Nandakumar publications

Spectroscopy
Instant reference handbook
Jogesh.S.Nanda

Jogesh.s.nanda@hotmail.com

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 1. Introduction
Spectroscopy was originally the study of the interaction
between radiation and matter as a function of wavelength. In fact historically,
spectroscopy is referred to the use of visible light dispersed according to its
wavelength. Later the concept was expanded greatly to comprise any
measurement of a quantity as function of either wavelength or frequency. Thus it
also can refer to interactions with particle radiation or to a response to an
alternating field or varying frequency. A further extension of the scope of the
definition added energy as a variable, once the very close relationship E=hν for
photons was realized. A plot of the response as a function of wavelength or more
commonly frequency is referred to as a spectrum.

Spectrometry is the spectroscopic technique used to

assess the concentration or amount of a given species. In those cases, the
instrument that performs such measurements is a spectrometer or spectrograph.
Spectroscopy or spectrometry is often used in physical and analytical chemistry
for the identification of substances through the spectrum emitted from or
absorbed by them.

Spectroscopy or spectrometry is also heavily used in

astronomy and remote sensing. Most large telescopes have spectrometers, which
are used either to measure the chemical composition and physical properties of
astronomical objects or to measure their velocities from the Doppler shift of their
spectral lines.

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 2. Atomic spectra
The atomic emission spectrum of an element is the set
of frequencies of the electromagnetic waves emitted by atoms of that element.
Each atom's atomic emission spectrum is unique and can be used to determine if
that element is part of an unknown compound.
Light consists of Electromagnetic radiation of different
wavelengths. Therefore, when the elements or their compounds are heated
either on a flame or by an electric arc they emit energy in form of light. Analysis,
of this light, with the help of spectroscope gives us a discontinuous spectrum. A
spectroscope or a spectrometer is an instrument which is used for separating the
components of light, which have different wavelengths. The spectrum appears in
a series of line called line spectrum. This line spectrum is also called the Atomic
Spectrum because it originates in the element. Each element has a different
atomic spectrum. The production of line spectra by the atoms of an element
indicates that an atom can radiate only certain amount of energy. This leads to
the conclusion that electrons cannot have any amount of energy but only a
certain amount of energy.

The emission spectrum characteristics of some elements

are plainly visible to the naked eye when these elements are heated. For example,
when platinum wire is dipped into a strontium nitrate solution and then inserted
into a flame, the strontium atoms emit a red color. Similarly, when copper is
inserted into a flame, the flame becomes green. These definite characteristics

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allow elements to be identified by their atomic emission spectrum. Not all lights
emitted by the spectrum are viewable to the naked eye; it also includes ultra
violet rays and infra red lighting.

The fact that only certain colors appear in an element's

atomic emission spectrum means that only certain frequencies of light are
emitted. This concludes that only photons having certain energies are emitted by
the atom. The principle of the atomic emission spectrum explains the varied
colors in neon signs, as well as chemical flame test results mentioned above.

The frequencies of light that an atom can emit are

dependent on states the electrons can be in. When excited, an electron moves to
a higher energy level/orbital. When the electron falls back to its ground level the
light is emitted. An emission spectrum is always the inverse of its absorption
spectrum.

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 3. Molecular spectra
The atomic spectra arise from the transitions of an
electron between the atomic energy levels whereas the molecular spectra arise
from three types of energy changes, via, molecular rotation, molecular vibration
and electronic transition. The rotational spectra of a molecule can be observed in
the microwave region, the irrational spectra in the infrared region and the
electronic spectra in the ultraviolet or visible regions of the electromagnetic
spectrum. The various types of spectra given by molecular species, the region in
which these spectra lie and the energy changes that occur in the molecules on
absorption of radiation, are summarized as follows :

1) Nuclear Magnetic Resonance (NMR) and Nuclear Quadrupole Resonance

(NQR) spectra – NMR spectra arise from transitions which are inducted
between the nuclear spin energy levels of a molecule in an applied
magnetic field. NQR spectra arise from the transition between the nuclear
spin energy levels of a molecule arising from the interaction of the
unsymmetrical charge distribution inn nuclei with the electric field
gradients (EFG) which arise from the bonding and non-bonding electrons in
the molecule. NMR and NQR spectra span the radio-frequency regions. 5-
100MHz.

2) Electron Spin Resonance (ESR) or Electron Paramagnetic Resonance (EPR)

Spectra - ESR spectra arise from transitions induced between the electron
spin energy levels of a molecule in an applied magnetic field. These spectra

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 can be exhibited by systems which are having odd (unpaired) electrons
such as free radicals and transition metal ions. Molecules such as nitric
oxide and oxygen and other paramagnetic system also exhibit ESR spectra.
This branch of spectroscopy comes in the microwave region 2000-9600
MHz

3) Pure Rotational (Microwave) Spectra – These spectra arise from transition

between the rotational energy levels of a gaseous molecule on absorbing
radiations falling in the microwave region. These spectra are shown by the
molecules which are having a permanent dipole moment, e.g., HCL, CO,
H2O vapour, NO, etc. Homonuclear diatomic molecules like H2, CL2, O2
and linear polyatomic molecules such as CO2 which are not having a dipole
moment do not show microwave spectra. Microwave spectra come in the
spectral range of 1-100 cm-1

4) Raman Spectra – These are related to irrational and rotational transitions in

molecules but in a different manner. In the case, what we measure is the
scattering and not the absorption of radiation. An intense beam of
monochromatic radiation in the visible region is allowed to fall on a sample
and the intensity of scattered light is having the same frequency as the
incident beam. Most of the scattering light as having the same amount as
light is having different frequencies than the incident beam. This is termed
as Raman scattering. The energy differences between these weak lies and
main Rayleigh line corresponds to irrational and rotational transitions in the
molecule under investigation. Raman spectra can be observed in the visible
region.

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5) Vibrational and Vibration- Rotational (Inferred) Spectra – These spectra
arises because of transitions induced between the irrational energy levels
of a molecule on absorbing radiation is accompanied by a change in the
dipole moment of the molecule. These spectra come in the range of 500 –
4000 MHz

6) Electronic Spectra – Electronic spectra arise due to the electronic transition

in a molecule by absorbing radiations falling in the visible and ultraviolet
regions. While electronic spectra in the visible region span 12,500-70,000
cm-1. As electronic transitions in a molecule have been invariably
accompanied by vibration and rotational transitions, the electronic spectra
of molecule would be highly complex.

7) Photoelectron Spectra (PES) – If a light photon falling on a molecule is

having very high energy, it can bring about ionization of the molecule i.e.,
the removal of the electron from the molecule. If the energy of the incident
proton is greater than the ionization energy, the ejected electron will have
excess kinetic energy. In PES, a beam of photon of known energy is made to
fall on the sample and the kinetic energy of the ejected electrons is
measured. The difference between the photon energy and the excess
kinetic would give the binding energy of the electron. PES provides one of
the most accurate methods for determining the ionization energies of
molecules. These values can provide a fairly good insight into the molecular
electronic structures.

8) Mossbauer Spectra or Gamma Ray Spectra – Mossbauer spectra refers to a

type of nuclear resonance spectra like nuclear magnetic resonance spectra.

