Mass spectrometry

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MASS SPECTROMETRY
By Mussarat Jabeen


MASS SPECTROMETRY

MASS SPECTROMETRY

Mass spectrometry is a powerful analytical technique that is used to identify unknown
compounds, to quantify known compounds, and to elucidate the structure and chemical
properties of molecules. It is the smallest scale in the world, not because of the mass
spectrometer’s size but because of the size what it weighs...molecules. According to the IUPAC
(International Union of Pure and Applied Chemistry), it is the branch of science dealing with all
aspects of mass spectroscopes and results obtained with these instruments. The information
given by mass spectrometry is sometimes sufficient, frequently necessary, and always useful for
identification of species.

History of Mass Spectrometry

 Mass Spectrometry was started by J.J. Thomson. Until 1897, scientists believed atoms
were indivisible; the ultimate particles of matter, but Thomson proved them wrong when
he discovered that atoms contained particles known as electrons. He concluded this by his
experiments on cathode rays. He found that the rays could be deflected by an electric
field (in addition to magnetic fields, which was already known). By comparing the
deflection of a beam of cathode rays by electric and magnetic fields he was able to
measure the particle's mass. This showed that cathode rays were matter, but he found that
the particles were about 2000 times lighter than the mass of the lightest atom, hydrogen.
He concluded that the rays were composed of very light negatively charged particles
which he called electron. He also concluded that neon is composed of two isotopes and
them which was the first example of mass spectrometry. On his discovery he was
awarded Nobel Prize in 1906.

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 In 1919, Thomson, with the help of his student Francis Aston (who would go on to
win his own Nobel Prize in Chemistry in 1922), built what later would be recognized
as the first mass spectrometer to measure the masses of charged atoms. In their first
mass spectrometer they measure the charge to mass ratio (z/m) for several ionic
species. In the expression z/m, z is the charge number, i.e. the total charge on an ion
divided by the elementary charge (e), and m is the nucleon number, i.e. the sum
of the total number of protons and neutrons in an atom, molecule or ion. In modern
mass spectrometry, the parameter measured is m/z, rather than z/m: the unit of m/z
was recently designated the Thomson (Th).This instrument used gas discharge tubes
to generate ions, which were then passed through parallel electric and magnetic fields.
The ions were deflected into parabolic trajectories and then detected on a
photographic plate.
 In 1934, First double focusing magnetic analyzer was invented by Johnson E.G., Nier
A.O.
 In 1939, Accelerator Mass Spectrometry was developed by Lawrence E.O., Alvarez
L.W., Brobeck W.M., Cooksey D., Corson D.R., McMillan E.M., Salisbury W.W.,
Thornton R.LAn important tool in trace biomolecule detection, still coming into its
own.
 In 1946, Time-of-Flight Mass Spectrometry was invented by W.Stephens the
significance of TOF mass analyzers has grown over the last 20 years especially in the
biomedical applications of mass spectrometry.
 In 1947, Preparative Mass Spectrometry was used to purify radioactive 235U by
Siuzdak G., Bothner B., Yeager M., Brugidou C., Fauquet C.M., Hoey K., Chang
C.M., which was then used to construct the first nuclear weapon. It was recently
demonstrated that viruses as well as other types of molecules could also be separated
and collected using electrospray ionization mass spectrometry.
 In 1953, Paul's invention of the Quadrupole and Quadrupole ion trap earned him the
Nobel Prize in Physics. These mass analyzers are the most widely used today and are
still being developed for an even wider range of applications.
 In 1956, Golhke R.S., McLafferty F., Wiley B., Harrington D invented GC/MS
instruments. A very powerful tool and still one of the most popular forms of doing
mass spectrometry.
 In 1956, Beynon J.H first time Identify Organic Compounds with Mass
Spectrometry.
 In 1966, Munson and Field described chemical ionization (CI). One of the first soft
ionization techniques

 In 1968, Electrospray Ionization was invented by Dole M., Mack L.L., Hines R.L.,
Mobley R.C., Ferguson L.D., Alice M.B.
 In 1975, Atmospheric Pressure Chemical Ionization (APCI) was developed by Carroll
D.I., Dzidic I., Stillwell R.N., Haegele K.D., Horning E.C.
 In 1976, Ronald MacFarlane and co-workers develop plasma desorption mass
spectrometry.

 In 1980, Inductively Coupled Plasma MS By Reed T.B. A very powerful tool for
elemental composition analysis
 In 1985, Franz Hillenkamp, Michael Karas and co-workers describe and coin the term
matrix-assisted laser desorption ionization (MALDI).

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 In 1989, Wolfgang Paul receives the Nobel Prize in Physics "for the development
of the ion trap technique"
 In 2002, John Bennett Fenn and Koichi Tanaka are awarded one-quarter of the
Nobel Prize in chemistry each "for the development of soft desorption ionisation
methods ... for mass spectrometric analyses of biological macromolecules."

The Five Mass Spectrometry Nobel Prize Pioneers

Joseph John Francis Wolfgang Paul John Bennet Koichi Tanaka
Thomson William 1989 Nobel Fenn 2002 Nobel
1906 Nobel Aston Prize for 2002 Nobel Prize Prize for
Prize for 1922 Nobel Physics for Chemistry Chemistry
Physics Prize for (for the (for the (mass
(theoretical Chemistry development of development of spectrometric
and (mass the ion trap Soft Desorption analyses of
experimental spectrograph, technique) ionization biological
investigations of isotopes, in Method) macromolecule)
on the a large
conduction of number of
electricity by non-
gases) radioactive
elements)

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MASS SPECTROMETRY

MASS SPECTROMETER

An instrument which measures the ratio of mass to the number of charges of ions produced
from elements and compounds. It is also of value in performing fundamental studies of the
properties of gaseous ions. A mass spectrometer is similar to a prism. In the prism, light is
separated into its component wavelengths which are then detected with an optical receptor,
such as visualization. Similarly, in a mass spectrometer the generated ions are separated in
the mass analyzer, digitized and detected by an ion detector.

Understanding Mass Spectrometry

To understand the basic principles of mass spectrometry, consider a person standing at the
top of a tower on a windy day. The person picks up various balls and drops them, one by one,
from the tower. As each ball falls, wind deflects it along a curved path. The masses of the
balls affect how they fall. A bowling ball, for example, is much heavier than a basketball and

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is therefore harder to move. As a result, a bowling ball follows a different path than a
baseball.

In a mass spectrometer, the same thing is happening, except it's atoms and molecules that are
being deflected, and it's electric or magnetic fields causing the deflection. It's also happening
in a cabinet that can be as small as a microwave or as large as a chest freezer.

Basic components of mass spectrometer

Four basic components are, for the most part, standard in all mass spectrometers: a sample
inlet, an ionization source, a mass analyzer and an ion detector. Some instruments combine
the sample inlet and the ionization source, while others combine the mass analyzer and the
detector. However, all sample molecules undergo the same processes. Sample molecules are
introduced into the instrument through a sample inlet. Once inside the instrument, the sample
molecules are converted to ions in the ionization source, before being electrostatically
propelled into the mass analyzer. Ions are then separated according to their m/z within the
mass analyzer. The detector converts the ion energy into electrical signals, which are then
transmitted to a computer.

