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Spectroscopic tools used for structural elucidation


Spectroscopy is the measurement of the absorption, emission, or scattering of

electromagnetic radiation by matter to qualitatively or quantitatively study the matter or physical

processes. The interaction of radiation with matter can cause redirection of the radiation and

absorption via transitions between the energy levels of the atoms or molecules. Spectroscopy is

widely used to study structural and dynamical aspect of molecular systems; it is a reliable tool

for the characterization of crystalline materials. Modern spectroscopic techniques such as FTIR,

FT-Raman, UV-visible, fluorescence and nuclear magnetic resonance (NMR) have proven to be

an exceptionally powerful technique for solving many drug molecules, pesticide molecules and

the natural products because of the availability of sophisticated instrumentation methods. This

chapter emphasizes the spectroscopic techniques used and the interpretation methods adopted

for systematic identification of pesticide active functional groups. Various spectroscopic

techniques used for the research work are explained briefly in this chapter. The names of

different regions along with the order of the range of frequencies and wavelength are shown in

fig. 2.1 [Donald et al. 2009].

Fig. 2.1 Electromagnetic spectrum

2.1 Infrared spectroscopy

Infrared spectroscopy is a branch of spectroscopy that deals about the vibration of atoms

in a molecule. Transition of atoms between the vibrational levels result in the vibrational spectra

which gives an insight in to the discrete motion of the atoms in the molecular system. Infrared

spectroscopy has been a workhorse technique for material analysis in the laboratory for over

seventy years. Functional groups have characteristic vibrational frequencies make the spectra as

one of the most reliable methods for understanding the structure of molecules [Sathyanarayana

1995]. IR region of electromagnetic spectrum is generally subdivided into 3 regions such as near

IR (12,500-4000 cm-1), mid IR (4000-400 cm-1) and far IR (400-50 cm-1). The mid IR is the

region most commonly employed in standard laboratory investigations, as it covers most of the

vibrational transitions. The mid IR region provides more information upon the structures of

compounds and consequently it is much used as a procedure for identifying organic compounds

for which it remains a form of functional group finger printing [Fayer & Fayer 2001; Bellamy

1980]. Because each different material is a unique combination of atoms, no two compounds

produce the exact same infrared spectrum [Gunzler & Gremlich 2002]. The main goal of IR

spectroscopic analysis is to identify the chemical functional groups in the sample. Thus, IR

spectroscopy is an important and popular tool for structural elucidation and compound

identification [Frank 1997].

2.1.1 Selection rules

In IR spectroscopy, the necessary condition for the absorption of a quantum of

radiation ‘h’ by the molecule should be equal to the energy difference between two states

represented by the wave functions ‘  i ’ and ‘  j ’. The transition between these states under the

influence of electromagnetic radiation depends on the interaction of the electric field of the

radiation with the electric dipole moment of the molecule.

According to quantum mechanical theory of a molecular system, the probability of transition

from a state ‘i’ to the state ‘j’ is proportional to the square of the transition moment.

ij   i *  j dτ (2.1)

where, ‘’ is the dipole moment of the molecule.

The dipole moment of a molecule is a function of the normal coordinates ‘Qk’ of the

vibrational mode and can be expanded in a Taylor series as:

  
   0    Qk  .....
 (2.2)
 Qk 0

On neglecting higher terms

  
 ij   0  i* j dτ     i* Qk  j d (2.3)
 Qk 0

The first term vanishes (Orthogonality condition) and the conditions for the second term to be

nonzero are:

  
(i)   must be finite at least for one component of the dipole moment. That is, for a mode

 Qk 0

of vibration to be infrared active; the vibrational motion of that mode must give rise to change in

dipole moment.

  i Qk  j d must be finite, which is possible only if the vibrational quantum

(ii) The integral

number change V   1 under harmonic approximation and for anharmonic oscillator

v   1,  2,  3...... [Colthup et al. 1990].