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 However , while NMR spectra results from absorption of low energy
photons of frequency around 60 MHz , Mossbauer spectra arise from
absorption of high energy γ photons of frequency around 1013 MHz by the
nuclei. Gamma ray spectra have been used specifically for studying
compounds of Iron and Tin. In this case γ radiations from Co source are
allowed to fall on a sample in which the iron nuclei are in an environment
identical with that of the source atoms. This causes in resonant absorption
of γ rays. The splitting in Mossbauer lines have been found to be of the
same order as in NMR spectroscopy.

This project deals with the detailed study of only NMR, ultraviolet, inferred and
Raman spectroscopy only and it is described in the following chapters.

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 4. Ultraviolet spectroscopy
Ultraviolet-visible spectroscopy or ultraviolet-visible
spectrophotometer (UV/ VIS) involves the spectroscopy of photons in the UV-
visible region. It uses light in the visible and adjacent near ultraviolet (UV) and
near infrared (NIR) ranges. In this region of the electromagnetic spectrum,
molecules undergo electronic transitions. This technique is complementary to
fluorescence spectroscopy, in that fluorescence deals with transitions from the
excited state to the ground state, while absorption measures transitions from the
ground state to the excited state.

General principles
Absorption of incident radiation by bonding/non-
bonding electrons represents a high energy (~100 kCal/ mole) transition. This
corresponds to a high frequency, i.e. low wavelength, absorption band which is
observed at 200 ~ 800 nm in the UV and visible range of detection. In solution,
electronic absorption spectra are found with broad, generally unresolved bands.
These contrast with the vibration fine structure in the vapour phase and with a
series of sharp peaks within a continuum in non-polar solvents.

For a solution of an absorbing substance, an absorptive

ratio at a monochromatic wavelength is defined as: (incident light,
Io)/(transmitted, I) and this is logarithmically related to concentration and optical
path-length by the Beer Lambert law: Absorbance (A) = log10(Io/I) = k.c.l., where c
mg/ml is the concentration of solute and 1 cm is the distance travelled between
parallel optical faces of a suitable cell, and k is a proportionality constant. It is

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frequently convenient to normalize to a concentration c = 10 mg/ml [i.e. 1%] and l
= 1 cm, which is expressed as the specific absorbance [A1%1cm]. Molar
absorptive is defined by the coefficient ε = Mr.(A/cl), and is related to the relative
molecular mass, Mr. This coefficient is computed for each wavelength maximum,
and also at minima if this is of diagnostic value. It may be useful in showing
relationships within a homologous series.

For light source emissivity, the common radiation source

is a deuterium lamp covering the operating range 180~ 350 nm and
supplemented by a tungsten filament lamp in the near UV, through the visible,
into the near-IR, i.e. over the range 320~1000 nm. Standardization of equipment
and monochromators is necessary to ensure the acceptability of data. The
wavelength scale is calibrated with a Holmium perchlorate solution, within a
tolerance of ±1 nm below 400 nm and ± 3 nm in the 400~600 range (see British
Pharmacopoeia, 1993). The absorbance may be checked with NPL calibrated
neutral density filters; or should agree within defined corresponding ‘windows’
with absorbance obtained with a potassium dichromate solution of specified
strength, at wavelengths 235, 257, 313 and 350 nm. Stray light is usually checked
with a 1.2% potassium chloride solution, where the absorbance for 1 cm path
length should exceed 2.0 at 200 nm against a water reference. This solution can
be replaced by 1% NaBr or NaI at the more accessible wavelengths of 215 or 240
nm respectively. Glass optics absorb UV light below about 300 nm and quartz
systems are used to extend the working range down to 200 nm, and even to 185
nm if there are high quality optics and stray light control. At lower wavelengths,
absorption of UV-radiation by air requires the use of vacuum systems in research

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instruments. For practical UV-vis spectrophotometry, the effective working range
is 200~800 nm.

Ultraviolet-visible spectrum
An ultraviolet-visible spectrum is essentially a graph of
light absorbance versus wavelength in a range of ultraviolet or visible regions.
Such a spectrum can often be produced directly by a more sophisticated
spectrophotometer, or the data can be collected one wavelength at a time by
simpler instruments. Wavelength is often represented by the symbol λ. Similarly,
for a given substance, a standard graph of the extinction coefficient (ε) vs.
wavelength (λ) may be made or used if one is already available. Such a standard
graph would be effectively "concentration-corrected" and thus independent of
concentration. For the given substance, the wavelength at which maximum
absorbance in the spectrum occurs is called λmax, pronounced "Lambda-max".

The Woodward-Fieser rules are a set of empirical

observations which can be used to predict λ max, the wavelength of the most
intense UV/Vis absorption, for conjugated organic compounds such as dienes and
ketones.

The wavelengths of absorption peaks can be correlated

with the types of bonds in a given molecule and are valuable in determining the
functional groups within a molecule. UV/Vis absorption is not, however, a specific
test for any given compound. The nature of the solvent, the pH of the solution,
temperature, high electrolyte concentrations, and the presence of interfering

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substances can influence the absorption spectra of compounds, as can variations
in slit width (effective bandwidth) in the spectrophotometer.

Ultraviolet-visible spectrophotometer
The instrument used in ultraviolet-visible spectroscopy is
called a UV/vis spectrophotometer. It measures the intensity of light passing
through a sample (I), and compares it to the intensity of light before it passes
through the sample (Io). The ratio I / Io are called the transmittance, and are
usually expressed as a percentage (%T). The absorbance, A, is based on the
transmittance:

A = − log(%T)

The basic parts of a spectrophotometer are a light

source (often an incandescent bulb for the visible wavelengths, or a deuterium
arc lamp in the ultraviolet), a holder for the sample, a diffraction grating or
monochromator to separate the different wavelengths of light, and a detector.
The detector is typically a photodiode or a CCD. Photodiodes are used with
monochromators, which filter the light so that only light of a single wavelength
reaches the detector. Diffraction gratings are used with CCDs, which collects light
of different wavelengths on different pixels.

A spectrophotometer can be either single beam or

double beam. In a single beam instrument (such as the Spectronic 20), all of the
light passes through the sample cell. Io must be measured by removing the
sample. This was the earliest design, but is still in common use in both teaching
and industrial labs.

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A spectrophotometer can be either single beam or double beam. In a single beam
instrument (such as the
Spectronic 20), all of the light
passes through the sample cell. Io
must be measured by removing
the sample.

This was the earliest design, but is

still in common use in both
teaching and industrial labs. (Diagram of a single-beam UV/vis spectrophotometer.)

In a double-beam instrument, the light is split into

two beams before it reaches the sample. One beam is used as the reference; the
other beam passes through the sample. Some double-beam instruments have two
detectors (photodiodes), and the sample and reference beam are measured at
the same time. In other instruments, the two beams pass through a beam
chopper, which blocks one beam at a time. The detector alternates between
measuring the sample beam and the reference beam.

Samples for UV/Vis spectrophotometry are most often

liquids, although the absorbance of gases and even of solids can also be
measured. Samples are typically placed in a transparent cell, known as a cuvette.
Cuvettes are typically rectangular in shape, commonly with an internal width of 1
cm. (This width becomes the path length, L, in the Beer-Lambert law.) Test tubes
can also be used as cuvettes in some instruments. The best cuvettes are made of
high quality quartz, although glass or plastic cuvettes are common. (Glass and

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most plastics absorb in the UV, which limits their usefulness to visible
wavelengths.)