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Sample Introduction Techniques

In order to perform mass analysis on a sample, which is initially at atmospheric pressure (760
mmHg), it must be introduced into the instrument in such a way that the vacuum inside the
instrument remains relatively unchanged (~10-6 torr). The most common methods of sample
introduction are direct insertion with a probe or plate commonly used with MALDI-MS,
direct infusion or injection into the ionization source such as ESI-MS.
Direct Insertion:

Using an insertion probe/plate is a very simple way to introduce a sample into an instrument.
The sample is first placed onto a probe and then inserted into the ionization region of the
mass spectrometer, typically through a vacuum interlock. The sample is then subjected to any
number of desorption processes, such as laser desorption or direct heating, to facilitate
vaporization and ionization

Direct Infusion:

A simple capillary or a capillary column is used to introduce a sample as a gas or in solution.
Direct infusion is also useful because it can efficiently introduce small quantities of sample
into a mass spectrometer without compromising the vacuum. Capillary columns are routinely
used to interface separation techniques with the ionization source of a mass spectrometer.
These techniques, including gas chromatography (GC) and liquid chromatography (LC), also
serve to separate a solution’s different components prior to mass analysis. In gas
chromatography, separation of different components occurs within a glass capillary column.
As the vaporized sample exits the gas chromatograph, it is directly introduced into the mass
spectrometer

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Ionization Methods

Ionization method refers to the mechanism of ionization while the ionization source is the
mechanical device that allows ionization to occur. The different ionization methods,
summarized here, work by either ionizing a neutral molecule through electron ejection,
electron capture, protonation, cationization, or deprotonation, or by transferring a charged
molecule from a condensed phase to the gas phase.

 Protonation

 Deprotonation

 Cationization

 Transfer of a charged molecule to the gas phase

 Electron ejection

 Electron capture

Protonation (positive ions)

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Protonation is a method of ionization by which a proton is added to a molecule, producing a
net positive charge of 1+ for every proton added. Protonation is used for basic compounds
such as amines, to form stable cations. Peptides are often ionized via protonation. Protonation
can be achieved via matrix-assisted laser desorption/-ionization (MALDI), electrospray
ionization (ESI) and atmospheric pressure chemical ionization (APCI).

Deprotonation (negative ions)

Deprotonation is an ionization method by which the net negative charge of 1- is achieved
through the removal of a proton from a molecule. This mechanism of ionization, commonly
achieved via MALDI, ESI, and APCI is very useful for acidic species including phenols,
carboxylic acids, and sulfonic acids.

Cationization (positive ions)

M + Cation+ → MCation+

Cationization is a method of ionization that produces a charged complex by non-covalently
adding a positively charged ion to a neutral molecule. While protonation could fall under this
same definition, cationization is distinct for its addition of a cation adduct other than a proton
(e.g. alkali, ammonium). Moreover, it is known to be useful with molecules unstable to
Protonation. The binding of cations other than protons to a molecule is naturally less

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covalent, therefore, the charge remains localized on the cation. This minimizes delocalization
of the charge and fragmentation of the molecule. Cationization is commonly achieved via
MALDI, ESI, and APCI. Carbohydrates are excellent candidates for this ionization
mechanism, with Na+ a common cation adduct.

Transfer of a charged molecule to the gas phase (positive or negative ions)

The transfer of compounds already charged in solution is normally achieved through the
desorption or ejection of the charged species from the condensed phase into the gas phase.
This transfer is commonly achieved via MALDI or ESI. The positive ion mass spectrum of
tetraphenylphosphine.

Electron ejection (positive ions)

As its name implies, electron ejection achieves ionization through the ejection of an electron
to produce a 1+ net positive charge, often forming radical cations. Observed most commonly
with electron ionization (EI) sources, electron ejection is usually performed on relatively
nonpolar compounds with low molecular weights and it is also known to generate significant
fragment ions. The mass spectrum resulting from electron ejection of anthracene.

Electron capture (negative ions)

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With the electron capture ionization method, a net negative charge of 1- is achieved with the
absorption or capture of an electron. It is a mechanism of ion-ization primarily observed for
molecules with a high electron affinity, such as halogenated compounds. The electron capture
mass spectrum of hexachloro-benzene.

Ionization Sources

 Electrospray Ionization (ESI)
 Nanoelectrospray Ionization (NanoESI)
 Atmospheric Pressure Chemical Ionization (APCI)
 Atmospheric pressure photoionization (APPI)
 Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
 Fast Atom Bombardment (FAB)
 Electron Ionization (EI)
 Chemical Ionization (CI)
 Thermal ionization (TI)

Types of Ionization Sources

 Hard ionization sources
 Soft ionization sources

Hard ionization sources

Leave excess energy in molecule and produced stable fragments which is not further
fragarmented.

Soft ionization sources

Little excess energy in molecule and produced unstable fragments which are again
fragmented.

Electrospray Ionization

Electrospray ionization (ESI) is a method routinely used with peptides, proteins,
carbohydrates, small oligonucleotides, synthetic polymers, and lipids. ESI produces gaseous
ionized molecules directly from a liquid solution. It operates by creating a fine spray of
highly charged droplets in the presence of an electric field. (An illustration of the electrospray
ionization process.

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The sample solution is sprayed from a region of the strong electric field at the tip of a metal
nozzle maintained at a potential of anywhere from 700 V to 5000 V. The nozzle (or needle)
to which the potential is applied serves to disperse the solution into a fine spray of charged
droplets. Either dry gas, heat, or both are applied to the droplets at atmospheric pressure thus
causing the solvent to evaporate from each droplet. As the size of the charged droplet
decreases, the charge density on its surface increases. The mutual Coulombic repulsion
between like charges on this surface becomes so great that it exceeds the forces of surface
tension, and ions are ejected from the droplet through a “Taylor cone”. Electrospray
ionization is conducive to the formation of singly charged small molecules, but is also well-
known for producing multiply charged species of larger molecules. This is an important
phenomenon because the mass spectrometer measures the mass-to-charge ratio (m/z) and
therefore multiple charging makes it possible to observe very large molecules with an
instrument having a relatively small mass range.

Many solvents can be used in ESI and are chosen based on the solubility of the compound of
interest, the volatility of the solvent and the solvent’s ability to donate a proton. Typically,
protic primary solvents such as methanol, 50/50 methanol/water, or 50/50 acetonitrile/H2O

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are used, while aprotic cosolvents, such as 10% DMSO in water, as well as isopropyl alcohol
are used to improve solubility for some compounds. Although 100% water is used in ESI,
water’s relatively low vapor pressure has a detrimental effect on sensitivity; better sensitivity
is obtained when a volatile organic solvent is added. Some compounds require the use of
straight chloroform with 0.1% formic acid added to facilitate ionization. This approach, while
less sensitive, can be effective for otherwise insoluble compounds.

Buffers such as Na+, K+, phosphate, and salts present a problem for ESI by lowering the
vapor pressure of the droplets resulting in reduced signal through an increase in droplet
surface tension resulting in a reduction of volatility (see Chapter 3 for quantitative
information on salt effects). Consequently, volatile buffers such as ammonium acetate can be
used more effectively.

Advantages

 practical mass range of up to 70,000 Da
 good sensitivity with femtomole to low picomole sensitivity typical
 softest ionization method, capable of generating noncovalent complexes in the gas
phase
 easily adaptable to liquid chromatography
 easily adaptable to tandem mass analyzers such as ion traps and triple quadrupole
instruments
 multiple charging allows for analysis of high mass ions with a relatively low m/z
range instrument
 no matrix interference

Disadvantages

 the presence of salts and ion-pairing agents like TFA can reduce sensitivity
 complex mixtures can reduce sensitivity
 simultaneous mixture analysis can be poor
 multiple charging can be confusing especially in mixture analysis
 sample purity is important
 carryover from sample to sample

Nanoelectrospray Ionization (NanoESI)

Low flow electrospray, originally described by Wilm and Mann, has been called
nanoelectrospray, nanospray, and micro-electrospray. This ionization source is a variation on
ESI, where the spray needle has been made very small and is positioned close to the entrance
to the mass analyzer. The end result of this rather simple adjustment is increased efficiency,
which includes a reduction in the amount of sample needed.

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The flow rates for nanoESI sources are on the order of tens to hundreds of nanoliters per
minute. In order to obtain these low flow rates, nanoESI uses emitters of pulled and in
some cases metallized glass or fused silica that have a small orifice (~5µ). The
approximate size of droplet in nanoESI is 0.2 micron diameter which is very small as
compared to normal ESI with droplet size 1 micron diameter.