2.1.2 Fourier transform infrared spectrometer

In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation

is absorbed by the sample and the remaining part is transmitted. The resulting spectrum

represents the molecular absorption and transmission, creating a molecular fingerprint of the

sample. Fourier transform infrared (FTIR) spectrometry was developed in order to overcome the

limitations encountered with dispersive instruments. The main difficulty in the dispersive

instrument was the slow scanning process. A method for measuring all of the infrared

frequencies simultaneously, rather than individually, was needed. A solution was developed

which employed a very simple optical device called an interferometer. Instrumentation

Fourier-transform infrared (FTIR) spectroscopy [Griffiths & Haseth 1986] is based on

the idea of the interference of radiation between two beams to yield an interferogram, in which a

signal is produced as a function of the change in path length between the two beams. The two

domains of distance and frequency are interconvertible by the mathematical method of Fourier-

transformation. The basic components of FTIR spectrometer are shown in fig. 2.2. The radiation

emerging from the source is passed through an interferometer to the sample before reaching a

detector. Upon amplification of the signal, in which high-frequency contributions have been

eliminated by a filter, the data are converted to digital form by an analog-to-digital converter

and transferred to the computer for Fourier-transformation. Source

In the mid IR several type of sources are used. They are either a lamp filament, or a

hollow rod, 1-3 mm in diameter and 2-4 cm long, made of fused mixtures of zirconium oxide or

rare earth oxides (Nernst source) heated by Joule effect by means of an internal resistor

(Globar). These sources are heated to 15000 C, without a protective shield. They dissipate power

of the order of a hundred watts by emitting radiation over a large domain ranging from visible to

thermal IR. Sample

For solid samples pellet technique is used. In Pellet technique first setup is to grind the

sample very finely with potassium bromide. The mixture is then pressed into transparent

pellets with the help of suitable dye. This is then placed in the IR beam in a suitable sample

holder [Silverstein et al. 2005]. Interferometer

The most common interferometer used in FTIR spectrometry is a Michelson

interferometer, which consists of two perpendicularly arranged plane mirrors, one of which can

travel in a direction perpendicular to the plane. A beam splitter (semi-reflecting film) bisects the

planes of these two mirrors. The beam splitter material has to be chosen according to the region

to be examined. Materials such as germanium or iron oxide are coated onto an ‘infrared-

transparent’ substrate such as potassium bromide or caesium iodide to produce beam splitters for

the mid-infrared regions. If a collimated beam of monochromatic radiation of wavelength (cm)

is passed into an ideal beam splitter, 50% of the incident radiation will be reflected to one of the

mirrors, while 50% will be transmitted to the other mirror. The two beams are reflected from

these mirrors, returning to the beam splitter, where they recombine and interfere. Fifty percent

of the beam reflected from the fixed mirror is transmitted through the beam splitter, while 50%

is reflected back in the direction of the source. The beam which emerges from the interferometer

at 90◦ to the input beam is called the transmitted beam, and this is the beam detected in FTIR

spectrometry. The moving mirror produces an optical path difference between the two arms of

the interferometer. For path differences of (n + 1/β) , the two beams interfere destructively in

the case of the transmitted beam and constructively in the case of the reflected beam. The

moving mirror is a crucial component of the interferometer. The beam enters the interferometer

where the “spectral encoding” takes place. The resulting interferogram signal then exits the

interferometer. Detector

There are two commonly used detectors employed for the mid-infrared region. The normal

detector for routine use is a pyroelectric device incorporating deuterium tryglycine sulfate

(DTGS) in a temperature-resistant alkali halide window. For more sensitive work, mercury

cadmium telluride (MCT) can be used, but this has to be cooled to liquid nitrogen temperature.

A detector must have adequate sensitivity to the radiation arriving from the sample and

monochromator over the entire spectral region required.

Fig. 2.2 Diagram of FTIR spectrometer

2.2 Raman spectroscopy


An electromagnetic radiation when passed through a transparent medium interacts with

the particles (e.g., molecules, atoms, or ions) constituting it. If the dimensions of the particles

are equal to or smaller than that of its wavelength then it undergoes scattering. Raman

spectroscopy is based on inelastic scattering of light by matter. It has been observed that most of

the scattered radiation has exactly the same wavelength as that of the incident radiation. Such a

scattering is referred to as Rayleigh scattering. However, a very small fraction (to the tune of

about 1 in 107) of the scattered radiation is found to have a wavelength different from that of the

incident radiation. This is called Raman scattering and the existence of Raman scattering is

called Raman effect. The intense peak in the centre of the spectrum having the same wavelength

(or wavenumber) as that of the incident radiation is called the Rayleigh peak and the other

signals on either side of the Rayleigh peak are the Raman lines. The lines to the left of Rayleigh

peak and having lower wavenumber (energy) are called Stokes lines, while the other on the right

having higher wavenumber (energy) are called anti-Stokes lines [Jacek 2005].