Operating conditions
Selection of a suitable solvent is influenced by the
wavelength expected to be studied. Water and the lower (polar) alcohols, through
diethyl ether and dioxin to nonpolar cyclohexane and light petroleum (‘aromatic
free’ in a spectroscopic grade) can be used above 190 nm, whereas chloroform
absorbs below ~245 nm. The table below provides a list of cut-off wavelengths.
Measured absorbances should be less than 0.4 relative to air using prism
monochromators; but higher absorptivity ratios are favoured with modern
instruments.
The choice of cells depends on the target range. Silica is
essential for measurements at UV wavelengths but glass is acceptable in the
visible region; air must be evacuated below ~200 nm. The matched pair required
for test solution and solvent reference path should demonstrate effectively
identical absorbance when filled with the same solvent. The optical faces of the
cells should be parallel; the absorbance of a matched pair of cells containing the
same solvent should not differ by more than 0.005 units. In quantitative work all
solutions should be at the same temperature; conveniently, they are transferred
from a water bath at, say, 20° and the absorbance measured immediately.
Sensitivity of the solution to laboratory and natural lighting should be established
in a pilot experiment. One test (British Pharmacopoeia, 1993) of Resolution Power
is the discrimination of adjacent maximum (269 nm) and minimum (266 nm) light
absorption of toluene with a ratio not less than 1.5.

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Quantitative procedures
UV photometry is a frequently used assay technique.
Provided that proper calibration checks are maintained, the UV-vis technique is
particularly useful for assay of formulations after extraction or separation of the
active substance by suitable chromatography. In the assay calibration, there may
be some deviation from Beer’s Law. This may be attributable to association in
solution or an effect of slit width. The latter should be large enough to gain a
reasonable I-value but remain small compared with the (half-) bandwidth for the
absorption measured. If in doubt, reduce the slit width slightly and check if the
apparent absorbance increases. UV photometric data can also be of value in
determining the kinetics of a process, or in following a reaction sequence, such as
the disappearance of an absorption peak representing starting material.
Light absorption’ measurements also provide a semi-
quantitative test of identity. This relies on the specific absorbance (defined above
as the A1cm1% value), or sometimes absorbance at a nominated concentration
and path length, either exactly at a specified wavelength, or at the absorption
maximum close to a named wavelength. If this test is used as the principal assay
of a substance in a formulation, it is advisable to use an authenticated reference
substance rather than rely on a published A11. The absorption spectrum may be
sensitive to control of pH. Chromophores involving an acidic or basic group will be
affected by pH, e.g. the bathochromic shift (to longer wavelength) and
hyperchromic peak (greater intensity) of phenates compared with their parent
phenol. This is a useful test for a phenolic system.
In subtractive spectrophotometry, the difference
between two (or more) spectra measures multicomponent mixtures and is

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especially useful in formulated product assays. This should be distinguished from
the use of second derivative spectroscopy, in which there is computer
differentiation of the algebraic function equivalent to the change of slope (i.e.
second differential) of the digitalized spectrum (British Pharmacopoeia, 1993).
This display sharpens separation of individual UV bands and thereby facilitates
lower levels of control. In other applications of computer-aided spectroscopy,
modern equipment will provide ‘smoothing’, deconvolution and regression (least
squares) analysis.
General chromophores
Absorption bands are particularly evident for conjugated
π-bond systems. Most single bond transitions are inaccessible, being derived from
higher energy σ-orbitals, with wavelengths below 185 nm, i.e. in the ‘vacuum
ultraviolet’. Many isolated triple bonds also absorb below 185 nm. The C C (~185)
and C N (~190) double bonds exhibit strong π–π* interactions but unless there is
very good control of stray light, measurement is still unreliable in this region. At
longer wavelengths there are rather weak n−π* interactions, such as N O and keto
C O in the range 280~300 nm. Simple benzene compounds show medium
intensity multiplets around 254 nm for non-conjugated derivatives, and shifted to
longer wavelengths when substituents are conjugated to the aromatic system. In
the table at the end of this section there are examples of commonly encountered
chromophores, including conjugated alkene, carbonyl and aromatic systems
which exhibit bathochromic (longer wavelength) and hyperchromic (enhanced
absorptivity) changes. For very extensive catalogues of individual UV spectra,
refer to DMS (1960–1971) (1160 substances), Hirayama (1967) (8500 selected
values) and Sadtler (1979) (2000 spectra for 1600 compounds). Older spectra of

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specifically aromatic compounds were collated by Friedel and Orchin (1951).
Schemes such as Woodward’s ‘Rules’ (1941, 1942), as further modified by L. and
M. Fieser, for conjugated polyenes and en-ones, have considerable predictive
power.

Beer-Lambert law
The method is most often used in a quantitative way to
determine concentrations of an absorbing species in solution, using the Beer-
Lambert law:
A= - log (I/ I0)
Where A is the measured absorbance, I0 is the intensity
of the incident light at a given wavelength, I is the transmitted intensity, L the
path length through the sample, and c the concentration of the absorbing species.
For each species and wavelength, ε is a constant known as the molar absorptive
or extinction coefficient. This constant is a fundamental molecular property in a
given solvent, at a particular temperature and pressure, and has units of 1 / M *
cm or often AU / M * cm. The absorbance and extinction ε are sometimes defined
in terms of the natural logarithm instead of the base-10 logarithm.
The Beer-Lambert Law is useful for characterizing many
compounds but does not hold as a universal relationship for the concentration
and absorption of all substances. A 2nd order polynomial relationship between
absorption and concentration is sometimes encountered for very large, complex
molecules such as organic dyes (Xylenol Orange or Neutral Red, for example)

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 Examples of π __> π* Excitation
Click on the Diagram to Advance
Solvent cut-off wavelengths
In this table, approximate wavelengths (nm) are
specified below which the solvent absorbance may be unacceptable. For
quantitative work, the cut-off may be set at a wavelength (L0) where the
absorbance for 10 mm pathlength of the solvent exceeds 0.05 absorbance unit
(relative to water), i.e. A1 cm > 0.05. For qualitative work, it may still be feasible
to work at significantly lower wavelengths and most analysts accept a cut-off
based on the wavelength (L1) for A1 cm > 1.0. However, if the UV absorption
curve rises steeply, the accessible wavelength range may not be greatly extended.

L0 L1 L0 L1

Alcohols Halocarbons (contd)

methanol 240 205 1,2-dichloroethane 250 230
ethanol 240 205 tetrachloroethylene 320 290
n-propanol 250 210 trichloroethylene >400
2-propanol 240 205
n-butanol 245 215 Miscellaneous
s-butanol 285 260 acetonitrile 200 190
isobutanol 250 200 NN-dimethylformamide 300 270
dimethylsulphoxide 330 285
Esters nitromethane >400 380
ethyl acetate 280 260 pyridine 345 325
n-butyl acetate 275 255 water 190 185

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Ethers Alkanes
diethyl ether 255 220 pentane 230 200
p-dioxane 290 220 hexane 225 195
tetrahydrofuran 280 220 heptane 230 200
2-methoxyethanol 270 200 cyclopentane 220 195
2-ethoxyethanol 280 210 cyclohexane 235 200
1,2-dimethoxyethane 300 220 2,2,4-trimethylpentane
[‘isooctane’] 230 210
Ketones decalin 250 230
acetone 340 330
butan-2-one [MEK] 345 330 Aromatic hydrocarbons
4-methylpentanone [MIBK] 375 335 benzene 295 280
5-methylhexanone [MIAK] 350 330 toluene 315 285
chlorobenzene 310 285
Halcocarbons 1,2-dichlorobenzene 350 295
chloroform 260 240 o-xylene 325 290
dichloromethane 245 230 1,2,4-trichlorobenzene 350 ?

