ST.

JOSEPH COLLEGE OF ENGINEERING

DEPARTMENT OF ELECTRICAL AND ELECTRONICS ENGINEERING

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OBT751 - ANALYTICAL METHODS AND
INSTRUMENTATION

UNIT - 3
LECTURE - 5

Presented by
J.Jayashree AP/EEE
SJCE
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of 1H NMR Spectrometers
Applications of Proton NMR:
• The most important chemical applications of proton NMR
spectroscopy is the have been to the identification and structure of
organic, metal-organic, and biochemical molecules.
Identification of Compounds :
• NMR is a powerful and indispensable tool for the characterization of
pure compounds.
Application of NMR to Quantitative Analysis :
• A unique aspect of proton NMR spectra is the direct proportionality
between peak areas and the number of nuclei responsible for the
peak.
• As a result, a quantitative determination of a specific compound does
not require pure samples for calibration.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of 1H NMR Spectrometers
• Thus, if an identifiable resonance for one of the constituents of a
sample does not overlap resonances of the other constituents, the
area of this peak can be used to establish the concentration of the
species directly, provided only that the signal area per proton is
known.
• This latter quantity can be obtained conveniently from a known
concentration of an internal standard.
• For example, if the solvent present in a known amount were
benzene, cyclohexane, or water, the areas of the single-proton peak
for these compounds could be used to give the desired information.
• One of the useful applications of NMR has been in the determination
of functional groups, such as hydroxyl groups in alcohols and phenols,
aldehydes, carboxylic acids, olefins, acetylenic hydrogens, amines,
and amides.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of 1H NMR Spectrometers
• NMR spectroscopy has also been used for elemental analysis. For
example, it is possible to make accurate quantitative determinations
of total hydrogen in organic mixtures.
Disadvantages of Proton Spectrometry:
• The widespread use of NMR spectroscopy for quantitative work has
been inhibited by the cost of the instruments.
• In addition, the probability of overlapping resonances becomes
greater as the complexity of the sample increases.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
Carbon-13 NMR :
• Usually the weak NMR signals from the 13C nucleus.
• These weak signals result from the low natural abundance of the
isotope (1.1%) and the small magnetogyric ratio, which is about 0.25
that of the proton.
• These factors combine to make 13C NMR about 6000 times less
sensitive than proton NMR.
• Carbon-13 NMR has several advantages over proton NMR in terms of
its power to find out its organic and biochemical structures.
• First, 13C NMR provides information about the backbone of molecules
rather than about the periphery.
• In addition, the chemical-shift range for 13C for most organic
compounds is about 200 ppm, compared with 10 to 15 ppm for the
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proton.
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
Proton Decoupling:
• Three primary types of proton decoupling experiments are used in 13C
NMR,
broadband decoupling;
off-resonance decoupling; and
pulsed, or gated, decoupling.
Broadband Decoupling :
• Broadband decoupling is a type of heteronuclear decoupling in which
spin-spin splitting of 13C lines by 1H nuclei is avoided by irradiating the
sample with a broadband RF signal that encompasses the entire
proton spectral region, whereas the 13C spectrum is obtained in the
usual way.
• Ordinarily, the proton signal is produced by a second coil located in
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the sample probe.
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
Off-Resonance Decoupling :
• Although broadband decoupling considerably simplifies most 13C
spectra, it also removes spin-spin splitting information that may be of
importance in structural assignments.
• In this technique, the decoupling frequency is set at 1000 to 2000 Hz
above the proton spectral region, which leads to a partially
decoupled spectrum in which all but the largest spinspin shifts are
absent.
• Under this circumstance, primary carbon nuclei (bearing three
protons) yield a quartet, secondary carbons give triplets, tertiary
carbon nuclei appear as doublets, and quaternary carbons exhibit a
single line.
• Figure below demonstrates the utility of this technique for identifying
the source of the resonances in a 13C spectrum. 8
UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers

Figure : Comparison of
(a) broadband and (b)
off-resonance
decoupling in 13C
spectra of
p‑ethoxybenzaldehyde.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
Pulsed Decoupling :
• Modern NMR spectrometers provide decoupling information through the
application of complex pulsing schemes, which yield higher signal-to-
noise ratios more rapidly than off-resonance decoupling.
Nuclear Overhauser Effect :
• Under conditions of broadband decoupling, it is found that the areas of
13C peaks are enhanced by a factor that is significantly greater than would