Advantages

 Very sensitive
 very low flow rates
 applicable to LC/MS
 has reasonable salt tolerance (low millimolar)
 multiple charging useful
 reasonable tolerance of mixtures
 Soft ionization (little fragmentation observed).

Disadvantages

 low flow rates require specialized systems
 significant suppression can occur with mixtures

Atmospheric Pressure Chemical Ionization

APCI has also become an important ionization source because it generates ions directly from
solution and it is capable of analyzing relatively nonpolar compounds. Similar to

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electrospray, the liquid effluent of APCI is introduced directly into the ionization source.
However, the similarity stops there. The droplets are not charged and the APCI source
contains a heated vaporizer, which facilitates rapid desolvation/vaporization of the droplets.
Vaporized sample molecules are carried through an ion-molecule reaction region at
atmospheric pressure.

Advantages

 As the solvent ions are present at atmospheric pressure conditions, chemical
ionization of analyte molecules is very efficient.
 At atmospheric pressure analyte molecules collide with the reagent ions frequently.
 Proton transfer (for protonation MH+ reactions) occurs in the positive mode
 electron transfer or proton loss, ([M-H]-) in the negative mode.
 Multiple charging is typically not observed presumably because the ionization process
is more energetic than ESI.

Atmospheric Pressure Photoionization

Atmospheric pressure photoionization (APPI) has recently become an important ionization
source because it generates ions directly from solution with relatively low background and is
capable of analyzing relatively nonpolar compounds. Similar to APCI, the liquid effluent of
APPI is introduced directly into the ionization source. The primary difference between APCI
and APPI is that the APPI vaporized sample passes through ultra-violet light (a typical
krypton light source emits at 10.0 eV and 10.6 eV). Often, APPI is much more sensitive than
ESI or APCI and has been shown to have higher signal-to-noise ratios because of lower
background ionization. Lower background signal is largely due to high ionization potential of
standard solvents such as methanol and water (IP 10.85 and 12.62 eV, respectively) which
are not ionized by the krypton lamp.

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In APPI Protonation, Deprotonation, Cationization reaction takes place.

Disadvantages

 It can generate background ions from solvents
 It requires vaporization temperatures ranging from 350-500° C, which can cause
thermal degradation.

Matrix-Assisted Laser Desorption/Ionization

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was first
introduced in 1988 by Tanaka, Karas, and Hillenkamp. It has since become a widespread
analytical tool for peptides, proteins, and most other biomolecules (oligonucleotides,
carbohydrates, natural products, and lipids).

While the exact desorption/ionization mechanism for MALDI is not known, it is generally
believed that MALDI causes the ionization and transfer of a sample from the condensed
phase to the gas phase via laser excitation and abalation of the sample matrix. In MALDI

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analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound,
usually a UV-absorbing weak organic acid. Irradiation of this analyte-matrix mixture by a
laser results in the vaporization of the matrix, which carries the analyte with it. The matrix
plays a key role in this technique. The co-crystallized sample molecules also vaporize, but
without having to directly absorb energy from the laser. Molecules sensitive to the laser light
are therefore protected from direct UV laser excitation.

MALDI matrix -- A nonvolatile solid material facilitates the desorption and ionization
process by absorbing the laser radiation. As a result, both the matrix and any sample
embedded in the matrix are vaporized. The matrix also serves to minimize sample damage
from laser radiation by absorbing most of the incident energy.

Advantages

 practical mass range of up to 300,000 Da. Species of much greater mass have been
observed using a high current detector;
 typical sensitivity on the order of low femtomole to low picomole. Attomole
sensitivity is possible;
 soft ionization with little to no fragmentation observed;
 tolerance of salts in millimolar concentrations;
 suitable for the analysis of complex mixtures.

Disadvantages

 Matrix background, which can be a problem for compounds below a mass of 700 Da.
This background interferences is highly dependent on the matrix material;

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 possibility of photo-degradation by laser desorption/ionization;
 Acidic matrix used in MALDI my cause degradation on some compounds.

Liquid SIMS (LSIMS) or Fast Atom Bombardment (FAB)

Fast atom ion bombardment, or FAB, is an ionization source similar to MALDI in that it uses
a matrix and a highly energetic beam of particles to desorb ions from a surface. It is
important, however, to point out the differences between MALDI and FAB. For MALDI, the
energy beam is pulsed laser light, while FAB uses a continuous ion beam. With MALDI, the
matrix is typically a solid crystalline, whereas FAB typically has a liquid matrix. It is also
important to note that FAB is about 1000 times less sensitive than MALDI.

Fast atom bombardment is a soft ionization source which requires the use of a direct insertion
probe for sample introduction, and a beam of Xe neutral atoms or Cs+ ions to sputter the
sample and matrix from the direct insertion probe surface. It is common to detect matrix ions
in the FAB spectrum as well as the protonated or cationized (i.e. M + Na+) molecular ion of
the analyte of interest.

FAB matrix -- Facilitating the desorption and ionization process, the FAB matrix is a
nonvolatile liquid material that serves to constantly replenish the surface with new sample as
it is bombarded by the incident ion beam. By absorbing most of the incident energy, the
matrix also minimizes sample degradation from the high-energy particle beam.

Two of the most common matrices used with FAB are m-nitrobenzyl alcohol and glycerol.

m-nitrobenzyl alcohol (NBA) glycerol

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The fast atoms or ions impinge on or collide with the matrix causing the matrix and analyte to
be desorbed into the gas phase. The sample may already be charged and subsequently
transferred into the gas phase by FAB, or it may become charged during FAB desorption
through reactions with surrounding molecules or ions. Once in the gas phase, the charged
molecules can be propelled electrostatically to the mass analyzer.

Electron Ionization

Electron ionization is one of the most important ionization sources for the routine analysis of
small, hydrophobic, thermally stable molecules and is still widely used. Because EI usually
generates numerous fragment ions it is a “hard” ionization source. However, the
fragmentation information can also be very useful. For example, by employing databases
containing over 200,000 electron ionization mass spectra, it is possible to identify an
unknown compound in seconds (provided it exists in the database). These databases,
combined with current computer storage capacity and searching algorithms, allow for rapid
comparison with these databases (such as the NIST database), thus greatly facilitating the
identification of small molecules.

Energetic process a heated filament emits electrons which are accelerated by a potential
difference of usually 70eV into the sample chamber.

Ionization of the sample occurs by removal of an electron from the molecule thus generating
a positively charged ion with one unpaired electron.

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Advantages

 Widely used technique when coupled to GC
 Suitable for volatile organic compounds
– eg hydrocarbons, oils, flavours, fragrances
 Not really coupled to LC today
 Also called electron impact
 Produces M+.radical cation giving molecular weight
 Produces abundant fragment ions

Chemical Ionization

Chemical Ionization (CI) is applied to samples similar to those analyzed by EI and is
primarily used to enhance the abundance of the molecular ion. Chemical ionization uses gas
phase ion-molecule reactions within the vacuum of the mass spectrometer to produce ions
from the sample molecule. The chemical ionization process is initiated with a reagent gas
such as methane, isobutane, or ammonia, which is ionized by electron impact. High gas
pressure in the ionization source results in ion-molecule reactions between the reagent gas
ions and reagent gas neutrals.

A possible mechanism for ionization in CI occurs as follows:

Reagent (R) + e- → R+ + 2 e-

R+ + RH → RH+ + R

RH+ + Analyte (A) → AH+ + R

In contrast to EI, an analyte is more likely to provide a molecular ion with reduced
fragmentation using CI. However, similar to EI, samples must be thermally stable since
vaporization within the CI source occurs through heating.

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Negative chemical ionization (NCI) typically requires an analyte that contains electron-
capturing moieties (e.g., fluorine atoms or nitrobenzyl groups). Such moieties significantly
increase the sensitivity of NICI, in some cases 100 to 1000 times greater than that of electron
ionization (EI). NCI is probably one of the most sensitive techniques and is used for a wide
variety of small molecules with the caveat that the molecules are often chemically modified
with an electron-capturing moiety prior to analysis.