Applications
UV/Vis spectroscopy is routinely used in the quantitative
determination of solutions of transition metal ions and highly conjugated organic
compounds.
• Solutions of transition metal ions can be coloured (i.e., absorb visible light)
because d electrons within the metal atoms can be excited from one electronic
state to another. The colour of metal ion solutions is strongly affected by the
presence of other species, such as certain anions or ligands. For instance, the
colour of a dilute solution of copper sulfate is a very light blue; adding ammonia
intensifies the colour and changes the wavelength of maximum absorption
(λmax).
• Organic compounds, especially those with a high degree of conjugation,
also absorb light in the UV or visible regions of the electromagnetic spectrum. The

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solvents for these determinations are often water for water soluble compounds,
or ethanol for organic-soluble compounds. (Organic solvents may have significant
UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol
absorbs very weakly at most wavelengths.) Solvent polarity and pH can effect the
absorption spectrum of an organic compound. Tyrosine, for example, increases in
absorption maxima and molar extinction coefficient when pH increases from 6 to
13 or when solvent polarity decreases.
• While charge transfer complexes also give rise to colours, the colours are
often too intense to be used for quantitative measurement.
The Beer-Lambert law states that the absorbance of a solution is directly
proportional to the solution's concentration. Thus UV/VIS spectroscopy can be
used to determine the concentration of a solution. It is necessary to know how
quickly the absorbance changes with concentration. This can be taken from
references (tables of molar extinction coefficients), or more accurately,
determined from a calibration curve.
A UV/Vis spectrophotometer may be used as a detector for
HPLC. The presence of an analyte gives a response which can be assumed to be
proportional to the concentration. For accurate results, the instrument's response
to the analyte in the unknown should be compared with the response to a
standard; this is very similar to the use of calibration curves. The response (e.g.,
peak height) for a particular concentration is known as the response factor.

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 5. Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical
phenomenon based upon the quantum mechanical magnetic properties of an
atom's nucleus. NMR also commonly refers to a family of scientific methods that
exploit nuclear magnetic resonance to study molecules.
All nuclei that contain odd numbers of protons or
neutrons have an intrinsic magnetic moment and angular momentum. The most
commonly measured nuclei are hydrogen-1 (the most receptive isotope at natural
abundance) and carbon-13, although nuclei from isotopes of many other
elements can also be observed.
NMR resonant frequencies for a particular substance are
directly proportional to the strength of the applied magnetic field, in accordance
with the equation for the Lamar precession frequency. NMR studies magnetic
nuclei by aligning them with an applied constant magnetic field and perturbing
this alignment using an alternating magnetic field, those fields being orthogonal.
The resulting response to the perturbing magnetic field is the phenomenon that is
exploited in NMR spectroscopy and magnetic resonance imaging, which use very
powerful applied magnetic fields in order to achieve high spectral resolution,
details of which are described by the chemical shift and the Zeeman Effect. NMR
phenomena are also utilized in low field NMR and Earth's field NMR
spectrometers, and some kinds of magnetometers.
Discovery
Nuclear magnetic resonance was first described and measured
in molecular beams by Isidor Rabi in 1938. Eight years later, in 1946, Felix Bloch

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and Edward Mills Purcell refined the technique for use on liquids and solids, for
which they shared the Nobel Prize in physics in 1952.
Purcell had worked on the development and application of
RADAR during World War II at Massachusetts Institute of Technology's Radiation
Laboratory. His work during that project on the production and detection of
radiofrequency energy, and on the absorption of such energy by matter,
preceded his discovery of NMR.
They noticed that magnetic nuclei, like 1H and 31P, could
absorb RF energy when placed in a magnetic field of a strength specific to the
identity of the nuclei. When this absorption occurs, the nucleus is described as
being in resonance. Different atoms within a molecule resonate at different
frequencies at a given field strength. The observation of the resonance
frequencies of a molecule allows a user to discover structural information about
the molecule.
The development of nuclear magnetic resonance as a
technique of analytical chemistry and biochemistry parallels the development of
electromagnetic technology and its introduction into civilian use.
Theory of nuclear magnetic resonance

Nuclear spin and magnets

The elementary particles, neutrons and protons, composing an
atomic nucleus, have the intrinsic quantum mechanical property of spin. The
overall spin of the nucleus is determined by the spin quantum number I. If the
number of both the protons and neutrons in a given isotope are even then I = 0,
i.e. there is no overall spin; just as electrons pair up in atomic orbital. Even

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numbers of protons and neutrons (which are also spin ½ particles and hence
fermions) pair up giving zero overall spin. In other cases, however, the overall spin
is non-zero. For example 27Al has an overall spin I = 5/2.
A non-zero spin, I, is associated with a non-zero magnetic
moment, µ, via

µ=γΙ
Where the proportionality constant γ is the gyromagnetic ratio. It is this magnetic
moment that is exploited in NMR.
Electron spin resonance is a related technique which exploits
the spin of electrons instead of nuclei. The basic principles are otherwise similar.

Values of spin angular momentum

The angular momentum associated with nuclear spin is
quantized. This means both that the magnitude of angular momentum is
quantized (i.e. I can only take on a restricted range of values), and also that the
'orientation' of the associated angular momentum is quantized. The associated
quantum number is known as the magnetic quantum number m and can take
values from +I to –I in integral steps. Hence for any given nucleus, there is a total
of 2I+1 angular momentum states.
The z component of the angular momentum vector, Iz, is
therefore:

Where Is Planck's constant.

The z component of the magnetic moment is simply

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Spin behavior in a magnetic field
Consider nuclei which have a spin of one-half, like 1H, 13C or
19F. The nucleus has two possible spin states: m = ½ or m = -½ (also referred to as
up and down or α and β, respectively). The energies of these states are
degenerate—that is to say that they are the same. Hence the populations of the
two states (i.e. number of atoms in the two states) will be approximately equal at
thermal equilibrium.
If a nucleus is placed in a magnetic field,
however, the interaction between the
nuclear magnetic moment and the
external magnetic field mean the two
states no longer have the same energy.
The energy of a magnetic moment µ
when in a magnetic field B0 (the zero subscript is used to distinguish this magnetic
field from any other applied field) is given by the negative scalar product of the
vectors:

Where the magnetic field has been oriented along the z axis.

Resonance
Resonant absorption will occur when electromagnetic
radiation of the correct frequency to match this energy difference is applied. The
energy of a photon is E = hν where ν is its frequency. Hence absorption will occur
when

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These frequencies typically correspond to the radio frequency range of the
electromagnetic spectrum.
It is this resonant absorption that is detected in NMR.