2.2.1 Classical theory of Raman Effect

According to Classical Theory of Raman Scattering, molecules placed in an electric field

under go polarization. The polarization ‘P’ so induced is proportional to the applied electric field


P = E (2.4)

where, ‘’ is the polarizability.

According to classical theory, when radiation of frequency ‘  0 ’ is allowed to fall on

molecules, each molecule experiences a varying electric field. This field depends on the incident

frequency given by:

E = E0 cos 2  0 t (2.5)

where, ‘E0’ is the vibrational amplitude.

Consider the vibrational motion of the molecule. If ‘q’ is the normal co-ordinate

associated with a particular mode of vibrational frequency ‘m’ of the molecule, the harmonic

approximation of ‘q’ can be written asμ

q  q0 cos 2 mt (2.6)

where, ‘ q0 ’ is the amplitude of vibration.

For small vibrational amplitudes, the polarizability ‘  ’ can be expanded using Taylor series


  
   0    q  ... (2.7)
 q 0

  
where, ‘  0 ’ is the polarizability at the equilibrium position. The term   is the rate of
 q  0

change of ‘  ’ with respect to ‘q’, evaluated at the equilibrium position. The induced dipole

moment is therefore:

1   
P =  0 E0 cos 2 0t    q0 E0 cos 2  0   m  t  cos 2  0   m  t  (2.8)
2  q 0

The first term represents Rayleigh scattering and the second term represents anti-Stokes

 0   m  and Stokes  0   m  lines of Raman scattering. For Raman active vibrations, the

rate of change of polarizability should be non zero,

  
i.e.    0 . (2.9)
 q  0

Thus a molecular vibration will be Raman active only if it causes a change in a

component of polarizability either in magnitude or in direction. Though the classical theory

correctly describes the frequencies of the Raman lines  0   m , it fails to predict correct

intensities. Quantum mechanical theory is therefore introduced to predict the intensities of

Raman lines [Aruldhas 2006].

2.2.2 Quantum theory of Raman effect

In explaining Raman scattering by the quantum theory, a sample is irradiated by an

intense source (usually a laser) in the UV-visible or IR region and the scattered light is

generally observed in a direction perpendicular to the incident beam. The scattered light consists

of two types. One is elastic Raleigh scattering, which is strong and has the same frequency as

incident beam (ν0). The other one is inelastic Raman scattering and is weak, approximately

105 times weaker than the incident beam, and has frequencies of (ν0νm) where νm is the

vibrational frequency of a given molecule. In Raman spectroscopy νm is measured as a shift

from the incident frequency (ν0) [Ferraro & Nakamoto 1994]. Stokes lines are those represented

by 0m shift while the anti-Stokes lines are those represented by 0m shift, the process

of Raman scattering can be explained using electromagnetic theory. In energy conservation

terms, for an incident photon of wavenumber (cm-1), the inelastic scattering processes are

represented by the following equations,

hc0 = hc (0m)  hcm, Stokes line (2.10)

hc0 hcm  hc (0m), anti Stokes line (2.11)

where, h is Planck’s constant, c is the velocity of light, 0 is expressed as a wavenumber (cm-1)

of the incident radiation, and νm is the wavenumber equivalent to the energy difference between

the lowest and first excited vibrational energy levels (fig. 2.3).

Fig. 2.3 Schematic representation of Raman lines

2.2.3 Fourier transform Raman spectrometer

Raman spectrometers basically employ one of the two techniques either dispersion or

Fourier Transform (FT) for the collection of spectral data. Dispersive Raman spectrometers

produce interfacing fluorescence due to incorporation of impurities with the samples.

Furthermore, the cost of this type of equipments and its maintenance are quite high. So FT-

Raman spectrometer replaces the dispersive Raman without producing sample fluorescence.