While most compounds will not produce negative ions using EI or CI, many important
compounds can produce negative ions and, in some cases, negative EI or CI mass
spectrometry is more sensitive and selective than positive ion analysis. In fact, compounds
like steroids are modified to enhance NCI.

Advantages

 Produces M+H+ ions or M - H-ions
 Gives molecular weight
 Softer ionization technique

Thermal ionization

Thermal ionization is based upon the generation of atomic or molecular ions at the surface of
an electrically heated filament. Samples are deposited on specially treated filaments (usually
rhenium or tantalum), then carefully dried. The filaments are heated slowly, leading to
evaporation and vaporization of the sample. It is useful for determining the elements that
evaporate at low temperature but require high ionization temperatures (Ca, for example). TI
is generally used for precise and accurate measurement of stable isotope ratio of inorganic
elements. It is also used to quantify toxic trace elements in foods.

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Mass Analyzers

Once ions have been formed and introduced into the vacuum, they are subjected to electrical
(DC and/or RF) or magnetic fields. Their motion under these conditions is a function of many
parameters but all include the mass-to-charge ratio. Ions can be ejected from the analyser one
m/z at a time or can be detected and measured, while trapped in the analyser.

Mass analyzer should have following properties

Accuracy

The accuracy of a mass measurement or concentration from a quantitative determination is a
measure of how close the value obtained is to the true value. The accuracy varies
dramatically from analyzer to analyzer depending on the analyzer type and resolution.

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Mass Range

The range over which a mass spectrometer analyzer can operate. Quadrupole, Paul and linear
ion traps tend to be limited to upper m/z values around 4000. Penning FTICR and TOF
analyzers have mass limits that can extend this to well over 200,000 but there is a resolving
power trade-off at high m/z values.

Resolution (Resolving Power)

Resolution is a measure of the ability of the mass spectrometer analyzer to separate two ions
of different, but defined, m/z value. For two overlapping singly- charged peaks m1 and m2 of
equal height, the resolving power is defined as m1/Δm, where m1 is the m/z value of one ion
and Δm is the mass difference between m1 and m2 such that the two peaks are resolved with
a defined interpeak valley. In the diagram, if h is 10% of the peak heights, then the method is
called the 10% valley method. Other definitions include the 50% valley method; where h are
50%, and the FWHM method. For two adjacent peaks at m/z values of 200.00 and 200.05
separated by a 10% valley, the resolving power is 200.00/0.05 = 4000.

In simple words we can say that resolution is a measure of how well a mass spectrometer
separates ions of different mass.

Low resolution - capable of distinguishing among ions of different nominal mass that is ions
that differ by at least one or more mass units

High resolution - capable of distinguishing among ions that differ in mass by as little as
0.0001 mass units

The resolving power A is defined as:

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– To resolve two mass e.g. 950 and 951 then a resolving power A needs to be 950

 Low resolution spectrometers A = 1-2000
 High resolution spectrometers A >100000

Scan Speed

Analyzers are scanned with a regular cycle time from low to high m/z or vice versa.
Quadrupole analyzers tend to be scanned linearly in mass while a magnetic analyzer is
scanned exponentially to provide peaks of equal width throughout the mass range. Fast scan
speeds are needed when a mass spectrometer is linked to a fast chromatographic system and
TOF analyzers are currently among the best for this.

Quadrupoles

Quadrupole mass analyzers have been used with EI sources since the 1950’s and are still the
most common mass analyzers in existence today. Interestingly, quadrupole mass analyzers
have found new utility in their capacity to interface with ESI and APCI.

Four parallel metal rods of circular cross section are electronically connected in pairs and a
combination of DC and RF applied. At a specific RF field, only ions of a specific m/z can
pass through the quadrupole.

In order to perform tandem mass analysis with a quadrupole instrument, it is necessary to
place three quadruples in series. Each quadrupole has a separate function: the first quadrupole
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(Q1) is used to scan across a preset m/z range and select an ion of interest. The second
quadrupole (Q2), also known as the collision cell, focuses and transmits the ions while
introducing a collision gas (argon or helium) into the flight path of the selected ion. The third
quadrupole (Q3) serves to analyze the fragment ions generated in the collision cell (Q2)

-Ions travel parallel to four rods

- Opposite pairs of rods have rapidly alternating potentials (AC)

- Ions try to follow alternating field in helical trajectories

- Stable path only for one m/z value for each field frequency

Small and low cost

Rmax ~ 500

Harder to push heavy molecule - m/zmax < 2000

Quadrupole Ion Trap

It is similar to Quadrupole analyzer but in an ion trap, rather than passing through a
quadrupole analyzer with a superimposed radio frequency field, the ions are trapped in a
radio frequency quadrupole field. The quadrupole ion trap typically consists of a ring
electrode and two hyperbolic endcap electrodes. The motion of the ions induced by the
electric field on these electrodes allows ions to be trapped or ejected from the ion trap. In the
normal mode, the radio frequency is scanned to resonantly excite and therefore eject ions
through small holes in the endcap to a detector. As the RF is scanned to higher frequencies,
higher m/z ions are excited, ejected, and detected.

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MASS SPECTROMETRY

Linear Ion Trap

The linear ion trap differs from the 3D ion trap as it confines ions along the axis of a
quadrupole mass analyzer using a two-dimensional (2D) radio frequency (RF) field with
potentials applied to end electrodes. The primary advantage to the linear trap over the 3D trap
is the larger analyzer volume lends itself to a greater dynamic ranges and an improved range
of quantitative analysis.

Double-Focusing Magnetic Sector

The earliest mass analyzers separated ions with a magnetic field. In magnetic analysis, the
ions are accelerated into a magnetic field using an electric field. A charged particle traveling
through a magnetic field will travel in a circular motion with a radius that depends on the
speed of the ion, the magnetic field strength, and the ion’s m/z. A mass spectrum is obtained
by scanning the magnetic field and monitoring ions as they strike a fixed point detector. A
limitation of magnetic analyzers is their relatively low resolution. In order to improve this,

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MASS SPECTROMETRY

magnetic instruments were modified with the addition of an electrostatic analyzer to focus the
ions. These are called double-sector or two-sector instruments. The electric sector serves as a
kinetic energy focusing element allowing only ions of a particular kinetic energy to pass
through its field irrespective of their mass-to-charge ratio. Thus, the addition of an electric
sector allows only ions of uniform kinetic energy to reach the detector, thereby decreasing the
kinetic energy spread, which in turn increases resolution. It should be noted that the
corresponding increase in resolution does have its costs in terms of sensitivity. These double-
focusing (Figure 2.7) mass analyzers are used with ESI, FAB and EI ionization, however they
are not widely used today primarily due to their large size and the success of time-of-flight,
quadrupole and FTMS analyzers with ESI and MALDI.

Quadrupole Time-of-Flight Tandem MS

Time-of-flight analysis is based on accelerating a group of ions to a detector where all of the
ions are given the same amount of energy through an accelerating potential. Because the ions
have the same energy, but a different mass, the lighter ions reach the detector first because of
their greater velocity, while the heavier ions take longer due to their heavier masses and
lower velocity. Hence, the analyzer is called time-of-flight because the mass is determined
from the ions’ time of arrival. Mass, charge, and kinetic energy of the ion all play a part in the
arrival time at the detector. Since the kinetic energy (KE) of the ion is equal to 1/2 mv2, the
ion’s velocity can be represented as v = d/t = (2KE/m)1/2. The ions will travel a given
distance d, within a time t, where t is dependent upon the mass-to-charge ratio (m/z). In this
equation, v = d/t = (2KE/m)1/2, assuming that z = 1. Another representation of this equation
to more clearly present how mass is determined is m = 2t2 KE/d2 where KE is constant.

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MASS SPECTROMETRY

It is now widely used for ESI, MALDI, and more recently for electron ionization in GC/MS
applications.