Nuclear shielding
It might appear from the above that all nuclei of the same
nuclide (and hence the same γ) would resonate at the same frequency. This is not
the case. The most important perturbation of the NMR frequency for applications
of NMR is the 'shielding' effect of the surrounding electrons. In general, this
electronic shielding reduces the magnetic field at the nucleus (which is what
determines the NMR frequency). As a result the energy gap is reduced, and the
frequency required to achieve resonance is also reduced. This shift of the NMR
frequency due to the chemical environment is called the chemical shift, and it
explains why NMR is a direct probe of chemical structure. If the nucleus is more
shielded, then it will be shifted upfield (lower chemical shift) and if it is more
deshielded, then it will be shifted downfield (higher chemical shift).
Unless the local symmetry is particularly high, the shielding
effect depends on the orientation of the molecule with respect to the external
field. In solid-state NMR, magic angle spinning is required to average out this
orientation dependence. This is unnecessary in conventional NMR of molecules in
solution since rapid molecular tumbling averages out the anisotropic component
of the chemical shift.

25
Relaxation
The process called population relaxation refers to nuclei that
return to the thermodynamic state in the magnet. This process is also called T1
relaxation, where T1 refers to the mean time for an individual nucleus to return to
its equilibrium state. Once the population is relaxed, it can be probed again, since
it is in the initial state.
The precessing nuclei can also fall out of alignment with each
other (returning the net magnetization vector to a non-precessing field) and stop
producing a signal. This is called T2 relaxation. It is possible to be in this state and
not have the population difference required to give a net magnetization vector at
its thermodynamic state. Because of this, T1 is always larger (slower) than T2. This
happens because some of the spins were flipped by the pulse and will remain so
until they have undergone population relaxation. In practice, the T2 time is the life
time of the observed NMR signal, the free induction decay. In the NMR spectrum,
meaning the Fourier transform of the free induction decay, the T2 time defines
the width of the NMR signal. Thus, a nucleus having a large T2 time gives rise to a
sharp signal, whereas nuclei with shorter T2 times give rise to more broad signals.
The length of T1 and T2 is closely related to molecular motion.

NMR spectroscopy
NMR spectroscopy is one of the principal techniques used to
obtain physical, chemical, electronic and structural information about molecules
due to the chemical shift and Zeeman Effect on the resonant frequencies of the
nuclei. It is a powerful technique that can provide detailed information on the
topology, dynamics and three-dimensional structure of molecules in solution and

26
the solid state. Also, nuclear magnetic resonance is one of the techniques that
have been used to build elementary quantum computers.

Continuous wave (CW) spectroscopy

In its first few decades, nuclear magnetic resonance
spectrometers used a technique known as continuous-wave (CW) spectroscopy.
Although NMR spectra could be obtained using a fixed magnetic field and
sweeping the frequency of the electromagnetic radiation, this more typically
involved using a fixed frequency source and varying the current (and hence
magnetic field) in an electromagnet to observe the resonant absorption signals.
(This is the origin of the now anachronistic but still common "high" and "low" field
terminology for low frequency and high frequency regions respectively of the
NMR spectrum.)
CW spectroscopy is inefficient in comparison to Fourier
techniques (see below) as it probes the NMR response at individual frequencies in
succession. As the NMR signal is intrinsically weak, the observed spectra suffer
from a poor signal-to-noise ratio (S/N). This can be mitigated by signal averaging
i.e. adding the spectra from repeated measurements. While the NMR signal is
constant between scans and so adds linearly, the random noise adds more slowly
— as the square-root of the number of spectra. Hence the overall ratio of the
signal to the noise increases as the square-root of the number of spectra
measured.

Fourier spectroscopy

27
 Most applications of NMR involve full NMR spectra, that is, the
intensity of the NMR signal as a function of frequency. Early attempts to acquire
the NMR spectrum more efficiently than simple CW methods involved irradiating
simultaneously with more than one frequency. It was soon realized however that
a simpler solution was to use short pulses of radio-frequency (centered at the
middle of the NMR spectrum). In simple terms, a short square pulse of a given
"carrier" frequency "contains" a range of frequencies centered about the carrier
frequency, with the range of excitation (bandwidth) being inversely proportional
to the pulse duration (the Fourier transform of an approximate square wave
contains contributions from all the frequencies in the neighborhood of the
principal frequency). The restricted range of the NMR frequencies made it
relatively easy to use RF pulses to excite the entire NMR spectrum.
Applying such a pulse to a set of nuclear spins simultaneously
excites all the NMR transitions. In terms of the net magnetization vector, this
corresponds to tilting the magnetization vector away from its equilibrium position
(aligned along the external magnetic field). The out-of-equilibrium magnetization
vector precesses about the external magnetic field at the NMR frequency of the
spins. This oscillating magnetization induces a current in a near by pickup coil,
creating an electrical signal oscillating at the NMR frequency. This signal is known
as the free induction decay (FID) and contains the sum of the NMR responses
from all the excited spins. In order to obtain the frequency-domain NMR
spectrum (intensity vs. frequency) this time-domain signal (intensity vs. time)
must be Fourier transformed. Fortunately the development of FT-NMR coincided
with the development of digital computers and Fast Fourier Transform
algorithms.

28
Richard R. Ernst was one of the pioneers of pulse (FT) NMR and won a Nobel Prize
in chemistry in 1991 for his work on FT-NMR and his development of multi-
dimensional NMR.

Multi-dimensional NMR Spectroscopy

The use of pulses of different shapes, frequencies and
durations in specifically-designed patterns or pulse sequences allows the
spectroscopist to extract many different types of information about the molecule.
Multi-dimensional nuclear magnetic resonance spectroscopy is
a kind of FT-NMR in which there are at least two pulses and, as the experiment is
repeated, the pulse sequence is varied. In multidimensional nuclear magnetic
resonance there will be a sequence of pulses and, at least, one variable time
period. In three dimensions, two time sequences will be varied. In four
dimensions, three will be varied.
There are many such experiments. In one, these time intervals
allow—among other things—magnetization transfer between nuclei and,
therefore, the detection of the kinds of nuclear-nuclear interactions that allowed
for the magnetization transfer. Interactions that can be detected are usually
classified into two kinds. There are through-bond interactions and through-space
interactions, the latter usually being a consequence of the nuclear Overhauser
effect. Experiments of the nuclear-Overhauser variety may establish distances
between atoms.
Although the fundamental concept of 2D NMR was proposed
by the Belgian scientist Jean Jeener, professor at the Free University of Brussels,

29
this idea was largely developed by Richard Ernst who won the 1991 Nobel prize in
Chemistry for his work in FT and multi-dimensional NMR. Multi-dimensional NMR
experiments were further developed into powerful methodologies for studying
biomolecules in solution, in particular for the determination of the structure of
biopolymers such as proteins or even small nucleic acids. Kurt Wüthrich shared
the 2002 Nobel Prize in Chemistry for his work in protein nuclear magnetic
resonance spectroscopy.

