Advance Analytical Course

Prof. Padma Vankar

Department of Chemistry
Indian Institute of Technology, Kanpur

Lecture No. # 26

Introduction to spectroscopy – spectroscopy is a very vast area, but proper understanding

of the electromagnetic radiation and the spectrum of race belonging to the
electromagnetic radiation is very important. Why, because each area of electromagnetic
radiation has to contribute something to molecular structural determination. And, that is
the advantage of using various regions of UV region, visible region, IR region, radio
frequency region for different types molecular structural detailing. And, it is the beauty
that each one contributes only a part of information about the molecule. So, let us try to
understand what spectroscopy has to offer.

(Refer Slide Time: 01:15)

We have been referring to very important two techniques in analytical chemistry: one is
the chromatographic technique and the other one is the spectroscopic technique. The
chromatographic technique is responsible for the separation of complex mixtures, so that
individual compounds can then be analyzed on spectroscopic methods.
(Refer Slide Time: 01:47)

If we try to look at the various forms of energy and particularly with respect to light, it
can be explained by wave theory or by particle theory – 2-dimensional form of
electromagnetic radiation; plane polar electromagnetic radiation travelling along an x-
axis. So, it is debatable whether the energy or the light is in form of a particle or a wave.
Nevertheless, we still consider it as a wave. The wave is characterized in three ways as
shown in the above figure. It can be described as a wavelength, that is, the difference
between the two humps; or, it is described as wave number, that is, the number of waves
in unit length or distance for cycle – reciprocal of wavelength. So, if we say wavelength,
then it is the reciprocal of wave number; or, it can be expressed in terms of frequency nu
– is the number of collisions of the field per second; that is expressed in hertz. It can also
be expressed in terms of velocity, c – independent of wavelength – in vacuum, the
velocity of light is considered as 3 into 10 to the power of 10 centimeters per second; or,
it can again be called as photon or quanta. Quantum mechanics nature of light to explain
the photoelectric effect is where the photon term is used. Most popularly, we talk about
the wavelength or the frequency. The wave number, the velocities are not so frequently
used when we are referring to different radiations of electromagnetic radiation.
(Refer Slide Time: 03:52)

Now, if you try to look at this particular slide, you will see that the areas are very clearly
demarked – the X ray area, the gamma ray area, the UV area. And, you will see a
very small visible region, which has been expanded at the bottom. And then, next
to the visible region is the IR region. Then, comes the microwave region; then, comes
the FM radio waves and the AM radio waves. And furthermore, are long radio waves,
which are responsible for the NMR analysis. So, you see that the entire spectrum, as I
mentioned a little while ago, different regions and their energy or their wavelength are
having certain properties, which can excite only some part of a molecular structure, and
therefore, imparts information only about that part. So, X rays will give inner bonding
and crystal lattice structures information; UV will give information related to
unsaturation in the molecule; IR will give information related to the functional groups
that are present in a molecule, and so on and so forth.
(Refer Slide Time: 05:17)

Now, what is meant by term spectroscopy and what is a spectrum? It should be clearly
understood. Spectroscopy is a technique that uses the interaction of energy with a sample
to perform an analysis. So, the technique is called spectroscopy. And, the data that is
obtained from the spectroscopy is called a spectrum. A spectrum is a plot of intensity of
energy detected versus the wavelength or mass or momentum or frequency of the energy.
So, spectrum is the graph that it is obtained out of this spectroscopic analysis.

(Refer Slide Time: 06:05)

Now, this is a very interesting slide, where I want to draw your attention to see how the
interaction of electromagnetic radiation can really affect our day-to-day life and our
molecular structures, and the molecules that we deal within day-to-day life. And, it is
measured by the characteristics, and the properties, and the quantity. Radio waves are
responsible for FM; radio waves are also responsible for NMR. Microwaves are
responsible for different types of digestion processes that I had covered a while ago; and,
they are also used in domestic purposes for baking cakes. Infrared is a region, where we
get functional, the thermometers; and, different types of infrared heating devices are
made out of it. Visible region is what we can see. Ultraviolet is related to sunlight. X
rays are related to the X rays that are taken for broken bones or even intact bones. And,
gamma rays of course, we know, are used in different other purposes. So, this was just to
give you an overall idea – how spectroscopy even plays a very important role in our
domestic life.