It combines time-of-flight technology with an electrostatic mirror. The reflectron serves to
increase the amount of time (t) ions need to reach the detector while reducing their kinetic
energy distribution, thereby reducing the temporal distribution Δt. Since resolution is defined
by the mass of a peak divided by the width of a peak or m/Δm (or t/Δt since m is related to t),
increasing t and decreasing Δt results in higher resolution. Therefore, the TOF reflectron
offers high resolution over a simple TOF instrument by increasing the path length and kinetic
energy focusing through the reflectron.

Quadrupole Time-of-Flight MS

Quadrupole-TOF mass analyzers are typically coupled to electrospray ionization sources and
more recently they have been successfully coupled to MALDI. It has high efficiency,
sensitivity, and accuracy as compared to Quadrupole and TOF analyzer.

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MASS SPECTROMETRY

Detectors

A device that detects the ions produced in the mass spectrometer and produces a measurable
signal, generally an electronic signal. In most detectors, this signal is amplified. Common
types include the Faraday cup, the electron multiplier, the microchannel plate detector and the
Daly photomultiplier detector.

 Faraday Cup
 Photomultiplier Conversion Dynode
 Array Detector
 Charge (or Inductive) Detector
 Electron Multiplier

Faraday Cup

A Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface which
causes secondary electrons to be ejected. This temporary electron emission induces a positive
charge on the detector and therefore a current of electrons flowing toward the detector. This
detector is not particularly sensitive, offering limited amplification of signal, yet it is tolerant
of relatively high pressure.

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MASS SPECTROMETRY

– Ions are accelerated toward a grounded “collector electrode”

– As ions strike the surface, electrons flow to neutralize charge, producing a small current
that can be externally amplified.

– Size of this current is related to # of ions in

– No internal gain → less sensitive

Photomultiplier Conversion Dynode

The photomultiplier conversion dynode detector is not as commonly used at the electron
multiplier yet it is similar in design where the secondary electrons strike a phosphorus screen
instead of a dynode. The phosphorus screen releases photons which are detected by the
photomultiplier. Photomultipliers also operate like the electron multiplier where the striking
of the photon on scintillating surface results in the release of electrons that are then amplified
using the cascading principle. One advantage of the conversion dynode is that the
photomultiplier tube is sealed in a vacuum, unexposed to the environment of the mass
spectrometer and thus the possibility of contamination is removed. This improves the
lifetimes of these detectors over electron multipliers. A five-year or greater lifetime is typical,
and they have a similar sensitivity to the electron multiplier.

Array Detector

An array detector is a group of individual detectors aligned in an array format. The array
detector, which spatially detects ions according to their different m/z, has been typically used
on magnetic sector mass analyzers. Spatially differentiated ions can be detected
simultaneously by an array detector. The primary advantage of this approach is that, over a
small mass range, scanning is not necessary and therefore sensitivity is improved.

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MASS SPECTROMETRY

Charge (or Inductive) Detector

Charge detectors simply recognize a moving charged particle (an ion) through the
induction of a current on the plate as the ion moves past. A typical signal is shown in Figure.
This type of detection is widely used in FTMS to generate an image current of an ion.
Detection is independent of ion size and therefore has been used on particles such as whole
viruses.

Electron Multiplier

Perhaps the most common means of detecting ions involves an electron multiplier which
is made up of a series (12 to 24) of aluminum oxide (Al2O3) dynodes maintained at ever
increasing potentials. Ions strike the first dynode surface causing an emission of electrons.
These electrons are then attracted to the next dynode held at a higher potential and therefore
more secondary electrons are generated. Ultimately, as numerous dynodes are involved, a
cascade of electrons is formed that results in an overall current gain on the order of one
million or higher.

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MASS SPECTROMETRY

Vacuum in the Mass Spectrometer

All mass spectrometers need a vacuum to allow ions to reach the detector without
colliding with other gaseous molecules or atoms. If such collisions did occur, the instrument
would suffer from reduced resolution and sensitivity. Higher pressures may also cause high
voltages to discharge to ground which can damage the instrument, its electronics, and/or the
computer system running the mass spectrometer. An extreme leak, basically an implosion,
can seriously damage a mass spectrometer by destroying electrostatic lenses, coating the
optics with pump oil, and damaging the detector. In general, maintaining a good vacuum is
crucial to obtaining high quality spectra.

STRUCTURAL ANALYSIS AND FRAGMENTATION PATTERNS

When the vaporized organic sample passes into the ionization chamber of a mass
spectrometer, it is bombarded by a stream of electrons. These electrons have a high enough
energy to knock an electron off an organic molecule to form a positive ion. This ion is called
the molecular ion - or sometimes the parent ion.

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MASS SPECTROMETRY

The molecular ion is often given the symbol M+ or - the dot in this second version
represents the fact that somewhere in the ion there will be a single unpaired electron. That's
one half of what was originally a pair of electrons - the other half is the electron which was
removed in the ionization process.

Fragmentation

The molecular ions are energetically unstable, and some of them will break up into smaller
pieces. The simplest case is that a molecular ion breaks into two parts - one of which is
another positive ion, and the other is an uncharged free radical.

The uncharged free radical won't produce a line on the mass spectrum. Only charged particles
will be accelerated, deflected and detected by the mass spectrometer. These uncharged
particles will simply get lost in the machine - eventually, they get removed by the vacuum
pump.

The ion, X+, will travel through the mass spectrometer just like any other positive ion - and
will produce a line on the stick diagram.

All sorts of fragmentations of the original molecular ion are possible - and that means that
you will get a whole host of lines in the mass spectrum. For example, the mass spectrum of
pentane looks like this:

It's important to realize that the pattern of lines in the mass spectrum of an organic compound
tells you something quite different from the pattern of lines in the mass spectrum of an
element. With an element, each line represents a different isotope of that element. With a
compound, each line represents a different fragment produced when the molecular ion breaks
up.

The tallest line in the stick diagram (in this case at m/z = 43) is called the base peak. This is
usually given an arbitrary height of 100, and the height of everything else is measured
relative to this. The base peak is the tallest peak because it represents the commonest
fragment ion to be formed - either because there are several ways in which it could be
produced during fragmentation of the parent ion, or because it is a particularly stable ion.

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MASS SPECTROMETRY

Fragmentation Patterns

By using fragmentation pattern we can easily study the structure of a compound.

 Stevenson’s Rule
 Homolytic bond cleavage
 Heterolytic fragmentation
 Alpha cleavage
 Beta-cleavage
 Inductive cleavage
 Retro Diels-Alder Cleavage
 McLafferty rearrangement
 Ortho effect
 Onimum Reaction
 CO Elimination

Stevenson’s Rule

When fragment ions form in the mass spectrometer, they almost always do so by means of
uni-molecular processes. The low pressure of the ionization chamber makes it unlikely a
significant number of bimolecular collisions could occur. The uni-molecular processes that
are energetically most favorable give rise to the most fragment ions. This is the idea behind
Stevenson’s Rule: The most probable fragmentation is the one that leaves the positive charge
on the fragment with the lowest ionization energy. In other words, fragmentation processes
that lead to the formation of more stable ions are favored over processes that lead to less-
stable ions. This idea is grounded in the same concepts as Markovnikov’s Rule, which states
that in the addition of a hydrogen halide to an alkene, the more stable carbocation forms the
fastest and leads to the major product of the addition reaction. In fact, a great deal of the
chemistry associated with ionic fragmentation can be explained in terms of what is known
about carbocations in solution. For example, alkyl substitution stabilizes fragment ions (and
promotes their formation) in much the same way that it stabilizes carbocations. Other familiar
concepts will help one predict likely fragmentation processes: electronegativity,
polarizability, resonance delocalization, the octet rule, and so on.

Often, fragmentation involves the loss of an electrically neutral fragment. This fragment does
not appear in the mass spectrum, but its existence can be deduced by noting the difference in
masses of the fragment ion and the original molecular ion. Again, processes that lead to the
formation of a more stable neutral fragment are favored over those that lead to less-stable
neutral fragments.