Sample is irradiated with longer wavelength excitation laser, so the virtual state is lower and it

will overlap with an upper electronic state. This greatly reduces fluorescence interferences and

therefore Raman signals are free from distortions. Conventional Raman spectroscopy measures

intensity versus frequency. But, FT-Raman is a time domain spectroscopy which measures the

intensity of light of many frequencies simultaneously. This spectrum is then converted to

conventional spectrum by applying Fourier transform. The FT-Raman spectrometer is shown in

fig. 2.4. The three basic spectrometer components in an FT system are source, interferometer

and detector [Douglas et al. 1998; Hobart et al. 1986]. Source

With the advent of lasers with highly desirable properties of high intensity, single

wavelength and coherence, the modern spectrometers almost exclusively use laser sources. The

intensity of Raman scattering is dependent on the fourth power of the frequency of the exciting

radiation, besides concentration and other factors. The argon and krypton ion laser sources are

more intense than the other lasers being used at the same power and accordingly give better

spectra. On the other hand the low frequency lasers i.e., diode laser and Nd/YAG laser can be

used at higher power and also have an added advantage that these do not generate the interfering

fluorescence spectral lines. Sample

A solid sample is ground to a fine powder and packed into a small cavity to be kept in the

path of incident radiation. When the sample is placed in the radiation path, the incident laser

beam enters the sample through the window and undergoes multiple reflections in the sample.

The scattered light at right angles to the sample is then suitably collected and measured. Detector

In initial versions of the spectrometers, photographic plates were used to detect the

scattered radiation. This was followed by more sensitive photomultiplier tubes (PMTs) that

allowed electronic data collection and manipulation. However they had the disadvantage for

they could count only one wavelength at a time. Today most of the Raman spectrometers are FT

instruments which use cooled germanium photoconductors as transducers. As the scattered

radiation has a major component of the Rayleigh scattering, this is eliminated by using a suitable
filtering device like holographic grating or interference filters before the detector. Therefore, the

radiation reaching the transducer is primarily the Stokes component.

Detector detects the interferogram signals and fed to the computer. It is the time domain

spectrum and records the detector response changes versus time within the mirror scan. If the

sample happens to absorb the signal at this frequency, the amplitude of the sinusoidal wave is

reduced by an amount proportional to the amount of sample in the beam. The interferogram

contains the information over the entire Raman region to which detector is responsive. The

required Raman shift is obtained after calibrating the original data with respect to the known

wavenumber relationship. The plot represents the wavenumber and the relative scattering

intensity in photon counts.

Fig. 2.4 Basic diagram of an FT-Raman spectrometer

2.3 Mutual exclusion principle

The IR and Raman activities of different modes of vibration of a molecule depend on its

symmetry properties. An analysis of the IR and Raman spectra of a large number of molecules

has lead to an extremely important general rule known as the rule of mutual exclusion. It states
that, “for a molecule having a centre of symmetry the Raman active vibrations are IR inactive

and vice versa”. However, if the molecule does not have a centre of symmetry then some

vibration may be Raman as well as IR active.

2.4 Electronic spectroscopy


Electronic spectroscopy is used to study the transition of electrons between different

electronic states which fall in the visible and ultraviolet regions of the electromagnetic spectrum

[Francis & Annick 2007]. UV-Visible region of the spectrum is conventionally divided into

three sub-domains termed as near UV (185-400 nm), visible (400-700 nm) and very near

infrared (700-1100 nm). Most commercial spectrophotometers cover the spectral range of 185 to

900 nm. The origin of absorption in this domain is the interaction of photons from a source with

ions or molecules of the sample. When a molecule absorbs a photon from the UV-Vis region,

the corresponding energy is captured by one (or several) of its outermost electrons. UV-Vis

spectrometers collate the data over the required range and generate the spectrum of the

compound under analysis as a graph representing the transmittance (or the absorbance) as a

function of wavelength along the abscissa, given in nanometres. In UV-visible spectroscopy, the

transmittance T is a measure of the attenuation of a beam of monochromatic light based upon

the comparison between the intensities of the transmitted light (I) and the incident light (I0)

according to whether the sample is placed, or not, in the optical pathway between the source and

the detector.

