RESEARCH  NEWS
Chemistry Nobel Prize 2002
Mass Spectrometry
M Vairamani
Introduction
Mass Spectrometry started with the discov-
ery of isotopic form of stable elements by
Thomson in the year 1911 during his out-
standing work on positive rays. The pioneer-
ing work by Thomson, Aston, Dempster and
Nier led to the construction of mass spec-
trometers for precise measurement of ionic
masses using magnetic and electrostatic
analysers. Today mass spectrometers are
widely used to identify unknown compounds
by way of determining their molecular weight
at the expense of negligible amount of sample.
The Nobel Prize in Chemistry for 2002 has
been awarded to John B. Fenn, USA, Koichi
Tanaka, Japan and Kurt Wuthrich, Ger-
many for pioneering in electrospray
ionisation mass spectrometry, soft laser des-
orption ionisation mass spectrometry and
nuclear magnetic resonance, respectively for
the analysis of biological macromolecules.
The mass spectrum of a compound is given
in the form of a bar graph representing the
abundance of various ions with respect to
their mass (m) to charge (z) ratio (m/z). In
addition to the molecular ion, which infers
the molecular weight of the sample, the other
ions present in the mass spectrum are very
characteristic of the compound, thus the mass
spectrum of a compound becomes the finger-
print of it in most of the cases.
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Principles
Mass spectrometry deals with the study of
gas-phase ions and the mass spectrometers
are used to determine the mass to charge
ratio (m/z) of ions and measure their concen-
tration. Hence, the basic principle in getting
a mass spectrum can be divided into three
parts, namely, production of ions, separation
of ions according to their mass to charge
ratio and their detection. A simple mass spec-
trometer, in general, consists of a source for
producing ions, an analyser for the separa-
tion of ions and a detector to measure their
abundance. The block diagram of a mass
spectrometer is given in Figure 1.
Positive and negative ions when produced in
the gas-phase will have the tendency to be
neutralised by collision with other species
around. Hence, the ions produced in gas-
phase are always analysed at low pressure
ranging from 10-6 Torr to 10–8 Torr (1 Torr =
1mm of Hg) in a mass spectrometer (Figure 1)
Ions can be produced by different ionisation
techniques and for the study of organic mol-
ecules of low molecular weight (~800 Da)
electron impact ionisation is more suitable.
Electron Impact Ionisation
In an electron impact (EI) source, ionisation
of neutral molecules (M) is effected by elec-
trons (e). Electrons are produced in vacuum
by thermionic emission from heated fila-
ments that are usually made of tungsten or
rhenium. Three processes can occur during
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Figure 1. Block diagram of a mass spectrometer.

electron impact and they are represented in under high vacuum (10–6 – 10–7 Torr). There-
the following equations. fore, the ions produced in the primary step
(1) undergo unimolecular fragmentation lead-
M+e– M+. + 2e (1)
ing to different ions (equations 5-10) for a
M+e– M–. (2)
hypothetical molecule ABCD.
M+e– Mn+ + (n+1)e (3)
ABCD+e– ABCD+. + 2e– (4)
The probability of the first reaction namely .
ABCD+. ABC+ + .D (5)
removal of one electron from the molecule
ABCD+. AB+ + CD (6)
(M) is very high compared to the other two .
ABCD+. A+ + BCD (7)
reactions and hence, singly charged positive
ABCD+. AD+. + BC (8)
ions are produced in high abundance when
ABC+ AB+ +. C (9)
compared to the negative (2) and multiply
AD+. A+ + D (10)
charged ions (3). The energy required for the
first reaction which equals the first ionisation Ionisation of neutral molecule (ABCD) by
potential of the molecule lies between 8 and removal of one electron (4) takes place in
12 eV for most of the organic compounds. 10–14–10–16 seconds and the other fragmenta-
Nevertheless, ionisation efficiency reaches a tion process occurs in 10–8 seconds. There-
steady state only with electrons of 50-90 eV fore, ions of different mass to charge ratio are
energy and hence, use of electrons of 70eV produced in the source of the mass spectrom-
energy is universally accepted for EI eter the moment sample is introduced into
ionisation. Hence, under EI molecular ions the source of the mass spectrometer and when
(M+·) are produced with high energy. Ions sample molecules collide in the high-energy
thus produced cannot dissipate their energy electrons. Ions thus formed are separated
in the EI source as the source is operated according to their mass to charge ratio in the