An OE• + can fragment in two ways: cleavage of a bond to create an EE+ and a radical (R•) or
cleavage of bonds to create another OE• + and a closed-shell neutral molecule (N). An EE+, on
the other hand, can only fragment in one way—cleavage of bonds to create another EE+ and
a closed-shell neutral molecule (N). This is the so-called even-electron rule. The most
common mode of fragmentation involves the cleavage of one bond. In this process, the OE• +
yields a radical (R•) and an EE+ fragment ion. Cleavages that lead to the formation of more
stable carbocations are favored. When the loss of more than one possible radical is possible, a
corollary to Stevenson’s Rule is that the largest alkyl radical to be lost preferentially. Thus,
ease of fragmentation to form ions increases in the order

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MASS SPECTROMETRY

Description of the fragmentation processes…

Fragmentation of an odd electron molecular ion (M+.) may occur by hemolytic or heterolytic
cleavage of sigma bond.

Homolytic bond cleavage

Homolytic bond cleavage is a type of ion fragmentation in which a bond is broken by the
transfer of one electron from the bond to the charged atom, the other electron remaining on
its starting atom. The movement of one electron is signified by a fishhook arrow. The
fragmentation of a ketone is shown in the figure.

Heterolytic bond cleavage

Heterolytic bond cleavage is a type of ion fragmentation in which a bond is broken by the
transfer of a pair of electrons from the bond to the charged atom. In alpha cleavage, a bond
alpha to the charged atom is broken and in beta cleavage, a bond two removed from the
charged atom is broken. The movement of 2 electrons is signified by a double-barbed arrow
and also referred to as charge-induced fragmentation. The fragmentation of an even-electron
ion is shown in the figure.

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MASS SPECTROMETRY

Alpha cleavage

Alpha cleavage occurs on α-bonds adjacent to heteroatoms (N, O, and S). Charge is stabilized
by heteroatom. Occurs only once in a fragmentation (cation formed is too stable to fragment
further) for example in alcohols, aliphatic ethers, aromatic ethers, cyclic compounds and
aromatic ketones etc.

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MASS SPECTROMETRY

Beta-cleavage

Fission of a bond two removed from a heteroatom or functional group, producing a radical
and an ion is called beta cleavage and also written as β-cleavage for or example allylic
fragmentation.

Inductive cleavage

If an electron pair is completely transferred towards a centre of positive charge as a result of
the inductive effect, shown schematically by the use of a double-headed arrow, then the ion
will fragment by inductive cleavage. The figure illustrates this for radical cation ether.

Retro Diels-Alder Cleavage

A multicentered ion fragmentation which is the reverse of the classical Diels-Alder reaction
employed in organic synthesis that forms a cyclic alkene by the cycloaddition of a substituted
diene and a conjugated diene. In the retro reaction, a cyclic alkene radical cation fragments to
form either a diene and an alkene radical cation or a diene radical cation and an alkene.
Depending on the substituents present in the original molecule, the more stable radical cation
will dominate.

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MASS SPECTROMETRY

McLafferty rearrangement

An ion fragmentation characterised by a rearrangement within a six-membered ring system.
The most usual configuration is for a radical cation formed by EI to undergo the transfer of a
γ- hydrogen atom to the ionisation site through a ring system as shown here. The distonic
radical cation so formed can break up by radical-site-induced (α), or charged site-induced
fragmentation as shown in the figure for example ketones, carboxylic acid and esters.

Ortho effect

The interaction between substituents oriented ortho, as opposed to para and meta, to each
other on a ring system, can create specific fragmentation pathways. This permits the
distinction between these isomeric species. The diagram shows a case in which only the ortho
isomer can undergo the rearrangement.

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MASS SPECTROMETRY

Onimum Reaction

Onium ion, A hypervalent species containing a non-metallic element such as the methonium
ion CH5+. It includes ions such as oxonium, phosphonium, and nitronium ions.

The onium reaction is not limited to alkyl substituents acyl groups can also can undergo the
onium reaction. An onium ion is a hypervalent species containing a non-metallic element
such as the methonium ion CH5+. It includes ions such as oxonium, phosphonium,
sulphonium and nitronium ions.

CO Elimination

From carbonyl compounds CO elimination reaction from α-cleavage takes place like in
aldehyde, ketones and phenols etc.

If there is more than one CO group present sequential elimination of all CO groups is
possible.

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MASS SPECTROMETRY

INTERPRETATION OF MASS SPECTRUM

Mass spectrum

It is a simple graph between the abundance of an ion along Y-axis against its mass-to-charge
ratio along X-axis. A mass spectrum contains a large number of peaks some are small and
some are large, these are

Molecular ion peak

The peak of an ion formed from the original molecule by electron ionization, by the loss of an
electron, or by addition or removal of an anion or cation and also known as parent peak,
radical peak.

Fragmentation peaks

The peaks observed by fragments of compounds.

Base peak

The most intense ion in a mass spectrum. The abundance of this ion is used as the base from
which to normalise the relative abundances of the remaining peaks in the spectrum and is
given a nominal value of 100%.

Isotopic peaks

Peaks observed due to isotopes like in case of carbon with M+. M+1 peak also observed.

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MASS SPECTROMETRY

Rules for interpretation of mass spectrum

Following are the main rules for interpretation

Molecular ion peak

If present it will be highest peak in all isotopic peaks.

DBR Calculations

Double bond or ring calculations tell us about how many rings or double bonds are present in
a compound.

DBR= C-H/2+N/2+1

C= number of carbon atoms

H= number of hydrogen atoms

N= number of nitogen atoms

Nitrogen Rule

The nominal molecular weight of a compound will have an even-number value if there are no
nitrogen atoms, or an even number of nitrogen atoms, present in the molecule. This holds for
compounds containing C, H, O, P, S, Si, or halogen atoms. Even-electron fragment ions
containing an even number of nitrogen atoms occur at odd-number m/z values. Conversely, if
there are an odd number of nitrogen atoms, the nominal molecular weight will be an odd
number and even-electron ions containing an odd number of nitrogen atoms occur at even-
number m/z values.

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MASS SPECTROMETRY

Isotopic effect

Mass spectrum can easily be drawn but there are some factors which make the spectra
complicated, one of these is isotopic effect.

Mass spectra (examples)

Alkanes

When an alkane is bombarded by high energy electrons it will lose an electron to form a
radical cation. This radical cation has the same mass as the parent compound and produces
the molecular ion (M+) peak. The type of radical formed follows the stability of radicals:

3o > 2o > 1o > methyl

The alkane molecular ion can further fragment to form a homologous series of neutral alkyl
radicals usually beginning with the methyl radical. The methyl radical has a mass of 15 and
the next largest peak in the mass spectrum usually corresponds to the loss of methyl radical
(M-15). Ethyl radical can also be lost (M-29) and so forth

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MASS SPECTROMETRY

Mechanism of fragmentation for butane

Mass spectrum for butane

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MASS SPECTROMETRY

Typical fragments lost from straight chain alkanes

Molecular Ion - Fragment Lost
1 H·
2 2 H·
15 CH3·
29 C2H5·
43 C3H7·
57 C4H9·
71 C5H11·

Peaks in the mass spectra of straight chain alkanes will usually appear in groups of 14 mass
unit intervals (corresponding to one CH2 group). The most intense fragmentation peak is
usually the 3 carbon fragment, with the intensities of the peaks decreasing with increasing
mass. Often, the M-15 peak (loss of methyl radical) will be absent. Fragments to look for in
these spectra correspond to CnH2n+1+, CnH2n+, and CnH2n-1+.

Branched alkanes tend to fragment very easily, due to the presence of 2o, 3o, and 4o carbon
atoms in the structure. When branched alkanes fragment, stable secondary and tertiary
carbocations are formed. For this reason the molecular ion peak is much less intense than in
straight chain alkanes. Figure 10 shows the mechanism of fragmentation for isobutane. The
mass spectrum for isobutane is contained in Figure 11. Notice the reduced intensity of the
molecular ion peak.

Mechanism of fragmentation for isobutane

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MASS SPECTROMETRY

Mass spectrum for isobutane

Summary

Strong M+ (but intensity decreases with an increase of branches.

Carbon-carbon bond cleavage

Loss of CH units in series: M-14, M-28, M-42 etc

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MASS SPECTROMETRY

Cycloalkanes

The fragmentation patterns of cycloalkanes may show mass clusters arranged in a
homologous series, as in the alkanes. However, the most significant mode of cleavage of the
cycloalkanes involves the loss of ethene from the parent molecule or from intermediate
radical-ions. Additionally, if the cycloalkane has a side chain, loss of that side chain is also a
favorable mode of fragmentation. The mass spectrum of cyclopentane has an intense peak at
m/e = 42 due to the loss of ethene.

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MASS SPECTROMETRY

Mechanism of fragmentation for cyclopentane

Mass spectrum for cyclopentane

In order for cycloalkanes to fragment, two carbon-carbon bonds must be broken. This process
may require a significant amount of time (relative to the amount of time it takes an ion to
reach the detector in a mass spectrometer), therefore, a significant amount of the molecular
ion will reach the detector resulting in a large molecular ion peak.

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MASS SPECTROMETRY

Cycloalkanes tend to cleave in CnH2n+, CnH2n-1+, and CnH2n-2+ fragments. The larger number
of even numbered mass fragments of cycloalkanes helps to distinguish this functional group
from the acyclic alkanes.

Summary

Strong M+, strong base peak at M-28 (loss of ethene)

A series of peaks: M-15, M-28, M-43 etc

Methyl, ethyl, propyl with an additional hydrogen give peaks

Alkenes

The mass spectra of most alkenes show distinct molecular ion peaks. This is probably due to
the loss of an electron in the p bond, leaving the carbon skeleton relatively undisturbed.

Alkenes usually form fragments corresponding to CnH2n+1+, CnH2n+, and CnH2n-1+ (the latter
two fragments are more intense). It is very difficult to locate the position of the double bond
in an alkene because of its facile migration in the fragments. For this reason, the mass spectra
of alkene isomers are nearly identical and almost impossible to discriminate between. The
only exception would be terminal alkenes which fragment to form an allylic carbocation with
m/e = 41

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MASS SPECTROMETRY

Mechanism of fragmentation for 1-butene

Mass spectrum for 1-butene

The mass spectra of cycloalkenes show distinct molecular ion peaks. It may be impossible to
locate the position of a double bond due to migration. The mechanism of fragmentation for
cyclic alkenes is virtually the same as for straight chain alkenes. One noteworthy
characteristic is the fragmentation of cyclohexenes which undergo a reverse Diels-Alder
reaction.

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MASS SPECTROMETRY

Mechanism of fragmentation for cyclohexene

Mass spectrum for cyclohexene

Summary

Strong M+

Fragmentation ion has formula CnH2n+ and CnH2n-1

α-Cleavage

A series of peaks: M-15, M-29, M-43, M-57 etc

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MASS SPECTROMETRY

Alkynes

The mass spectra of alkynes are virtually identical to those of alkenes. The molecular ion
peak is intense, and fragmentation parallels that of the alkenes.

Two differences are worth mentioning: terminal alkynes fragment to form propargyl ions
(m/e = 39), and can also lose the terminal hydrogen, yielding a strong M-1 peak.

Mechanism of fragmentation for 1-butyne

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MASS SPECTROMETRY

Mass spectrum for 1-butyne

Summary

Strong M+

Strong base peak at M-1 peak due to the loss of terminal hydrogen

Alpha cleavage

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MASS SPECTROMETRY

Aromatic Hydrocarbons

The mass spectra of most aromatic compounds show distinct molecular ion peaks. This is
probably due to the loss of an electron in the p system, leaving the carbon skeleton relatively
undisturbed.

When an alkyl side-chain is attached to the ring, fragmentation usually occurs at the benzylic
position, producing the tropylium ion (m/e = 91)

Formation of tropylium ion

However, fragmentation can also occur at the attachment point to the ring producing the
phenyl cation (m/e = 77) as shown in figure 21.

Formation of phenyl cation

If the side-chain is a propyl group or larger, then the McLafferty rearrangement is a
possibility, producing a fragment of m/e = 92.

Mechanism of McLafferty rearrangement

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MASS SPECTROMETRY

Formation of a substituted tropylium ion is typical for alkyl-substituted benzenes producing a
peak at m/e = 105.

Formation of substituted tropylium ion

The complete mass spectrum for propyl benzene is given in figure 24, which illustrates all of
these points.

Mass spectrum for propyl benzene

Summary

Strong M+

Loss of hydrogen gives base peak


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MASS SPECTROMETRY

Formation of benzyl cation or tropylium ion

Alcohols

M+ weak or absent

Loss of alkyl group via a-cleavage

Dehydration (loss of water) gives peak at M-18

Loss of alkyl group in alcohol fragmentation

A second common mode of fragmentation involves dehydration. The importance of this
fragmentation process increases with increasing chain length. Loss of water (M - 18) is very
indicative of an alcohol functionality (see figure 26).

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MASS SPECTROMETRY

Dehydration of an alcohol

Phenols can lose the elements of carbon monoxide to give strong peaks at M - 28.

Phenols can also lose the elements of the formyl radical (HCO·) to give strong peaks at M -
29.

Mass spectrum for 2-pentanol

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MASS SPECTROMETRY

Phenols

Strong M+

M-1 due to hydrogen elimination

M-28 due to loss of CO

M-29 due to loss of HCO (formyl radical)

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MASS SPECTROMETRY

Ethers

M+ weak but observable

Loss of alkyl radical due to a-cleavage

B-cleavage( formation of carbocation fragments through loss of alkoxy radicals)

C-O bond cleavage next to double bond

Peaks at M-31, M-45, M-59 etc

Cleavage of the C-C bond to the a-carbon

A second common mode of fragmentation involves cleavage of the C-O bond (see figure 29).

Cleavage of the C-O bond of ether

Hydride transfer from a b-carbon is an important rearrangement process in ethers as shown in
figure 30.

Rearrangement of an ether

Peaks usually occur in CnH2nOH+ increments for ethers.

.

Page 58

MASS SPECTROMETRY

Mass spectrum for n-butyl ether

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MASS SPECTROMETRY

Aldehydes

M+ weak, but observable (aliphatic)

Aliphatic : M-29, M-43 etc

McLafferty rearrangement is common gives the base peak

α-cleavage

β-cleavage

a-cleavage

A second common mode of fragmentation involves b-cleavage

b-cleavage

McLafferty rearrangement can take place for aldehydes with at least 4 carbons. A fragment
ion of m/e = 44 is formed, and is considered quite characteristic of an aldehyde

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MASS SPECTROMETRY

McLafferty rearrangement of an aldehyde

Mass spectrum for hexanal

Page 61

MASS SPECTROMETRY

M+ strong (aromatic)

Aromatic: M-1 (loss of hydrogen)

M-29 (loss of HCO)

McLafferty rearrangement is common

α-cleavage

β-cleavage

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MASS SPECTROMETRY

Ketones

Strong M+

A series of peaks M-15, M-29, M-43 etc

Loss of alkyl group attached to the carbonyl group by a-cleavage

Formation of acylium ion (RCO+)


a-cleavage

McLafferty rearrangement can take place for ketones with at least 3 carbons

McLafferty rearrangement of a ketone

For aromatic ketones, a-cleavage usually occurs, which is followed by loss of carbon
monoxide as indicated in figure 38.