T is expressed as a fraction or a percentage: T = I / I0

The absorbance (optical density) is defined by: A= - log T

2.4.1 Instrumentation

An UV-Vis spectrophotometer consists of source, dispersive system (combined in a

monochomator), and detection system as shown in fig. 2.5. More than one type of source can be

used in the same instrument, which automatically swaps lamps when scanning between the UV

and visible region. For the visible region of the spectrum, an incandescent lamp fitted with a

tungsten filament housed in a silica glass and for the UV region a deuterium arc lamp working

under a slight pressure to maintain an emission continuum (< 350nm) and alternatively, for the

entire region 200 to 1100 nm, a xenon arc lamp can be used for routine apparatus.

Fig. 2.5 Basic diagram of an UV-Vis spectrometer

The light emitted by the source is dispersed through either a planar or concave grating,

which forms part of a monochromator assembly. This device permits the extraction of a narrow

interval of the emission spectrum. The wavelength or more precisely the width of the spectral

band, which is a function of the slit width, can be varied gradually by pivoting the grating.

Optical paths with long focal lengths (0.2 to 0.5 m) yield the best resolution. A sample

compartment is inserted into the optical path either before or after the dispersive system

depending upon the design of the instrument.

The detector converts the intensity of the light reaching it to an electrical signal. It is by

nature a single channel device. Two types of detector are used, either a photomultiplier tube or a

semiconductor. For both of which, the sensitivity depends upon the wavelength. For

‘simultaneous’ instruments which do not possess a monochromator but possess a dispersive

system, the light intensity at all wavelengths is measured practically at the same instant by

aligning a large number of detectors in the form of a diode array.

2.5 Fluorescence spectroscopy


Some organic or inorganic compounds, liquids or solids (molecular or ionic crystals),

whether pure or in solution, emit light when they are excited by photons from the visible or the

near ultra-violet regions. Among the analytical applications of this phenomenon, known as

photoluminescence, is fluorimetry, a selective and highly sensitive method for which a wide

range of measurements are accessible. The intensity of the fluorescence is proportional to the

concentration of the analyte and the measurements are made with the aid of spectrofluorometers.

The extremely rapid extinction of the light intensity when excitation ceases is the object of

analysis. By contrast, phosphorescence is characterized by a more gradual diminution during

time. Many compounds, when excited by a light source in the visible or near ultraviolet region

absorb energy which is nearly instantaneously re-emitted in the form of radiation. According to

Stokes’ law, the maximum of the spectral emission band is located at a longer wavelength than

that of the original excitation light. These are fluorescent compounds [David & Churchill 1999].

In addition to fluorescence (the desired decay route), there are nonradiative decay processes,

leading to release of energy in the form of heat rather than light. Other sample constituents may

interact with an excited analyte molecule in such a way as to prevent it from fluorescing; such

processes are called quenching.

2.5.1 Instrumentation

A fluorescence spectrometer consists of source, wavelength selector, sample illumination,

sample and detector. A shimadzu F-4500 fluorescence spectrometer is shown in fig. 2.6. In

addition to the optical components shown, most fluorometers have dedicated computers, which

control instrumental operating parameters and the acquisition of spectral data, and also may be

used for post processing of the data.

Fig. 2.6 Schematic diagram of a fluorescence spectrometer Source

The signal produced by an analyte is proportional to the number of excited analyte

molecules formed per unit time. Thus, the source must produce high optical power. Because

molecular absorption spectra usually are broad, a highly monochromatic source is not generally

needed; an intense continuum source that emits throughout the UV, visible and near infrared

regions is adequate. The source used in most commercial fluorometers is the xenon arc lamp

[Phillips 1983; Hofstraat et al. 1993; Brina & Miller 1992]. Wavelength Selector

Portable, inexpensive fluorescence spectrometers use filters as wavelength selectors.

Such instruments are used when it is sufficient to measure fluorescence intensity at a single

excitation and emission wavelength [Alarie et al. 1993; Brennan et al. 1993]. Filters can

transmit a very large number of photons from source to sample and from sample to detector.

Most fluorometers in laboratory environments use grating monochromators as excitation and

emission wavelength selectors [Parker 1968].