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Box 1. Time-of-Flight (TOF)

From the equation it is clear that high mass ions take

longer time to reach the detector than the low mass ions.
The resolution of TOF instruments is controlled by the
spread in time, space, and kinetic energy of the group of
ions formed. Increasing the flight time by increasing the
length of the drift tube alone will not lead to high
resolution because of the kinetic energy spread in the
ions of same mass produced in the ion source. Increased
flight time and partial compensation of kinetic energy Schematic diagram of a time of flight analyzer
spread can be achieved by using a device called ions are separated according to their velocity
and the m/z ratio is determined from a measure-
reflectron. Reflectron TOF instruments can give a reso- ment of the time taken to traverse a specified
lution greater than 10000 under full width at half height flight path (L) to the detector using the equa-
tion, t = [m/2zeV]/1/2 L. TOF analyzers are ca-
maximum (FWHM) condition. TOF analyzers have
pable of analyzing ions with m/z values in ex-
high sensitivity when compared to sector and quadurpole cess of 500000 generated by MALDI.
instruments because all the ions accelerated out of the
ion source will reach the detector in the pulsed mode operation and also due to the absence of beam defining
slits. The mass range of the TOF analyzers is unlimited.

analyser part of the mass spectrometer and a) Chemical Ionisation (CI)

detected. Magnetic, quadrupole and time-of-
flight analysers (Box 1) are some of the most
commonly used analysers. For detection of
ions, secondary electron multipliers are nor-
mally used. Thus, the mass spectrum of a
compound represents the abundance of vari-
ous ions against their respective mass to
charge ratio (m/z) (Figure 2).
In a normalised mass spectrum, the most
abundant ion is taken as 100% (base peak)
and the abundance of the other ions are cal-
culated relative to the base peak. EI tech-
nique is only suitable for volatile and ther-
mally stable compounds and hence, it is rec-
ommended only for analysing low molecular
weight (~ 800 Da) compounds.
In general majority of the low molecular
weight organic compounds give stable mo-
lecular ions under EI ionisation. But there
are compounds that are volatile and ther-
mally stable, but electron impact unstable.
Such compounds do not give molecular ion
under EI and hence, knowing the correct
molecular weight of such compounds be-
comes difficult. Also at times it becomes
necessary to confirm the molecular ion ob-
tained under EI by some other techniques.
Under such circumstances the technique
namely, Chemical Ionisation (CI) is very use-
ful. CI is an outcome of extensive research on
ion-molecule reactions. Ion-molecule reac-
tions play an important role in all the soft-
ionisation techniques. Ion-molecule reactions