Solid-state NMR spectroscopy

This technique complements biopolymer X-ray crystallography
in that it is frequently applicable to biomolecules in a liquid or liquid crystal phase,
whereas crystallography, as the name implies, is performed on molecules in a
solid phase. Though nuclear magnetic resonance is used to study solids, extensive
atomic-level biomolecular structural detail is especially challenging to obtain in
the solid state. There is no signal averaging by thermal motion in the solid state,
where molecules are held still, each in a slightly different electronic environment,
giving a different signal. This variation in electronic environment lowers resolution
greatly and makes interpretation more difficult. Raymond Andrew was a pioneer
in the development of high-resolution solid-state nuclear magnetic resonance. He
introduced the magic angle spinning (MAS) technique and allowed for an increase
in resolution by several orders of magnitude. In MAS, the sample is spun at
several kilohertz around an axis that makes the so-called magic angle with the
static magnetic field and the spin interactions are averaged to their isotropic
values.

30
 A concept developed by Sven Hartmann and Erwin Hahn was
utilized in transferring magnetization from protons to less sensitive nuclei
(popularly known as cross-polarization) by Gibby Alex Pines and John S. Waugh.
Schaefer and coworkers demonstrated the powerful use of cross-polarization
under MAS which is now routinely used to detect low-abundance and low-
sensitivity nuclei.

Applications
MEDICINE

The use of nuclear magnetic resonance best known to the

general public is magnetic resonance imaging for medical diagnosis and MR
Microscopy in research settings; however, it is also widely used in chemical
studies, notably in NMR spectroscopy such as proton NMR, carbon-13 NMR,
deuterium NMR and phosphorus-31 NMR. Biochemical information can also be
obtained from living tissue (e.g human brain tumors) with the technique known as
in vivo magnetic resonance spectroscopy or chemical shift NMR Microscopy.
These studies are possible because nuclei are surrounded by
orbiting electrons, which are also spinning charged particles such as magnets and,
so, will partially shield the nuclei. The amount of shielding depends on the exact
local environment. For example, hydrogen bonded to oxygen will be shielded
differently than hydrogen bonded to a carbon atom. In addition, two hydrogen
nuclei can interact via a process known as spin-spin coupling, if they are on the
same molecule, which will split the lines of the spectra in a recognizable way.
CHEMISTRY

31
 By studying the peaks of nuclear magnetic resonance spectra,
skilled chemists can determine the structure of many compounds. It can be a very
selective technique, distinguishing among many atoms within a molecule or
collection of molecules of the same type but which differ only in terms of their
local chemical environment. See the articles on carbon-13 NMR and proton NMR
for detailed discussions.
By studying T2 information a chemist can determine the
identity of a compound by comparing the observed nuclear precession
frequencies to known frequencies. Further structural data can be elucidated by
observing spin-spin coupling, a process by which the precession frequency of a
nucleus can be influenced by the magnetization transfer from nearby nuclei. Spin-
spin coupling is most commonly observed in NMR involving common isotopes,
such as Hydrogen-1 (HNMR).
T2 information can give information about dynamics and
molecular motion. Because the nuclear magnetic resonance timescale is rather
slow, compared to other spectroscopic methods, changing the temperature of a
T2 experiment can also give information about fast reactions, such as the Cope
rearrangement or about structural dynamics, such as ring-flipping in cyclohexane.
At low enough temperatures, a distinction can be made between the axial and
equatorial hydrogen in cyclohexane.
An example of nuclear magnetic resonance being used in the
determination of a structure is that of buckminsterfullerene. This now famous
form of carbon has 60 carbon atoms forming a sphere. The carbon atoms are all in
identical environments and so should see the same internal H field.
13
Unfortunately, buckminsterfullerene contains no hydrogen and so C nuclear

32
 13
magnetic resonance has to be used. C spectra require longer acquisition times
since carbon-13 is not the common isotope of carbon (unlike hydrogen, where 1H
is the common isotope). However, in 1990 the spectrum was obtained by R.
Taylor and co-workers at the University of Sussex and was found to contain a
single peak, confirming the unusual structure of C60
NON-DESTRUCTIVE TESTING
Nuclear magnetic resonance is extremely useful for analyzing
samples non-destructively. Radio waves and static magnetic fields easily
penetrate many types of matter and anything that is not inherently
ferromagnetic. For example, various expensive biological samples, such as nucleic
acids, including RNA and DNA, or proteins, can be studied using nuclear magnetic
resonance for weeks or months before using destructive biochemical
experiments. This also makes nuclear magnetic resonance a good choice for
analyzing dangerous samples.
DATA ACQUISITION IN THE PETROLEUM INDUSTRY

Another use for nuclear magnetic resonance is data acquisition

in the petroleum industry for petroleum and natural gas exploration and
recovery. A borehole is drilled into rock and sedimentary strata into which nuclear
magnetic resonance logging equipment is lowered. Nuclear magnetic resonance
analysis of these boreholes is used to measure rock porosity, estimate
permeability from pore size distribution and identify pore fluids (water, oil and
gas). These instruments are typically low field NMR spectrometers.
Process control
NMR has now entered the arena of real-time process control
and process optimization in oil refineries and petrochemical plants. Two different

33
types of NMR analysis are utilized to provide real time analysis of feeds and
products in order to control and optimize unit operations. Time-domain NMR (TD-
NMR) spectrometers operating at low field (2-20 MHz for 1H) yield free induction
decay data that can be used to determine absolute hydrogen content values,
rheological information, and component composition. These spectrometers are
used in mining, polymer production, cosmetics and food manufacturing as well as
coal analysis. High resolution FT-NMR spectrometers operating in the 60 MHz
range with shielded permanent magnet systems yield high resolution 1H NMR
spectra of refinery and petrochemical streams. The variation observed in these
spectra with changing physical and chemical properties is modeled utilizing
chemometrics to yield predictions on unknown samples. The prediction results
are provided to control systems via analogue or digital outputs from the
spectrometer.
EARTH'S FIELD NMR
In the Earth's magnetic field, NMR frequencies are in the audio
frequency range. EFNMR is typically stimulated by applying a relatively strong dc
magnetic field pulse to the sample and, following the pulse, analyzing the
resulting low frequency alternating magnetic field that occurs in the earth's
magnetic field due to free induction decay (FID). These effects are exploited in
some types of magnetometers, EFNMR spectrometers, and MRI imagers. Their
inexpensive portable nature makes these instruments valuable for field use and
for teaching the principles of NMR and MRI.

34
 6. Mass spectrometry
Mass spectrometry is an analytical technique that identifies
the chemical composition of a compound or sample based on the mass-to-charge
ratio of charged particles. A sample undergoes chemical fragmentation forming
charged particles (ions). The ratio of charge to mass of the particles is calculated
by passing them through
electric and magnetic
fields in a mass
spectrometer.

The design of a mass

spectrometer has three
essential modules: an ion
source, which transforms
the molecules in a sample into ionized fragments; a mass analyzer, which sorts
the ions by their masses by applying electric and magnetic fields; and a detector,
which measures the value of some indicator quantity and thus provides data for
calculating the abundances of each ion fragment present. The technique has both
qualitative and quantitative uses, such as identifying unknown compounds,
determining the isotopic composition of elements in a compound, determining
the structure of a compound by observing its fragmentation, quantifying the
amount of a compound in a sample, studying the fundamentals of gas phase ion

35
chemistry (the chemistry of ions and neutrals in a vacuum), and determining
other physical, chemical, or biological properties of compounds.