5.1.3 Sample Illumination

The most common arrangement is the right-angle geometry, where in fluorescence is

viewed at a 90° angle relative to the direction of the exciting light beam. This geometry is
suitable for weakly absorbing solution samples. For solutions that absorb strongly at the

excitation wavelength, and for solids, a front surface geometry is preferable. For solution

samples, rectangular 1cm glass or fused silica cuvettes with four optical windows are usually

used [Harris 1982]. Detector

The fluorescence signal for an analyte present at low concentration is small; thus, a key

requirement for a detector is its ability to detect weak optical signals. A photomultiplier tube

(PMT) is used as the detector in most fluorescence spectrometers. PMTs used in fluorometry are

chosen for low noise and high sensitivity, and are sometimes operated at subambient

temperatures to improve their signal-to-noise ratios. Working

Spectrofluorometers have software which can determine automatically the excitation-

emission pair to get the best results. Knowing the UV-Vis spectrum of the compound, the

procedure can be decomposed in the following steps such as the excitation monochromator is set

to the value corresponding to the maximum of the UV absorption spectrum, the fluorescence

spectrum is recorded and the emission monochromator is set to the wavelength of maximum

fluorescence and the excitation wavelength is varied. The excitation spectrum is obtained

allowing the best final choice of the excitation radiation [Francis & Annick 2007].

2.6 Nuclear magnetic resonance spectroscopy


NMR Nuclear magnetic resonance (NMR) spectroscopy is a powerful and

theoretically complex analytical tool that allows the study of compounds in either solution or in

the solid state and serves equally in quantitative as in structural analysis, it is very efficient in

gathering structural information concerning molecular compounds. NMR spectroscopy is one of

the methods of spectroscopic technique in which under appropriate conditions in a magnetic

field, a sample can absorb electromagnetic radiation in the radio frequency (r.f) region at
frequencies governed by the characteristics of the sample [Francis & Annick 2007; David &

Churchill 1999.]

All nuclei carry a charge, in some nuclei this charge "spins" on the nuclear axis, and this

circulation of nuclear charge generates a magnetic dipole along the axis. The angular

momentum of the spinning charge can be described in terms of quantum spin numbers I: these

numbers have values of 0,1/2,1,3/2 and so on (I=0 denotes no spin). The intrinsic magnitude of

the generated dipole is expressed in terms of nuclear magnetic moment, . The fundamental

NMR equation correlating the applied radio frequency radiation (  1 ) with the magnetic field

strength ( B0 ) is

1  B0 (2.12)
where  is called magnetogyric ratio.

A-Sample container, B-Transmitter coil, C-Sweep generator coils, D-Receiver coil, E- Magnet

Fig. 2.7 Continuous wave NMR spectrometer

2.6.1 Instrumentation

The basic layout of a continuous wave NMR spectrometer is shown in fig. 2.7. NMR

spectrometer consists of Sample container, Transmitter coil, Sweep generator coils, Receiver

coil, Magnet, detector and recorder. These instruments were the original type of instrument and

have largely been replaced by Fourier transform instruments.

An electromagnet is one which gives a powerful, stable and homogeneous magnetic field

[Herald 1994]. The field must be constant over the area of the sample and over the period of

time of the experiment. A sweep generator supplies a variable current to a secondary magnet so

that the total applied magnetic field can be varied over a small range. The sample container is

usally a glass tube (5 mm OD) spun by an air-driven turbine to average the magnetic field over

the sample container. The process is often referred to as the spinning of the sample. A radio

frequency oscillator is connected to a coil, called the transmitter coil, which transmits the energy

to the sample. The axis of the coil has to be perpendicular to the magnetic field. A radio

frequency receiver is connected to a coil, called the receiver coil, which encircles the sample. Its

axis has to be perpendicular to both the magnetic field and the axis of the transmitter coil. The

transmitter and receiver coils and the sample holder are constructed in to a single unit called

probe. In addition to the major components there is a read out system consisting of an radio

frequency amplifier, recorder and other accessories to increase the sensitivity, resolution and


The sample is placed in the sample container and is spun in the fixed magnetic field at ca

30 revolutions/s by means of an air turbine thus ensuring uniformity of the magnetic field across

the sample in a horizontal direction. The sample is analysed in a deuterated solvent to ensure

there is no interference from protons in the relatively much larger amount of solvent with the

signal from the sample protons. The reference point of 0 parts per million (ppm) is determined

by the frequency at which the protons in tetramethyl silane (TMS) absorb. Sometimes residual
protons in the solvent are used to lock the protons in a spectrum, e.g. the residual proton in

deuterated chloroform is at 7.25 ppm relative to TMS. The receiver coil measures the absorption

of radiation as the frequency is swept over the range being examined. As well as determining the

frequency at which protons in the molecule absorb, the instrument determines the area of each

signal, which is proportional to the number of protons absorbing radiation. Modern instruments,

rather than being based on a continuous wave, are based on a pulsed wave. The data in the form

of pulses are collected in the computer and the mathematical manipulations (Fourier

transformation) are carried out. Then it is combined to produce a spectrum in which the signal to

noise characteristics are much improved.

