Mass Spectrometry

I GAA Septiari
Introduction
▪ MS is a technique for measuring masses of atoms, molecules, or fragments of
molecules and is performed in a mass spectrometer.
▪ A mass spectrometer is built from three components, an ion source, a mass
analyser, and a detector.
▪ The analytes are ionized in the ion source and are separated according to their
mass-to-charge ratio (m/z) in the mass analyser.
▪ The abundance of each ion with a different m/z value is finally measured by the
detector.
▪ The result is displayed as a mass spectrum showing the number of ions detected
at each value of the m/z ratio.
Introduction
• In LC-MS, the chromatographic
system is linked to the mass
spectrometer by an interface. In this
interface, the sample constituents in
the mobile phase are ionized and
vapourized before they enter the
mass spectrometer

• In GC-MS, the sample constituents

enter the mass spectrometer in the
carrier gas directly inside the
vacuum region of the mass
spectrometer and an interface is not
necessary.
Basic Theory of Mass Spectrometry

• Mass spectra display all isotopes of the molecule.

• All elements frequently found in drug substances
exist as more than one isotope, except for fluorine,
iodine, and phosphorus, which are monoisotopic.
Basic Theory of Mass Spectrometry
Instrumentation of Mass Spectrometry
▪ All mass spectrometers comprise four main components.
▪ion source, where sample molecules may be ionized by a variety of means,
▪ a mass analyzer that separates ions according to their mass-to-charge ratio,
m/z,
▪ a detector that measures the abundances of the separated ions as an
electrical signal
▪ a recording device that converts the detector signal into a form suitable for
further study and processing.
Ionization Techniques – Organic Mass
Spectrometry
▪ The first step in the MS analysis is ionization of the sample constituents.
▪ The ionization methods are different in GC-MS and LC-MS.

Electron Ionization (EI) application in GC-MS

▪ The sample constituents eluting from the GC column are bombarded with electrons inside
the mass spectrometer under vacuum conditions.
▪ This is a hard ionization method termed electron ionization (EI) (or electron impact
ionization), in which sample constituents are often degraded to fragments.

▪ The analyte is vaporized into the EI source and bombarded with 70-eV electrons.

▪ The ion source chamber is maintained at a vacuum of 10 -3 Pa.

Electron Ionization (EI) application in GC-MS

▪ Electron bombardment leads to some of the molecules being ionized, in most

cases by loss of a single electron per molecule.

▪ A proportion of the energy of the electron beam is transferred to the molecule,

ejecting an electron to generate a molecular ion (M+•), the most important ion in
the mass spectrum.
Electron Ionization (EI) application in GC-MS

▪ Excess electronic energy is also transferred to the molecule during the ionization
process and is rapidly converted to internal vibrational energy.

▪ This energy may be sufficient to induce bond cleavage in some ions, generating
fragment ions that may also decompose.
Electron Ionization (EI) application in GC-MS

▪ Most ions formed by EI have a single positive charge (z=1), and m/z is then
equivalent to the mass of the ion.

▪ Mass spectra are generally recorded in the form of a line diagram (bar graph).

▪ Ion intensities are normalized so that the largest peak in the mass spectrum, the
base peak, has an intensity of 100 units, and all other ion abundances are
expressed as a percentage of this value.
• A molecular ion can be seen at
m/z 194, and a number of
abundant fragment ions are also
present.
• The molecular ion is also the
base peak, although this is not
always the case; strongly
favored fragmentations may yield
abundant fragment ions that are
more intense than the molecular
ion
Electron Ionization (EI) application in GC-MS
▪ Some molecules are difficult to analyze by EI mass spectrometry

▪ The molecular ion is of very low abundance and therefore (in the case of an
unknown) difficult to assign.

▪ The molecules may be involatile or thermally labile and difficult or impossible to

vaporize without inducing thermolytic decomposition.
Electrospray ionization (ESI) in LC-MS

• In LC-MS, the chromatographic system is linked to the mass spectrometer by an interface.

• In this interface, sample constituents are ionized and vapourized before they enter the mass
spectrometer.
• In ESI, the analyte is dissolved in a suitable solvent, for example a 50:50 mixture of acetonitrile
and water, and is pumped through a narrow diameter stainless steel capillary
• In the ESI interface, the mobile phase is converted to a fine spray and exposed to an electric
potential of 2–5 kV, resulting in a fine aerosol of charged droplets.
• A heated (usually nitrogen) gas aids evaporation of the charged droplets and helps break up
clusters of analyte and solvent ions.
• Analyte ions are generated from the charged droplets by an evaporation mechanism.
▪ ESI forms positive or negative ions depending on applied voltage.