Etymology
The word spectrograph has been used since 1884 as an
"International Scientific Vocabulary’’. A mass spectroscope configuration was
used in early instruments when it was desired that the effects of adjustments be
quickly observed. Once the instrument was properly adjusted, a photographic
plate was inserted and exposed. The term mass spectroscope continued to be
used even though the direct illumination of a phosphor screen was replaced by
indirect measurements with an oscilloscope. The use of the term mass
spectroscopy is now discouraged due to the possibility of confusion with light
spectroscopy. Mass spectrometry is often abbreviated as mass-spec or simply as
MS. Thomson has also noted that a mass spectroscope is similar to a mass
spectrograph except that the beam of ions is directed onto a phosphor screen.
The suffix -scope here denotes the direct viewing of the spectra (range) of
masses.

History
Francis William Aston won the 1922 Nobel Prize in Chemistry
for his work in mass spectrometry. In 1886, Eugen Goldstein observed rays in gas
discharges under low pressure that travelled through the channels in a perforated
cathode toward the anode, in the opposite direction to the negatively charged

36
cathode rays. Goldstein called these positively charged anode rays
"Kanalstrahlen"; the standard translation of this term into English is "canal rays".
Wilhelm Wien found that strong electric or magnetic fields deflected the canal
rays and, in 1899, constructed a device with parallel electric and magnetic fields
that separated the positive rays according to their charge-to-mass ratio (Q/m).
Wien found that the charge-to-mass ratio depended on the nature of the gas in
the discharge tube. English scientist J.J. Thomson later improved on the work of
Wien by reducing the pressure to create a mass spectrograph.

Some of the modern techniques of

mass spectrometry were devised by Arthur Jeffrey
Dempster and F.W. Aston in 1918 and 1919
respectively. In 1989, half of the Nobel Prize in Physics
was awarded to Hans Dehmelt and Wolfgang Paul for
the development of the ion trap technique in the
1950s and 1960s. In 2002, the Nobel Prize in
Chemistry was awarded to John Bennett Fenn for the
development of electro spray ionization (ESI) and
Koichi Tanaka for the development of soft laser desorption (SLD) in 1987.
However earlier, matrix-assisted laser desorption/ionization (MALDI), was
developed by Franz Hillenkamp and Michael Karas this technique has been widely
used for protein analysis.

Simplified example
Schematics of a simple mass spectrometer with sector type
mass analyzer. The following example describes the operation of a spectrometer

37
mass analyzer, which is of the sector type. (Other analyzer types are treated
below.) Consider a sample of sodium chloride. In the ion source, the sample is
vaporized (turned into gas) and ionized (transformed into electrically charged
particles) into sodium (Na+) and chloride (Cl-) ions. Sodium atoms and ions are
mono-isotopic, with a mass of about 23 amu. Chloride atoms and ions come in
two isotopes with masses of
approximately 35 amu (at a natural
abundance of

About 75 percent) and approximately 37

amu (at a natural abundance of about
25 percent). The analyzer part of the
spectrometer contains electric and
magnetic fields, which exert forces on
ions traveling through these fields. The
speed of a charged particle may be
increased or decreased while passing through the electric field, and its direction
may be altered by the magnetic field. The magnitude of the deflection of the
moving ion's trajectory depends on its mass-to-charge ratio. By Newton's second
law of motion, lighter ions get deflected by the magnetic force more than heavier
ions. The streams of sorted ions pass from the analyzer to the detector, which
records the relative abundance of each ion type. This information is used to
determine the chemical element composition of the original sample (i.e. that both
sodium and chlorine are present in the sample) and the isotopic composition of
its constituents (the ratio of 35Cl to 37Cl).

38
Instrumentation
Ion source technologies
The ion source is the part of the mass spectrometer that
ionizes the material under analysis (the analyte). The ions are then transported by
magnetic or electric fields to the mass analyzer.

Techniques for ionization have been key to determining what types of samples
can be analyzed by mass spectrometry. Electron ionization and chemical
ionization are used for gases and vapors. In chemical ionization sources, the
analyte is ionized by chemical ion-molecule reactions during collisions in the
source. Two techniques often used with liquid and solid biological samples include
electrospray ionization (invented by John Fenn) and matrix-assisted laser
desorption/ionization (MALDI, developed by K. Tanaka and separately by M. Karas
and F. Hillenkamp). Inductively coupled plasma (ICP) sources are used primarily
for metal analysis on a wide array of sample types. Others include glow discharge,
field desorption (FD), fast atom bombardment (FAB), thermospray,
desorption/ionization on silicon (DIOS), Direct Analysis in Real Time (DART),
atmospheric pressure chemical ionization (APCI), secondary ion mass
spectrometry (SIMS), spark ionization and thermal ionization (TIMS). Ion
Attachment Ionization is a newer soft ionization technique that allows for
fragmentation free analysis.

Spectrometry
A tandem mass spectrometer is one capable of multiple
rounds of mass spectrometry, usually separated by some form of molecule

39
fragmentation. For example, one mass analyzer can isolate one peptide from
many entering a mass spectrometer. A second mass analyzer then stabilizes the
peptide ions while they collide with a gas, causing them to fragment by collision-
induced dissociation (CID). A third mass analyzer then sorts the fragments
produced from the peptides. Tandem MS can also be done in a single mass
analyzer over time, as in a quadruple ion trap. There are various methods for
fragmenting molecules for tandem MS, including collision-induced dissociation
(CID), electron capture dissociation (ECD), electron transfer dissociation (ETD),
infrared multiphoton dissociation (IRMPD) and blackbody infrared radioactive
dissociation (BIRD). An important application using tandem mass spectrometry is
in protein identification.

Tandem mass spectrometry enables a variety of experimental

sequences. Many commercial mass spectrometers are designed to expedite the
execution of such routine sequences as single reaction monitoring (SRM), multiple
reaction monitoring (MRM), and precursor ion scan. In SRM, the first analyzer
allows only a single mass through and the second analyzer monitors for a single
user defined fragment ion. MRM allows for multiple user defined fragment ions.
SRM and MRM are most often used with scanning instruments where the second
mass analysis event is duty cycle limited. These experiments are used to increase
specificity of detection of known molecules, notably in pharmacokinetic studies.
Precursor ion scan refers to monitoring for a specific loss from the precursor ion.
The first and second mass analyzers scan across the spectrum as partitioned by a
user defined m/z value. This experiment is used to detect specific motifs within
unknown molecules.

40
 An important type of Tandem mass spectrometry is
Accelerator Mass Spectrometry (AMS), which uses very high voltages, usually in
the mega-volt range, to accelerate negative ions into a type of tandem mass
spectrometer. One of the most important applications of this technique is
radiocarbon dating.

Common mass spectrometer configurations and techniques

When a specific configuration of source, analyzer, and detector
becomes conventional in practice, often a compound acronym arises to designate
it, and the compound acronym may be better known among nonspectrometrists
than the component acronyms. The epitome of this is MALDI-TOF, which simply
refers to combining a Matrix-assisted laser desorption/ionization source with a
Time-of-flight mass analyzer. The MALDI-TOF moniker is more widely recognized
by the non-mass spectrometrist scientist than MALDI or TOF individually. Other
examples include inductively coupled plasma-mass spectrometry (ICP-MS),
accelerator mass spectrometry (AMS), and Thermal ionization-mass spectrometry
(TIMS) and spark source mass spectrometry (SSMS). Sometimes the use of the
generic "MS" actually connotes a very specific mass analyzer and detection
system, as is the case with AMS, which is always sector based.