2.6.2 Chemical shift

Proton (1H) NMR is the most commonly used form of NMR because of its sensitivity

and the large amount of structural information it yields. The exact absorption or resonance

frequency of a proton depends on its environment. For example, a proton attached to carbon

atom is affected predominantly by the groups which are separated from the carbon atom to

which it is attached by one bond or to a lesser extent two bonds. As discussed earlier the

chemical shift of a proton is determined in relation to the protons of tetramethylsilane, which are

arbitrarily assigned a shift of 0 ppm. Shift values for individual protons in a molecule are

expressed in ppm and the value of 1 ppm in Hertz depends on the strength of the applied

magnetic field which determines the energy required to excite a proton. The chemical shift is

determined by the extent to which a proton is deshielded by the group to which it is attached.

The more a proton is shielded by the electron density around it, the lower its chemical shift


2.7 Electron displacement effects which affect the spectral nature of compounds

2.7.1 Hybridization

Hybridization is the process of change from atomic orbitals to bonding orbitals.

Hybridization is one of the most important concepts of quantum chemistry, since it provides a
basis for correlating many physical properties of molecules with electronic structure. This

concept was originally introduced by Pauling 1931, who suggested that a mixture of s and p

atomic orbitals would provide a better function for bond formation than either an s or p orbital

alone. The bond is described by a pair of electrons in atomic orbitals, one on each of the two

centers involved. Thus, hybridization is a valence bond concept. When a carbon atom bonds to

other atoms, the four orbitals in the second shell are somehow mixed together and rearranged to

give four new orbitals of equal energy. These four new orbitals are called hybrid orbitals. Each

of the four hybrid orbitals is equivalent to the others and each contains one electron. These

hybrid orbitals resulted from the combination of a s orbital and three p orbitals are called sp 3

hybrid orbitals or simply sp3 orbitals [Bernard et al. 1977]. There are three types of


(i) sp3 hybridization (involved in saturated organic compounds containing only single

covalent bonds),

(ii) sp2 hybridization (involved in organic compounds having carbon atoms linked by

double bonds) and

(iii) sp hybridization (involved in organic compounds having carbon atoms linked by

triple bonds).

2.7.2 Back donation

When the orbital of a bond is in the trans position with the lone pair of the adjacent

electronegative atom, the electronic charge is back donated from the lone pair to the * orbital.

This will increase the electronic charge in the orbital, and make the bond weaker, decreases the

stretching force constant of the bond, and increases the bond distance and the stretching

vibrational intensity.

2.7.3 Induction

The presence of an electron attracting constituents attached to the carbon atom in the

molecule causes the shifting of bonding electron pair, inducing polarity in the carbon atom as

well as on the substituent [Gussoni & Castiglioni 2000]. The electron attracting substituent

develops negative charge in it and produces negative inductive effect whereas the electron

releasing subsituent causes positive inductive effect. In C-H bond, stronger polarization occurs

due to induction in molecules and the presence of electronegative atom. This effect reduces with

the distance of C-H bond from the electronegative atom. Stronger polarization makes the charge

on hydrogen atom to increase. The C-H stretching force constant increases whereas the C-H

bond distance and C-H stretching bond intensity decreases.

2.7.4 Conjugation

The conjugation effect operates on systems with conjugate -bonds, alternate - and -

bonds, in which the electron displacement is relayed through the -electron system. The

vibrational spectral influence of conjugation differs for various systems. For an aliphatic

conjugation of carbon-carbon double bonds, the splitting of C=C stretching band is resulted

[Bellamy 1980]. The conjugation of the carbonyl group causes appreciable shift in C=O

stretching band position and hence most of the aryl ketones produce C=O absorption band in the

lower wavenumber range, 1680-1690 cm-1. Unsaturated aldehydes containing double bond in

the  or -positions show a fall in the carbonyl frequency due to conjugation. Aromatic ring in

conjugation with the aldehyde groups also show marked effect on band position and C=O

absorption occurs in the range 1710-1695 cm-1.