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Figure 2. Hypothetical electron impact mass spectrum of com-
pound ABCD.
are in general absent under EI condition be-
cause of the high vacuum maintained in the
source of the mass spectrometer. However,
the primary ions formed under EI condition
can be made to undergo ion-molecular reac-
tions in the source of the mass spectrometer
by increasing the pressure inside the source
of the mass spectrometer. This can be achieved
by suitably modifying the source of the in-
strument and the vacuum system. Methane
for example, under high pressure (1mm of
Hg) gives CH5 + as the major ion. Similarly,
isobutane gives C4H10+ and ammonia gives
NH4+ ions under high pressure CI condi-
tions. These ions are Bronsted acids and they
have the tendency to donate a proton when
they collide with the substrate molecules of
high proton affinity. Under CI condition the
reagent (CI gas) to the substrate ratio is kept
approximately as 1000:1 and hence, the sub-
strate undergoes ion molecular reaction with
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the reagent ion of choice
produced in the source.
The proton transfer results
in the formation of
[M+H]+ ion as the more
abundant one and hence,
the molecular ion can be
easily identified and also
confirmed. Thus CI works
as a complementary tech-
nique to EI and has now
become more routine.
b) Fast Atom Bombard-
ment (FAB)
EI and CI techniques are only suitable for
volatile and thermally stable compounds and
hence, they are not useful for analysing high
molecular weight compounds that are in
general non-volatile and thermally unstable.
This limitation was overcome by the intro-
duction of another popular soft ionisation
technique known as fast atom bombardment
(FAB) ionisation in the year 1982 by Barber
and others. In this technique sample is
dissolved in a suitable matrix such as glyc-
erol or m-nitrobenzyl alcohol and coated on
a metal (usually stainless steel or copper)
target. The sample coated surface of the tar-
get is bombarded with high energy (in kilo-
volts) atoms (Argon or Xenon) or ions (Ce-
sium) in the source of the mass spectrom-
eter. High energy impact causes sputtering
of atoms, ions and neutral molecules from
the surface. Complex ion-molecule reactions
occur in the selvedge region above the sur-
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Figure 3. Schematic diagram of electrospry ionization process.
face and results in the formation of (M+H)+
ions for most of the molecules. This tech-
nique is useful for analysing polar and non-
volatile compounds of molecular weight upto
10000 Da and thus suitable for analysing
amino acids, peptides, glyco peptides, sugars
etc..
c) Electrospray ionisation (ESI)
The principle of electrospraying liquids was
studied by Zeleny and Dole in the years 1917
and 1968 respectively. But the real break-
through came in the year when John Fenn
(Nobel Laureate 2002) of Virginia Com-
monwealth University, Richmond, USA
showed that polypeptides and proteins of
molecular weight 40 kDa can be successfully
analysed by combining electrospray
ionisation (Figure 3) and mass spectrometry.
ESI consists in flowing a few microliters a
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minute of dilute analyte solution through a
stainless steel needle on the axis of a chamber
containing a bath gas such as dry nitrogen. A
potential difference of several kilovolts be-
tween the needle and the chamber walls pro-
duces an intense electric field at the needle
exit that disperses the emerging liquid into a
fine spray of charged droplets. These charged
droplets move towards the sampling cone of
the mass spectrometer analyzer under suit-
able electric field in presence of desolvation
gas nitrogen. Solvent evaporation occurs and
finally only de-solvated ions enter the ana-
lyzer and the m/z value of these ions mea-
sured as usual, resulting in the electrospray
ionisation mass spectrum of the analyte.
Multiple charging is inherent property of
electrospray ionisation process and hence,
results in ions with varying charges (z) in the
range +2 up to +40 or even higher depend-
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Figure 4. ESI mass spectrum of Cytochrome C (mol.wt 12360.1 Da)
ing on the nature of protonation sites in the
molecule. Thus macromolecules under
electrospray ionisation give a series of mul-
tiple charged ions with m/z values which are
easily measurable by most of the commer-
cial mass spectrometers. The ESI mass spec-
trum of cytochrome-C with a series of multi-
ply charged ions is shown in the Figure 4.
The m/z values of the multiple charged ions
can be deconvoluted to give the molecular
weight of the compound under study. This
ionisation process is the least invasive of all
the ionisation methods and hence, most of
the biological samples such as proteins, and
DNA oligomers could be studied at
attomolar (10-18 M) concentraion. Non-co-
valent interactions such as protein-protein,
enzyme-substrate or protein-ligand com-
plexes can also be studied by this technique.
In addition to this it has become the most
rugged interface for connecting high pres-
sure liquid chromatograph (HPLC) with
74