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MASS SPECTROMETRY

Aromatic ketone fragmentation

Mass spectrum for 2-pentanone

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MASS SPECTROMETRY

Esters

M+ weak but generally observable

Loss of alkyl group attached to the carbonyl group by a-cleavage

Formation of acylium ion (RCO+)


Acyl portion of ester OR+

Methyl esters: M-31 due to loss of OCH3

Higher esters: M-32, M-45, M-46, M-59, M-60, M-73 etc

a-cleavage

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MASS SPECTROMETRY

McLafferty rearrangement can take place for esters in the alkyl portion

McLafferty rearrangement of an ester

Mass spectrum for ethyl acetate

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MASS SPECTROMETRY

Carboxylic Acids

Aliphatic carboxylic acids:


A-cleavage on either side of C=O

M-17 due to loss of OH

M-45 due to loss of COOH

McLafferty rearrangement gives base peak

a-cleavage

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MASS SPECTROMETRY

Loss of the alkyl group as a free radical, leaving CO2H+, also occurs as shown in figure 44.

Loss of alkyl radical

With acids having g hydrogens, the principal pathway for rearrangement is the McLafferty
rearrangement

McLafferty rearrangement of an acid

Mass spectrum for pentanoic acid

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MASS SPECTROMETRY

Aromatic carboxylic acids:

M+ Strong

A-cleavage on either side of C=O

M-17 due to loss of OH

M-18 due to loss of HOH

M-45 due to loss of COOH

McLafferty rearrangement gives base peak

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MASS SPECTROMETRY

Amines

M+ weak or absent

Nitrogen rule obey

α-cleavage

a-cleavage

Loss of hydrogen radical, is quite common in amines

Loss of hydrogen radical

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MASS SPECTROMETRY

Mass spectrum for diethyl amine

Nitriles


M-1 visible peak due to loss of termiminal hydrogen

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MASS SPECTROMETRY

Loss of hydrogen radical

McLafferty rearrangement can take place for nitriles

McLafferty rearrangement of a nitrile

Mass spectrum for pentanenitrile

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MASS SPECTROMETRY

Amides

The molecular ion peak is usually observable, and will be a good indication of the presence
of an amide (nitrogen rule).

a-cleavage

McLafferty rearrangement can take place for amides in the alkyl portion

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MASS SPECTROMETRY

McLafferty rearrangement of an amide

Mass spectrum for propanamide

Nitro Compounds

M+ seldom observed

Loss of NO+ gives visible peak

Loss of NO2+ gives peak

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MASS SPECTROMETRY

Alkyl Chlorides and Alkyl Bromides

Strong M+ 2 peaks

For Cl M/M+2 = 3:1

F or Br M/M+2 = 1:1

A-cleavage

Loss of Cl or Br

Loss of HCl or HBr

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MASS SPECTROMETRY

APPLICATIONS OF MASS SPECTROMETRY

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. Followings are the main
applications

Toxicity of Toothpastes

In some Chinese toothpastes, a toxic compound known as DEG is sometimes used as a
sweetener. The compound is banned, but it is difficult to truly enforce the ban, since
toothpaste is very difficult to test. It can be done, but it takes a lot of time. Until now, A
Chinese scientist, Huanwen Chen, has come up with a way of using mass spectrometry to
quickly screed for toxins.

DEG (diethylene glycol)

Looking for pesticides

Nutritional supplements are often touted as "natural" ways to boost health. However, the fact
of the matter is that pesticides can find themselves in supplements and food. Unfortunately,
testing for multiple pesticides is difficult and is practically impossible without mass
spectrometry. Douglas Hayward and Jon Wong at the U.S. FDA have developed a mass
spectrometry method that can identify multiple compounds at once, hoping to reduce the
amount of pesticides that enter the food supply.

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MASS SPECTROMETRY

Measuring nanoparticle size

Usually, nanoparticles are measured with the use of transmission electron microscopy or x-
ray diffraction. Interestingly, it appears that you can use a MALDI-TOF spectrometer to
measure the size of nanoparticles. Once size of a sphere is measured, its density is also
calculated.

Isotope Ratio Data

a. Carbon, Hydrogen, Nitrogen, and Oxygen

We must remember that molecular species exist that contain less abundant isotopes of carbon,
hydrogen, nitrogen, and oxygen which will give rise to isotope peaks at M+1, M+2, etc.

In above Figure, you will notice additional peaks at 122 (M+1) and 123 (M+2). These are
peaks due to the presence of isotopes of carbon, hydrogen, nitrogen, and oxygen in
benzamide, and are not to be confused with the molecular ion peak. Sometimes, the intensity
of the M+1 and M+2 peaks can lead to valuable information about the molecular formula of
the compound.

To calculate the intensity of the M+1 peak (with respect to the M+ peak), use the following
equation:

%(M+1) = (1.1 * #C atoms) + (0.016 * #H atoms) + (0.38 * #N atoms)

To calculate the intensity of the M+2 peak, use the following equation:

%(M+2) = [(1.1 * #C atoms)2 / 200] + [(0.016 * #H atoms)2 / 200] + (0.38 * #O
atoms)

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MASS SPECTROMETRY

The following example shows how the above equations can be used to help confirm the
formula for a chemical compound.

The above calculations will only approximate the sizes of the M+1 and M+2 peaks. Also,
these formulas are really only useful if the molecular formula is already known, but they
provide a good check on the validity of a proposed molecular formula.

b. Bromine and Chlorine

When bromine or chlorine is present in a compound, the M+2 peak becomes very significant.
This is due to the fact that, for bromine, two isotopes (79Br and 81Br) are present in a 1:1 ratio
in naturally occurring substances, and, for chlorine, two isotopes (35Cl and 37Cl) are present in
a 3:1 ratio in naturally occurring substances. If a compound contains bromine, the M+ and
M+2 peaks are present in equal intensities. Additionally, if a compound contains chlorine, the
M+ and M+2 peaks will be present in a 3:1 ratio. The presence of these M+ and M+2 peaks is
very indicative of brominated and chlorinated compounds.

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MASS SPECTROMETRY

Pharmacokinetics

Pharmacokinetics is often studied using mass spectrometry because of the complex nature of
the matrix (often blood or urine) and the need for high sensitivity to observe low dose and
long time point data.

Protein characterization

Mass spectrometry is an important emerging method for the characterization of proteins. The
two primary methods for ionization of whole proteins are electrospray ionization (ESI) and
matrix-assisted laser desorption/ionization (MALDI).

Space exploration

Mass spectrometers are also widely used in space missions to measure the composition of
plasmas. For example, the Cassini spacecraft carries the Cassini Plasma Spectrometer
(CAPS), which measures the mass of ions in Saturn's magnetosphere.

Environmental Chemistry

Mass spectrometry is a powerful tool in environmental chemistry for the analysis of trace
elements and compounds in environmental samples like air, water, soil etc because of its
detection power, specificity and structural analysis functions. Techniques for the
determination of the degradation products and metabolites of chemicals in practically all the
relevant matrices, since no comparable tool developed are available, although the technical
problems can occur. Generally, sample preparation is at least one type of chromatography
coupled with MS either offline or online. From the online combinations ("hyphenated
techniques"), GCMS is the most successful; eventhough LC / MS is rapidly catching up in
this area. The development of gum and Benchtop LC / MS instruments has made it possible
to mass spectrometry in routine laboratory and in field measurements.

Species Analysis

Storage, transportation, and the action of metals in the environment are largely dependent on
the chemical form and oxidation state as the association with organic ligands. Heavy metals
in the environment are stored in complexes with humic acids, can be converted by microbes
in different complexes, and can be transported in live animals and humans. This applies to
many elements such as lead, mercury, arsenic, astatine, tin and platinum. Several mass
spectrometric techniques have been employed in the study of the fate of metals in the human
body and other organic environments by determining the species formed in the biological or
environmental matrices.
For example, tin and lead alkylates established in soil, water or muscle tissue by GC / MS
after exhaustive alkylation or thermal spray, and ICP-LC/MS API methods. The APIM
techniques have proven very successful, even with metal bound to proteins and enzymes, and
an interface with micro-separation techniques, even elements such as iodine or phosphorus
can be quantitatively determined.

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Mass spectrometry

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