▪ Ions are produced based on acid–base chemistry, by proton uptake, or release

from the analyte molecule, resulting in [M + H]+ ions and [M−H]− ions,
respectively.

▪ ESI is a soft ionization technique, which leaves the ions stable without, or only
limited, fragmentation.
• ESI has revolutionized mass
spectrometry by enabling
mass spectra to be obtained
routinely on polar, involatile
molecules.
• Even high-molecular-weight
biomolecules can be
analyzed by ESI.
• The y-axis in a mass spectrum shows the
abundance or intensity of the different ions.
• The x-axis displays the m/z value.
• The signal for the most intense ion is termed
the base peak and the intensity of the base
peak is scaled to 100%.
• The other fragment ions are then scaled
relative to the base peak.
• The base peak in the EI mass spectrum
appears at m/z 254.
• The EI mass spectrum shows only a small
signal for the molecular ion at m/z 303, as the
molecular ion is extensively fragmented.
• In the ESI mass spectrum, the base peak is
the protonated molecule, which appears as [M
+ H]+ at m/z 304.
Mass analyzer
▪ The mass analyzer of the mass spectrometer separates ionized atoms and
molecules according to their mass-to-charge ratio, m/z.
▪ Several different types of mass analyzer are available.
▪Magnetic sector mass spectrometers
▪Isotope ratio mass spectrometers
▪Quadrupole mass analyzers
▪FTICR mass spectrometry
▪Ion Traps
FTICR mass spectrometry
▪ FTICR (sometimes simply known as Fourier transform mass spectrometry) is a
Penning trap in which ions confined by a strong magnetic field, B, move in circular
orbits of characteristic frequency.
▪ A broad-band ‘chirp’ of electromagnetic radiation is applied to excite all ions in the
trapping cell to their cyclotron frequency.
▪ The orbiting ions induce an alternating ‘image current’ in the walls of the cell
▪ This signal is complex because each m/z value generates its own characteristic
frequency.
▪ A Fourier transform of this complex signal yields the masses of all ions in the
spectrum.
▪ Frequencies can be measured with great accuracy, and so very high resolving
powers, over 1 000 000 in some cases, are attainable.
▪ However, resolution does fall off with increasing m/z.
Ion detection and recording

▪ The most common detector in mass spectrometry is the electron multiplier; this
amplifies the weak ion beam signal greatly, yielding gains of up to the order of
106.

▪ Multipliers can be either a discrete dynode type or a continuous dynode,

generally known as a channel electron multiplier or channeltron
Data acquisition and processing

▪ Computer forms part of the system used to digitize the mass spectrum, calibrate the mass
scale, enhance the quality of data, and, ultimately, to aid in the interpretation of data.

▪ Library searching of a large database of the mass spectra of known compounds is now
routine as an aid to interpreting EI spectra and identifying components in (for example) a
complex gas chromatography – mass spectrometry chromatogram.

▪ Computer systems are now an integral component of mass spectrometers and have
greatly simplified the process of instrument control, data acquisition, and interpretation.
Identification by MS
Structural Information from Isotopes
Structural Information from Isotopes
CH2Cl from the molecular ion
(C14H19Cl2NO2 −CH2Cl = C13H17ClNO2).
▪ Many drug substances contain nitrogen, and for these compounds the nitrogen rule
applies. How to apply the nitrogen rule is dependent on the ionization method.

▪ For EI the rule is as follows:

▪ If a compound has an odd number of nitrogen atoms then the molecular ion M•+
has an odd nominal m/z value.
▪ If a compound has an even number of nitrogen atoms, or no nitrogen atoms, the
molecular ion M•+ has an even nominal m/z value.
▪ For ESI (and other ionization methods generating (M + H)+ or (M − H)−ions) the rule
should be applied as follows:
▪ If a compound has an odd number of nitrogen atoms then the molecular ion (M +
H)+ or (M − H)− has an even nominal m/z value.
▪ If a compound has an even number of nitrogen atoms, or no nitrogen atoms, the
molecular ion (M + H)+ or (M − H)− has an odd nominal m/z value.
If the elemental composition of the
analyte is known, the number of rings
(R) + double bonds (DB) can be
calculated from the following equation: