a hydrogen atom via a 6-member transition state to the formal radical/charge site from a carbon atom four atoms removed from the charge/radical site (the γ-carbon); subsequent rearrangement of electron density leads to expulsion of an olefin molecule. This term was originally applied to ketone ions where the charge/radical site is the carbonyl oxygen, but it is now more widely applied.
Metastable Ion : An ion that is formed with internal energy higher than the threshold for dissociation but with a lifetime great enough to allow it to exit the ion source and enter the mass analyzer where it dissociates before detection.
Molecular Ion : An ion formed by the removal of one or more electrons to form a positive ion or the addition of one or more electrons to form a negative ion.
MSn : This symbol refers to multi-stage MS/MS experiments designed to record product ion spectra where n is the number of product ion stages (progeny ions). For ion traps, sequential MS/MS experiments can be undertaken where n > 2 whereas for a simple triple quadrupole system n = 2. Synonymous with multiple-stage mass spectrometry.
m/z – The three-character symbol m/z is used to denote the dimensionless quantity formed by dividing the mass of an ion by the unified atomic mass unit and also by its charge number (regardless of sign). The symbol is written in italicized lower case letters with no spaces.
Negative Ion : An atomic or molecular species having a net negative electric charge.
Neutral Loss : The loss of an uncharged species from an ion during either a rearrangement process or direct dissociation.
Nominal Mass : The mass of an ion or molecule calculated using the mass of the most abundant isotope of each element rounded to the nearest integer value. It is in effect the sum of the mass numbers of all constituent atoms.
Peak : A localized region of relatively large ion signal in a mass spectrum. Although peaks are often associated with particular ions, the terms peak and ion should not be used interchangeably.
Peak Intensity : The height or area of a peak in a mass spectrum.
Peak Matching : A method for measuring the accurate mass of an ion using scanning mass spectrometers, in which the peak corresponding to the unknown ion and that for a reference ion of known m/z are displayed alternately on a display screen and caused to overlap by adjusting appropriate electric fields.
Positive Ion : An atomic or molecular species having a net positive electric charge.
Precursor Ion : An ion that reacts to form particular product ions. The reaction can be unimolecular dissociation, ion/molecule reaction, isomerization, or change in charge state.
The term parent ion is not recommended.
Product Ion : An ion or ions arising from the precursor ion by collisionally induced dissociation under tandem mass spectrometry.
Proton Affinity : The proton affinity of a species M is defined as the negative of the enthalpy change for the reaction M+H+ → [M+H]+, where all species are in their ground rotational, vibrational and electronic states.
Protonated Molecule : An ion formed by interaction of a molecule with a proton, and represented by the symbolism [M+H]+. The terms protonated molecular ion, quasi-molecular ion and pseudo-molecular ion are not recommended.
Radical Ion : An ion, either a cation or anion, containing unpaired electrons in its ground state. The unpaired electron is denoted by a superscript dot alongside the superscript symbol for charge, such as for the molecular ion of a molecule M, that is, M+•.
Reagent Ion : An ion produced in large excess in a chemical ionization source that reacts with neutral sample molecules to produce an ionized form of the molecule through an ion/molecule reaction.
Selected Ion Monitoring (SIM) : The operation of a mass spectrometer in which the abundances of several ions of specific m/z values are recorded rather than the entire mass spectrum.
Selected Reaction Monitoring (SRM) : Data acquired from specific product ions corresponding to m/z selected precursor ions recorded via two or more stages of mass spectrometry. Selected reaction monitoring can be preformed as tandem mass spectrometry in time or tandem mass spectrometry in space.
Soft Ionization : The formation of gas-phase ions without extensive fragmentation.
8. Conclusion
Mass spectrometry is one of the most sensitive analytical technique used for structure elucidation of chemicals. It is very useful tool for:
♥ Identification of unknown compounds;
♥ Quantification of known materials; and ♥ Structure elucidation of molecules
All the above can be accomplished with very small quantities 9. Bibliography
1. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997).
2. Griffiths, Jennifer (2008). "A Brief History of Mass Spectrometry". Analytical Chemistry. 80 (15): 5678–5683.
3. Dass, Chhabil. Fundamentals of Contemporary Mass Spectrometry - Dass - Wiley Online Library (http://doi.wiley.com/10.1002/0470118490). doi:10.1002/0470118490 (https://doi.org/10.1002%2F0470118490).
4. R. Davis, M. Frearson, (1987). Mass Spectrometry – Analytical Chemistry by Open Learning, John Wiley & Sons, London.
5. Murray, K.K., Boyd, R.K., Eberlin, M.N., Langley, G.J., Li, L., and Naito, Y. (2013) Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013).
Pure Appl. Chem., 85, 1515–1609.
6. Scott E. Van Bramer, An Introduction to Mass Spectrometry, 1998, http://science.widener.edu/~svanbram
