Mass spectrometric methodsMass spectrometric methods

Assistant of the pharmaceutical chemistry department

Burmas Nataliya Ivanivnae-mail: Natashenka-Burmas@rambler.ru

PlanPlan

1. The concept of mass-spectrometric analysis.

2. The mass spectrometer3. Quantitative and qualitative analysis4. Applications of mass-spectrometry

1.1. The concept of mass-spectrometry analysisThe concept of mass-spectrometry analysis

Mass spectrometry (Mass spectrometry (MSMS) is an ) is an analytical technique for the analytical technique for the determination of the elemental determination of the elemental composition of a sample or molecule. It composition of a sample or molecule. It is also used for elucidating the chemical is also used for elucidating the chemical structures of molecules, such as peptides structures of molecules, such as peptides and other chemical compounds. and other chemical compounds.

In a typical MS procedure:In a typical MS procedure:a)a) a sample is loaded onto the MS instrument, and a sample is loaded onto the MS instrument, and

undergoes vaporization.undergoes vaporization.b)b) the components of the sample are ionized by one of a the components of the sample are ionized by one of a

variety of methods (e.g., by impacting them with an variety of methods (e.g., by impacting them with an electron beam), which results in the formation of electron beam), which results in the formation of charged particles (ions)charged particles (ions)

c)c) the positive ions are then accelerated by an electric the positive ions are then accelerated by an electric fieldfield

d)d) computation of the mass-to-charge ratio (computation of the mass-to-charge ratio (m/zm/z) of the ) of the particles based on the details of motion of the ions as particles based on the details of motion of the ions as they transit through electromagnetic fields, andthey transit through electromagnetic fields, and

e)e) detection of the ions, which in step 4 were sorted detection of the ions, which in step 4 were sorted according to according to m/zm/z..

Analysers characteristicsAnalysers characteristicsThe mass resolving powerThe mass resolving power - - is the measure of the is the measure of the

ability to distinguish two peaks of slightly different ability to distinguish two peaks of slightly different m/z.m/z.

The mass accuracyThe mass accuracy - is the ratio of the m/z - is the ratio of the m/z measurement error to the true m/z. Usually measurement error to the true m/z. Usually measured in ppm or milli mass units.measured in ppm or milli mass units.

The mass rangeThe mass range - is the range of m/z amenable to - is the range of m/z amenable to analysis by a given analyzer.analysis by a given analyzer.

The linear dynamic rangeThe linear dynamic range - is the range over which - is the range over which ion signal is linear with analyte concentration.ion signal is linear with analyte concentration.

2. The mass spectrometer2. The mass spectrometer

In order to measure the characteristics In order to measure the characteristics of individual molecules, a mass of individual molecules, a mass spectrometer converts them to ions so spectrometer converts them to ions so that they can be moved about and that they can be moved about and manipulated by external electric and manipulated by external electric and magnetic fields. magnetic fields.

The word The word spectrographspectrograph has been used since 1884 as has been used since 1884 as an "an "International Scientific VocabularyInternational Scientific Vocabulary". The ". The linguistic roots are a combination and removal of linguistic roots are a combination and removal of bound morphemes and free morphemes which relate bound morphemes and free morphemes which relate to the terms to the terms spectrspectrumum and and photphotographographic plateic plate. Early . Early spectrometryspectrometry devices that measured the mass-to- devices that measured the mass-to-charge ratio of ions were called charge ratio of ions were called mass spectrographsmass spectrographs which consisted of instruments that recorded a which consisted of instruments that recorded a spectrum of mass values on a photographic plate. A spectrum of mass values on a photographic plate. A mass spectroscopemass spectroscope is similar to a is similar to a mass spectrographmass spectrograph except that the beam of ions is directed onto a except that the beam of ions is directed onto a phosphor screen. phosphor screen.

The three essential functions of a mass The three essential functions of a mass spectrometer, and the associated components, spectrometer, and the associated components, are:are:

1.1. A small sample of compound is ionized, usually A small sample of compound is ionized, usually to cations by loss of an electron.   to cations by loss of an electron.   ((the ion the ion source)source)

2.2. The ions are sorted and separated according to The ions are sorted and separated according to their mass and charge.   their mass and charge.   ((the mass analyzer)the mass analyzer)..