(Refer Slide Time: 07:34)

Now, when we try to look at the spectroscopy and the relationship between them, the
wavelength is directly proportional to the frequency. And, when we try to find out the
sign of proportionality, it is always replaced by a constant. And, here c is the velocity of
light. So, lambda, which is the wavelength, is equal to c by v. And, the units of
wavelength are normally angstrom, which is 10 to the power of minus 10 meters; or, 10
to the power of minus 9 if it is nanometer; if it is micrometer, it is 10 to the power of
minus 6 meters. So, these are certain relationships, which I wanted to make it very clear
to you, because time and again, we will be referring to angstrom or nanometers, and one
should know where does it stand in a meter scale.

(Refer Slide Time: 08:37)

Now, light energy could have a longer wavelength, but that longer wavelength light will
have lower energy; or, it may have shorter wavelength and light will have higher energy.

(Refer Slide Time: 08:47)

The De-Broglie relationship link between particle and wave behavior should also be
understood, because whether it is a particle or a wave, it must be understood very clearly.
E is equal to h nu; where, h is Planck’s constant and it also a sign of proportionality; or,
when we can say that nu is c by lambda. So, it can be substituted that E is equal to h c by
lambda. So, these are different relationships of energy with Planck’s constant with
velocity of light and wavelength and frequency. So, how they are interrelated must be
understood.

(Refer Slide Time: 09:33)

The De-Broglie relation with E is equal to m c square is also very well-known.

Combining with the previous equation, that is, c is equal to m c square, that will be equal
to h c by lambda; or, that gives that lambda is equal to h by m c. So, these are different
types of energy, mass, and wavelength, and velocity of light, Planck’s constant. How
they are connected to each other – De-Broglie relationship gives this equation very
clearly.
(Refer Slide Time: 10:12)

Now, when we look at the different regions of electromagnetic spectrum, we see that
radio frequencies are responsible for the NMRs. Similarly, the microwave is responsible
for the rotational power of a bond. Then, comes the far infrared, infrared and near
infrared. They are responsible for causing vibration in a bond. And similarly, the visible,
and the ultraviolet, and the vacuum ultraviolet are responsible for the electronic
excitation; that is, pi to pi star or n to pi star excitation of electrons are brought about by
these particular electromagnetic radiation areas. Then, comes the X ray, which excites
the inner shell electrons. And then, comes the gamma rays, which cause nuclear reaction
and give out alpha, beta, gamma rays. So, that is how the regions of electromagnetic
spectrum are divided and each area contributes to its particular functioning.
(Refer Slide Time: 11:29)

Now, when we try to look an electron distribution in an orbital, the sigma and the sigma
star, and the pi and the pi star orbitals look like this.

(Refer Slide Time: 11:37)

Molecules – when they are radiated, what exactly happens? When molecule is excited
with h nu – h nu is an energy source, what exactly happens to that? It forms M dot. That
means it is in an excited state; the mass has now become excited. Change in nuclear
energy level X rays is very high. Change in nuclear spin state lower energy and causes
magnetic field. Radiation excited species have a life time of 10 to the power of minus 8
to the 10 to the power of minus 9 seconds. So, what it happens that this excited molecule
either gives then the normal molecule and heat by collision with the neighbors or it may
give h nu by emission, highly probable at high frequency. So, either it can give some
heat and come back to its normal state or the excited molecule, that is, the M star will
emit some energy.

(Refer Slide Time: 12:51)

Now, these are certain molecular electronic transitions that are permitted. You will see
that from the transition of pi to pi star, it is the most fissile energy exchange that can
occur; or, n to pi star. But, n to sigma star or sigma to sigma star requires the highest
energy. So, depending on what kind of UV light is shown on the molecule, the most
fissile exchange or the most fissile excitation would be pi to pi star; whereas, the next
fissile exchange would be n to pi star. However, n to sigma star or sigma to sigma star
requires still higher energy.
(Refer Slide Time: 13:43)

Now, energy transitions, that is, sigma to sigma star – observed only in highest energy,
that is, vacuum ultraviolet. Rarely, never observed in the normal UV and visible range.
Whereas, n to sigma star – saturated compounds containing atoms with unshared electron
pairs, that is, nonbonding electrons capable of this transition. This transition requires less
energy – happens in 150 to 250 nanometer range and the absorption below – mostly 200
nanometer is what is observed. The molar absorptivity are fairly low; that is, 100 to 3000
liters per centimeter for mole. So, this is why these two transitions – energy transitions
are not so popular, when we are talking about electronic excitations.