Certain applications of mass spectrometry have developed

monikers that although strictly speaking they would seem to refer to a broad
application, in practice have come instead to connote a specific or a limited
number of instrument configurations. An example of this is isotope ratio mass
spectrometry (IRMS), which refers in practice to the use of a limited number of

41
sectors, based mass analyzers; this name is used to refer to both the application
and the instrument used for the application.

Applications
Isotope ratio MS: isotope dating and tracking

12
Mass spectrometer to determine the 16O/18O and C/13C
isotope ratio on bigamous carbonate. Mass spectrometry is also used to
determine the isotopic composition of elements within a sample. Differences in
mass among isotopes of an element are very small, and the less abundant
isotopes of an element are typically very rare, so a very sensitive instrument is
required. These instruments, sometimes referred to as isotope ratio mass
spectrometers (IR-MS), usually use a single magnet to bend a beam of ionized
particles towards a series of Faraday cups which convert particle impacts to
electric current. A fast on-line analysis of deuterium content of water can be done
using flowing afterglow mass spectrometry, FA-MS. Probably the most sensitive
and accurate mass spectrometer for this purpose is the accelerator mass
spectrometer (AMS). Isotope ratios are important markers of a variety of
processes. Some isotope ratios are used to determine the age of materials for
example as in carbon dating. Labeling with stable isotopes is also used for protein
quantification.

Trace gas analysis

Several techniques use ions created in a dedicated ion source

injected into a flow tube or a drift tube: selected ion flow tube (SIFT-MS), and
proton transfer reaction (PTR-MS), are variants of chemical ionization dedicated
for trace gas analysis of air, breath or liquid headspace using well defined reaction

42
time allowing calculations of analyte concentrations from the known reaction
kinetics without the need for internal standard or calibration.

Atom probe

An atom probe is an instrument that combines time-of-flight

mass spectrometry and field ion microscopy (FIM) to map the location of
individual atoms.

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. The most
common instrumentation used in this application is LC-MS with a triple quadruple
mass spectrometer. Tandem mass spectrometry is usually employed for added
specificity. Standard curves and internal standards are used for quantization of
usually a single pharmaceutical in the samples. The samples represent different
time points as a pharmaceutical is administered and then metabolized or cleared
from the body. Blank or t=0 samples taken before administration are important in
determining background and insuring data integrity with such complex sample
matrices. Much attention is paid to the linearity of the standard curve; however it
is not uncommon to use curve fitting with more complex functions such as
quadratics since the response of most mass spectrometers is less than linear
across large concentration ranges.

43
 There is currently considerable interest in the use of very high
sensitivity mass spectrometry for micro dosing studies, which are seen as a
promising alternative to animal experimentation.

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). In keeping with the performance and mass range
of available mass spectrometers, two approaches are used for characterizing
proteins. In the first, intact proteins are ionized by either of the two techniques
described above, and then introduced to a mass analyzer. This approach is
referred to as "top-down" strategy of protein analysis. In the second, proteins are
enzymatically digested into smaller peptides using proteases such as trypsin or
pepsin, either in solution or in gel after electrophoretic separation. Other
proteolytic agents are also used. The collection of peptide products are then
introduced to the mass analyzer. When the characteristic pattern of peptides is
used for the identification of the protein the method is called peptide mass
fingerprinting (PMF), if the identification is performed using the sequence data
determined in tandem MS analysis it is called de novo sequencing. These
procedures of protein analysis are also referred to as the "bottom-up" approach.

Space exploration

As a standard method for analysis, mass spectrometers have

reached other planets and moons. Two were taken to Mars by the Viking
program. In early 2005 the Cassini-Huygens mission delivered a specialized GC-MS

44
instrument aboard the Huygens probe through the atmosphere of Titan, the
largest moon of the planet Saturn. This instrument analyzed atmospheric samples
along its descent trajectory and was able to vaporize and analyze samples of
Titan's frozen, hydrocarbon covered surface once the probe had landed. These
measurements compare the abundance of isotope(s) of each particle
comparatively to earth's natural abundance.

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.

Respired gas monitor

Mass spectrometers were used in hospitals for respiratory gas

analysis beginning around 1975 through the end of the century. Some are likely
still in use but none are currently being manufactured.

Found mostly in the operating room, they were a part of a

complex system in which respired gas samples from patients undergoing
anesthesia were drawn into the instrument through a valve mechanism designed
to sequentially connect up to 32 rooms to the mass spectrometer. A computer
directed all operations of the system. The data collected from the mass
spectrometer was delivered to the individual rooms for the anesthesiologist to
use.

This magnetic sector mass spectrometer's uniqueness may

have been the fact that a plane of detectors, each purposely positioned to collect

45
the entire ion species expected to be in the samples, allowed the instrument to
simultaneously report all of the patient respired gases. Although the mass range
was limited to slightly over 120 u, fragmentation of some of the heavier
molecules negated the need for a higher detection limit.

46
 7. Conclusion
Spectroscopic techniques have been applied in virtually all
technical fields of science and technology. Radio-frequency spectroscopy of nuclei
in a magnetic field has been employed in a medical technique called magnetic
resonance imaging (MRI) to visualize the internal soft tissue of the body with
unprecedented resolution. Microwave spectroscopy was used to discover the so-
called three-degree blackbody radiation, the remnant of the big bang (i.e., the
primeval explosion) from which the universe is thought to have originated. The
internal structure of the proton and neutron and the state of the early universe
up to the first thousandth of a second of its existence is being unraveled with
spectroscopic techniques utilizing high-energy particle accelerators. The
constituents of distant stars, intergalactic molecules, and even the primordial
abundance of the elements before the formation of the first stars can be
determined by optical, radio, and X-ray spectroscopy. Optical spectroscopy is
used routinely to identify the chemical composition of matter and to determine
its physical structure.

Spectroscopic techniques are extremely sensitive. Single atoms

and even different isotopes of the same atom can be detected among 1020 or
more atoms of a different species. Trace amounts of pollutants or contaminants
are often detected most effectively by spectroscopic techniques. Certain types of
microwave, optical, and gamma-ray spectroscopy are capable of measuring
infinitesimal frequency shifts in narrow spectroscopic lines. Frequency shifts as
small as one part in 1015 of the frequency being measured can be observed with

47
ultrahigh resolution laser techniques. Because of this sensitivity, the most
accurate physical measurements have been frequency measurements.

48
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110 002.
2) A Text Book Of Organic chemistry, Arun Bahl, B.S. Bhal, S.Chand and
Company Ltd. ,New Delhi 110 055.
3) Principles of Chemistry , M.Viswanthan, jai Sai Publications ,kollam.
4) Essential Of Physical Chemistry , B.S.Bhal ,Arun Bhal ,S.Chand and company
Ltd. Ram Nagar ,new Delhi 110 055.
5) Elementary Organic Absorption Spectroscopy ,Y.R. Sharma S.Chand and
company ,New Delhi 110 055.
6) Principles of Physical chemistry , B.R.Puri ,L.R.Sharma , Vishal Publishing Co.
Jahlandhar 114 008.

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