3.3. The separated ions are then detected and tallied, The separated ions are then detected and tallied, and the results are displayed on a chart.   and the results are displayed on a chart.   ((the the detector)detector)

The analyser and detector of the mass spectrometer, and The analyser and detector of the mass spectrometer, and often the ionisation source too, are maintained under high often the ionisation source too, are maintained under high vacuum to give the ions a reasonable chance of travelling vacuum to give the ions a reasonable chance of travelling from one end of the instrument to the other without any from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction mass spectrometer, and often the sample introduction process also, is under complete process also, is under complete data system data system control on control on modern mass spectrometers.modern mass spectrometers.

The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum).

Replica of an early mass spectrometer

Because ions are very reactive and short-lived, Because ions are very reactive and short-lived, their formation and manipulation must be their formation and manipulation must be conducted in a vacuum. Atmospheric pressure is conducted in a vacuum. Atmospheric pressure is around 760 torr (mm of mercury). The pressure around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10under which ions may be handled is roughly 10 -5-5 to to 1010-8-8 torr (less than a billionth of an atmosphere). torr (less than a billionth of an atmosphere). Each of the three tasks listed above may be Each of the three tasks listed above may be accomplished in different ways. In one common accomplished in different ways. In one common procedure, ionization is effected by a high energy procedure, ionization is effected by a high energy beam of electrons, and ion separation is achieved beam of electrons, and ion separation is achieved by accelerating and focusing the ions in a beam, by accelerating and focusing the ions in a beam, which is then bent by an external magnetic field. which is then bent by an external magnetic field. The ions are then detected electronically and the The ions are then detected electronically and the resulting information is stored and analyzed in a resulting information is stored and analyzed in a computer. computer.

When a high energy electron collides with a When a high energy electron collides with a molecule it often ionizes it by knocking away one molecule it often ionizes it by knocking away one of the molecular electrons (either bonding or non-of the molecular electrons (either bonding or non-bonding). This leaves behind bonding). This leaves behind a a molecular ionmolecular ion (colored red in the following diagram). Residual (colored red in the following diagram). Residual energy from the collision may cause the molecular energy from the collision may cause the molecular ion to fragment into neutral pieces (colored green) ion to fragment into neutral pieces (colored green) and smaller and smaller fragment ionsfragment ions (colored pink and (colored pink and orange). The molecular ion is a radical cation, but orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations the fragment ions may either be radical cations (pink) or carbocations (orange), depending on the (pink) or carbocations (orange), depending on the nature of the neutral fragment. nature of the neutral fragment.

Many mass spectrometers work in either Many mass spectrometers work in either negative ion modenegative ion mode or or positive ion modepositive ion mode. It is . It is very important to know whether the very important to know whether the observed ions are negatively or positively observed ions are negatively or positively charged. This is often important in charged. This is often important in determining the neutral mass but it also determining the neutral mass but it also indicates something about the nature of indicates something about the nature of molecules.molecules.

4-methyl-3-pentene-2-one

N, N-diethylmethylamine

bromine

Bromine:   50.50% 79Br and 49.50% 81Br

IsotopesIsotopesSince a mass spectrometer separates and detects ions of slightly Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a different masses, it easily distinguishes different isotopes of a given element. This is manifested most dramatically for given element. This is manifested most dramatically for compounds containing bromine and chlorine, as illustrated by compounds containing bromine and chlorine, as illustrated by the following examples. Since molecules of bromine have only the following examples. Since molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a two atoms, the spectrum on the left will come as a surprise if a single atomic mass of 80 amu is assumed for Br. The five peaks single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate clearly that natural bromine in this spectrum demonstrate clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively. Thus, the bromine masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two 79Br atoms (mass 158 amu), molecule may be composed of two 79Br atoms (mass 158 amu), two 81Br atoms (mass 162 amu) or the more probable two 81Br atoms (mass 162 amu) or the more probable combination of 79Br-81Br (mass 160 amu). Fragmentation of combination of 79Br-81Br (mass 160 amu). Fragmentation of Br2 to a bromine cation then gives rise to equal sized ion peaks Br2 to a bromine cation then gives rise to equal sized ion peaks at 79 and 81 amu.at 79 and 81 amu.