mass spectrometric detectors because the tech-
nique is very ideal for handling reverse phase
solvents such as water, methanol, acetoni-
trile, etc..
d) Matrix Assisted Laser Desorption
(MALDI)
Use of laser light, as a source for ionizing high
molecular weight biological molecules was
being tested by different groups since 1980.
In the year 1987, Koichi Tanaka (Nobel Lau-
reate 2002) of Shimadzu corporation in Kyoto,
Japan showed that gaseous macromolecular
ions such as chymotrypsin (25,717 Da), car-
boxypeptidase-A (34,472 Da), and cyto-
chrome-c (12,360 Da) can be formed using
soft laser desorption technique. His success
was mainly due to the proper combination of
laser energy and wavelength with the absor-
bance of the matrix and the molecular struc-
ture of the analyte in the matrix. In this
technique, low energy laser for example, ni-
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Figure 5. Schematic diagram of laser des-
orption/ionization process.
trogen laser beam of 337 nm wavelength is
used for ionizing macromolecules dissolved
and co-crystallized in presence of a laser ab-
sorbing matrix such as substituted cinnamic
acids, nicotinic acid, dihydroxy benzoic ac-
ids, etc. This process permits intact low-
charge transfer of the macromolecule to the
gas phase. MALDI with time-of-flight mass
(TOF) spectrometric analyzer has become
the most important technique for the mo-
lecular weight
Figure 6. MALDI mass spectrum of a mixture of three pro-
teins.
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determination of biological macromolecules.
It is today a cornerstone of proteomics.
Ions produced by MALDI technique (Fig-
ure 5) are accelerated through a voltage V and
the resultant velocity v is characteristic of
the mass-to-charge ratio. In the TOF mass
spectrometer ( Figure 6).
Applications
The most important application of mass spec-
trometry is the unequivocal identification of
unknown compounds from their mass spec-
tra. Because of its high sensitivity, it has
become powerful analytical tool for moni-
toring environmental pollutants. Pesticide
and insecticide residues in food, water and
soil can be easily analysed by mass spectrom-
etry at parts per billion and trillion levels
(conc. Range 10–9 -10 –12. This is a method
chosen for analysing narcotics and drugs of
abuse in forensic science. Mass spectrometry
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 RESEARCH  NEWS
has become more powerful because of its
capability to integrate with other separation
science techniques such as gas chromatogra-
phy ( GC), high pressure liquid chromatog-
raphy (HPLC), etc.. GC and HPLC are two
powerful chromatographic techniques for the
separation of samples containing mixture of
compounds. When these techniques are hy-
phenated with mass spectrometer one gets
the advantage of separation science and the
sensitivity and accuracy from the mass spec-
trometry techniques. The introduction of
capillary columns for high resolution separa-
tion has made the coupling of GC to the mass
spectrometer easy. The low flow rate (1-2ml
of helium) required for capillary GC work is
ideal for the EI source of the mass spectrom-
eter, that is operated under low pressure.
Thus complex mixtures which are volatile
and thermally stable can be very efficiently
analysed by GC/MS technique. GC/MS is a
method of choice in trace analysis of pesti-
cides, toxic components, drugs, narcotics,
pollutants, etc.. On the other hand HPLC
coupled with mass spectrometer with the
atmospheric ionisation technique such as
electrospray ionisation has become more pow-
erful for analysing polar compounds espe-
cially pharmaceuticals in quality control and
pharmacokinetic studies. Electrospray
ionisation and laser desorption ionisation
techniques are complimentary to one an-
other in proteomic and genomic research
because of their capability in analysing bio-
logical macromolecules in very low concen-
trations.
76
Acknowledgements
Some of the figures represented in this paper
have been taken from the Vanderbilt Univer-
sity Mass Spectrometry Research Center web
site.
The author wishes to thank Prof. Richard
Capriole, Director of the Mass Spectrometry
Center at Vanderbilt University for giving
permission to use some of the figures from
his tutorial.
Suggested Reading
[1] M E Rose and R A W Johnston, Mass spectro-
metry for chemists and biochemists, Cambridge
University Press, Cambridge, 1982.
[2] F W McLafferty and PTurecek, Interpritation
of Mass Spectra, University Science Press,
Mill Valley California,1993.
[3] J R Chapman, Practical Organic Mass Spec-
trometry, John Wiley and Sons, New York,
1995.
[4] M S B Munson and F H Field J. Am. Chem.
Soc., Vol. 88, p. 2622, 1966.
[5] M Barber, R S Bordoli, R D Sedgwick and A N
Tyler, J. Chem. Commun., Vol. 325, 1981.
[6] M Yamashita and J B Fenn, J. Phys.Chem., Vol.
88, p. 4451,1984.
[7] K Tanaka, H Waki, Y Ido, S Akita, Y Yoshida
and T Yoshida, Rapid Commun. Mass Spectrom,
Vol. 2, p. 151, 1988.
[8] T W D Chan, A W Coleburn, and P J Derrick,
Org. Mass Spectrum, Vol. 27, p. 53 ,1992.
M. Vairamani, National Center for Mass Spectrom-
etry, Indian Institute of Chemical Technology,
Hyderabad 500 007, India.
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