be expected from the collapse of the multiple structures into single lines.
• This phenomenon is a manifestation of the nuclear Overhauser effect
(NOE), which is a general effect encountered in decoupling experiments.
• The enhancement arises from direct magnetic coupling between a
decoupled proton and a neighboring 13C nucleus that results in an
increase in the population of the lower energy state of the 13C nucleus
over that predicted from the Boltzmann relation.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• Although the NOE does increase the sensitivity of 13C
measurements, it has the disadvantage that the proportionality
between peak areas and number of nuclei may be lost.
Application of 13C NMR to Structure Determination :
• As with proton NMR, the most important and widespread
applications of 13C NMR are for the determination of structures of
organic and biochemical species.
• Such determinations are based largely on chemical shifts, with spin-
spin data playing a lesser role than in proton NMR.
Application of 13C NMR to Solid Samples :
• NMR spectra for solids18 is not very useful for structural studies
because of line broadening.
• Since, line broadening eliminates or obscures the characteristic
sharp individual peaks of NMR. 11
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• Much of this broadening is attributable to two causes:
 static dipolar interactions between 13C and 1H and
 anisotropy in 13C-shielding tensors.
• In isotropic liquids, these effects are averaged to zero because of the
rapid and random motion of molecules.
• In solids, heteronuclear dipolar interactions between magnetic
nuclei, such as 13C and protons, result in characteristic dipolar line
splittings, which depend on the angle between CiH bonds and the
external field.
• In an amorphous solid, there is a large number of fixed orientations
of these bonds and therefore a large number of splittings can occur.
• The broad absorption bands in this instance are made up of the
numerous lines arising from these individual dipolar interactions.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• It is possible to remove dipolar splitting from a 13C spectrum
by irradiating the sample at proton frequencies while the
spectrum is being obtained.
• This procedure, called dipolar decoupling, is similar to spin
decoupling.
• A second type of line broadening for solids is caused by
chemical-shift anisotropy.
• The broadening produced here results from changes in the
chemical shift with the orientation of the molecule or part
of the molecule with respect to an external magnetic field.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• From NMR theory, it is known that the chemical shift △ brought
about by magnetic anisotropy is given by the equation

where
 is the angle between the double bond and the applied field,
△ is the difference in magnetic susceptibilities between the parallel
and perpendicular orientation of the double bond and
R is the distance between the anisotropic functional group and the
nucleus.
When  is exactly 54.7°, △, as defined by above Equation, is zero.
Experimentally, line broadening due to chemical-shift anisotropy is
eliminated by magic angle spinning, which involves rotating solid
samples rapidly at a frequency greater than 2 kHz in a special sample
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• at an angle of 54.7° with respect to the applied field. In effect, the solid
then acts like a liquid being rotated in the field.
• One further limitation in 13C FT-NMR of solids is the long spin-lattice
relaxation time for excited 13C nuclei.
• The rate at which the sample can be pulsed depends on the relaxation
rate.
• That is, after each excitation pulse, enough time must elapse for the
nuclei to return to the equilibrium ground state.
• Unfortunately, spin-lattice relaxation times for 13 C nuclei in solids are
often several minutes, which means that several hours or even days of
signal averaging would be required to give a good spectrum.
• The problem caused by slow spin-lattice relaxation times is overcome by
cross polarization, a complicated pulsed technique that causes the
Larmor frequencies of proton nuclei and 13C nuclei to become identical
—that is, CB1C =  HB1H. 15
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.4 Applications of Carbon-13 NMR Spectrometers
• Under these conditions, the magnetic fields of the precessing proton
nuclei interact with the fields of the 13C nuclei, causing the latter to relax.
• Instruments are available commercially that incorporate dipolar
decoupling, magic angle spinning, and cross polarization, thus making
possible the acquisition of high-resolution 13C spectra from solids.
• Figure below, which shows spectra for crystalline adamantane collected
under various conditions, illustrates the power of these instruments.

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Figure : Carbon-13 spectra of
crystalline adamantane: (a)
nonspinning and with no proton
decoupling; (b) nonspinning but
with dipolar decoupling and cross
polarization; (c) with magic angle
spinning but without dipolar
decoupling or cross polarization;
and (d) with spinning, decoupling,
and cross polarization.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.5 Molecular Mass Spectrometry
• Mass spectrometry is perhaps the most widely applicable of all the
analytical tools available because the technique can provide
information about
(1) the elemental composition of samples of matter;
(2) the structures of inorganic, organic, and biological molecules;
(3) the qualitative and quantitative composition of complex mixtures;
(4) the structure and composition of solid surfaces; and
(5) isotopic ratios of atoms in samples.
Molecular Mass Spectra :
• Figure below illustrates how mass spectral data are usually presented.
• The analyte was ethyl benzene, which has a nominal molecular mass
of 106 daltons (Da).
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UNIT – 3 NMR AND MASS SPECTROMETRY
3.5 Molecular Mass Spectrometry

FIGURE : Mass spectrum of ethyl benzene.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.5 Molecular Mass Spectrometry
• To obtain this spectrum, ethyl benzene vapor was bombarded with a
stream of electrons that led to the loss of an electron by the analyte
and formation of the ion C6H5CH2CH3 .1 as shown by the reaction:

• The charged species C6H5CH2CH3 .+ is the molecular ion.

• As indicated by the dot, the molecular ion is a radical ion that has the

same molecular mass as the molecule.

• The collision between energetic electrons and analyte molecules
usually imparts enough energy to the molecules to leave them in an
excited state.
• Relaxation then often occurs by fragmentation of part of the
molecular ions to produce ions of lower masses.
• For example, a major product in the case of ethyl benzene is 20
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.5 Molecular Mass Spectrometry
• The positive ions produced in electron ionization (EI) are attracted
through the slit of a mass spectrometer where they are sorted according
to their mass-to-charge ratios and displayed in the form of a mass
spectrum.
• Note that the plot shown in Figure below is in the form of a bar graph
that relates the relative intensity of mass peaks to their mass-to-charge
ratio.
• The largest peak in a spectrum, termed the base peak, is arbitrarily
assigned a value of 100.
• The heights of the remaining peaks are then computed as a percentage
of the base-peak height.
• Modern mass spectrometers are programmed to automatically recognize
the base peak.
• They then normalize the remaining peaks in the spectrum relative to the
base peak. 21
THANK YOU
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