vinyl chloride

Chlorine:   75.77% 35Cl and 24.23% 37Cl

methylene chloride

3. Quantitative and qualitative analysis3. Quantitative and qualitative analysisMass analyzers separate the ions according to their mass-Mass analyzers separate the ions according to their mass-to-charge ratio. The following two laws govern the to-charge ratio. The following two laws govern the dynamics of charged particles in electric and magnetic dynamics of charged particles in electric and magnetic fields in vacuum: fields in vacuum: (Lorentz force (Lorentz force law)law)

wherewhereF is the force (in newtons)F is the force (in newtons)E is the electric field (in volts per meter)E is the electric field (in volts per meter)B is the magnetic field (in teslas)B is the magnetic field (in teslas)qq is the electric charge of the particle (in coulombs) is the electric charge of the particle (in coulombs)v is the instantaneous velocity of the particle (in meters per second)v is the instantaneous velocity of the particle (in meters per second)× is the vector cross product× is the vector cross product

Newton's second law of motion in non-Newton's second law of motion in non-relativistic case, i.e. valid only at ion relativistic case, i.e. valid only at ion velocity much lower than the speed of velocity much lower than the speed of light.light.

Here Here FF is the force applied to the ion, is the force applied to the ion, mm is the mass of the ion, is the mass of the ion, aa is the acceleration is the acceleration

Many mass spectrometers work in either Many mass spectrometers work in either negative ion modenegative ion mode or or positive ion modepositive ion mode. It is . It is very important to know whether the observed very important to know whether the observed ions are negatively or positively charged. ions are negatively or positively charged. This is often important in determining the This is often important in determining the neutral mass but it also indicates something neutral mass but it also indicates something about the nature of molecules. Different about the nature of molecules. Different types of ion source result in different arrays types of ion source result in different arrays of fragments produced from the original of fragments produced from the original molecules.molecules.

By understanding the origin of a sample, By understanding the origin of a sample, certain expectations can be assumed as to certain expectations can be assumed as to the component molecules of the sample and the component molecules of the sample and their fragmentations. A sample from a their fragmentations. A sample from a synthesis/manufacturing process will synthesis/manufacturing process will probably contain impurities chemically probably contain impurities chemically related to the target component. A relatively related to the target component. A relatively crudely prepared biological sample will crudely prepared biological sample will probably contain a certain amount of salt, probably contain a certain amount of salt, which may form adducts with the analyte which may form adducts with the analyte molecules in certain analyses.molecules in certain analyses.

4. 4. Application of mass-spectrometryApplication of mass-spectrometryMass spectrometry is also used to determine Mass spectrometry is also used to determine the isotopic composition of elements within a the isotopic composition of elements within a sample. Differences in mass among isotopes of sample. Differences in mass among isotopes of an element are very small, and the less an element are very small, and the less abundant isotopes of an element are typically abundant isotopes of an element are typically very rare, so a very sensitive instrument is very rare, so a very sensitive instrument is required. These instruments, sometimes required. These instruments, sometimes referred to as isotope ratio mass referred to as isotope ratio mass spectrometers (IR-MS), usually use a single spectrometers (IR-MS), usually use a single magnet to bend a beam of ionized particles magnet to bend a beam of ionized particles towards a series of Faraday cups which towards a series of Faraday cups which convert particle impacts to electric current. convert particle impacts to electric current.

A fast on-line analysis of deuterium content of A fast on-line analysis of deuterium content of water can be done using Flowing afterglow water can be done using Flowing afterglow mass spectrometry, FA-MS. Probably the most mass spectrometry, FA-MS. Probably the most sensitive and accurate mass spectrometer for sensitive and accurate mass spectrometer for this purpose is the accelerator mass this purpose is the accelerator mass spectrometer (AMS). Isotope ratios are spectrometer (AMS). Isotope ratios are important markers of a variety of processes. important markers of a variety of processes. Some isotope ratios are used to determine the Some isotope ratios are used to determine the age of materials for example as in carbon age of materials for example as in carbon dating. Labeling with stable isotopes is also dating. Labeling with stable isotopes is also used for protein quantification.used for protein quantification.

Thank you for attention!