2.7.5 Hyperconjugation

It is the interaction of orbital of a single bond with the -orbital of an adjacent double /

triple bond. In C-H bonds, the hyperconjugation causes the releasing of electronic charge from a
CH to the C-C bond [Gussoni & Castiglioni 2000]. As a result, C-C bond length decreases and

the bond strength increase. Due to the release of electronic charge from CH, the hydrogen

becomes more acidic due to increase of charge on hydrogen. This will increases the C-H bond

ionicity and decreases the C-H bond strength. But due to the decrease of electronic charge in

CH bonding orbital, there should be decrease in C-H bond strength. Due to suitable balance

between these two opposing effects, the bond length and spectral characteristics of C-H bonds of

the hyperconjugated systems do not show direct correlation with the charge on hydrogen.

2.7.6 Intramolecular charge transfer (ICT)

The compounds having the electron donor and electron acceptor at the end positions of the

π-conjugated system is related to the existence of large intramolecular charge transfer

responsible for pesticide activity. The ICT causes the electron releasing effect in the acceptor

moiety, affecting the spectral modes. C=C stretching mode of acceptor subunit occurs at lower

wavenumber compared to the corresponding mode of the donor subunit [Ruiz et al. 2003]. For

the conjugated path, ICT induces large variation in the dipole moment as well as in the

molecular polarizability simultaneously during vibration. This produces the IR and Raman

activity for the same mode and hence comparable intensities of IR and Raman bands arise from

the vibrations of conjugated system. The electron donating effect of donor unit also causes

wavenumber shifting in the vibrations of the donor group. The existence of a large ICT at

relatively low energy values depends on the efficiency of the π-electron delocalization through

the bridge and of the electron donor and the electron-acceptor strength of the end groups.

2.7.7 Hydrogen bonding

Hydrogen bond formation is an important phenomenon that determines essential

properties of many chemical and biological systems. The H-bond plays a key role in chemistry,

physics, and biology, and its consequences, such as the properties of solid, liquid and gas. The
importance of H-bonds is enormous. They are responsible [Scheiner 1997] for the structure and

properties of water, an essential compound for life, as a solvent and in its various phases.

Further, H-bonds also play a key role in determining the shapes, properties, and functions of

biomolecules. This is the basis for several spectroscopic, structural, and thermodynamic

techniques. The characteristic features of X-H∙ ∙ ∙Y-H bond are as follows: (i) the X-H covalent

bond stretches in correlation with the strength of the H-bond; (ii) a small amount of electron

density (0.01-0.03 e) is transferred from the proton-acceptor (Y) to the proton-donor molecule

(X-H); (iii) the band which corresponds to the X-H stretch shifts to lower frequency (red shift),

increases in intensity, and broadens. The value of the red shift and the strength of the H-bond are

correlated. Frequency shifts correlate with various characteristics of the H-bonded system

[Morokuma et al. 1981; Kollman 1981; Singh & Kollman 1984; Silverstein 2005].

Intramolecular hydrogen bonds are formed when the proton donor and acceptor are

present in a single molecule under spatial conditions that allow the required overlap of orbitals,

for example, the formation of a five-or six-membered ring. Intramolecular bonding involving

the same bonding groups is stronger when a six-membered ring is formed than when a smaller

ring results from bonding. Intramolecular hydrogen bonds are formed in all three phases, but in

crystalline state, their strength competes with that of intermolecular hydrogen bonds. The

carbohydrates are rich in intramolecular hydrogen bonding due to the presence of hydroxyl

groups attached to the adjacent carbon atoms. The intramolecular hydrogen bonding is an

internal effect and persists at very low concentrations. When the A-(H)…B distance is very

short, i.e. O…O  2.5Å and N…N  2.6 Å, the strong hydrogen bonds are formed. When the

distances are between 2.5 and 3.2 Å, normal hydrogen bonds may be formed. [George 1997].

Intermolecular hydrogen bonding interaction plays an important part in determining the

structures and activities of organic, organometallic and biological molecules [Takhashi et al.

2003; Reynisson & Steenken 2003]. Intermolecular hydrogen bonding involves association of

two or more molecules of the same or different compounds.