(Refer Slide Time: 14:49)

Adsorption maxima of certain solvents are given here. You will see some examples
adsorption due to n to sigma star transition, has been shown here. And, water
particularly, which adsorbs at 167 nanometers having lambda max of 167, shows a molar
adsorptivity – 1480; whereas, methanol shows lambda max at 184 and molar adsorptivity
at 150. Similarly, methylene chloride shows lambda max at 173 and has a molar
adsorptivity at 200. Iodomethane shows lambda max at 258 and has a molar adsorptivity
at 365, and so on. One can go on looking at the list and see that the lambda maxs are
much lower and the molar adsorptivity of diethyl ether is very high; otherwise, most of
these compounds have very low molar adsorptivity. And, these are typical examples of n
to sigma star transitions and the samples are all taken in vapor state.

(Refer Slide Time: 16:15)

Energy transitions, which are more popular are the n to pi star and pi to pi star. And, they
have molar adsorptivity, which are in the range of 10 to 100 liters per centimeter per
mole, and therefore, pi to pi star is still more popular, which has a molar adsorptivity
between 1000 to 10,000 liters per centimeter per mole. Applications of these two
particular energy transitions are what we are going to study in adsorption spectroscopy to
understand the chemistry of organic compounds, and it is based on this transition to
excited state of pi star. Energy required for those exited states is within 200 to 700
nanometers. So, it is well within the UV region. Chromophores involved are unsaturated
functional groups to provide the pi orbitals. n to pi star transitions are hypsochromic; that
means, they shift towards blue light with increasing polarity of the solvent. And, pi to pi
star transition is bathochromic, which shifts to red with increasing solvent polarity.

Now, let me explain you a little bit about these things, because it is now getting a bit
more complicated. What is a chromophore? A chromophore is a part of a molecule,
which has the pi and the n bonds, and is in conjugation with single bond. When the
double bond, single bond, double bond are in line with each other, one after the other, we
say that the system is conjugated. And, the more the number of pi bonds, the more will
be the excitation of these pi electrons to pi star; or, n electrons from the hetero atoms like
nitrogen, oxygen, chlorine, sulfur, phosphorous and so on to the pi star. Now, these cause
increasing of wavelength or decreasing of wavelength, and that is what is known as the
hypsochromic and bathochromic changes in polarity.

(Refer Slide Time: 18:52)

When try to look at the interactions of molecules with this radiation, it is very interesting
to see that the energy levels can create different types of interaction. It can either cause
rotational energy levels or it can create vibrational energy levels or it can finally, cause
electronic energy. Now, the rotational and the vibrational are brought about by the IR
energy or IR lights; whereas, the electronic energy transitions, which are much higher in
magnitude are brought about by the UV light or the visible light.

Energy level diagram associated with absorption of electromagnetic radiation; that is, the
rotational changes are shown by the far infrared region; the rotational-vibrational
changes are shown by near infrared region; the rotational-vibrational-electronic
transitions are given by the visible and UV regions, and therefore, from the electronic
ground state to the electronic first excited state are depicted by E 0 and E 1. So, you can
get a relative idea – what are the energy contents, whether the IR energy is stronger than
the UV energy? The immediate answer will be that IR energies are causing rotation or
vibration. So, they are smaller in their energy content; whereas, the UV lights excite the
electron from one level to another orbital, and therefore, they have larger energy content.

(Refer Slide Time: 20:42)

Miscellaneous information that is related to conjugation – little while ago, I was talking
about single bond, double bond, single bond being in reputation. Now, when such a
system exists in a molecule, then we call it that the molecule is conjugated or the system
has conjugation. Electrons in that are delocalized; lower energy is required to excite
electrons. Absorption maxima will shift to longer wavelength; that means, because I just
told you a while ago, longer wavelength, shorter energy; shorter wavelength, higher
energy. So, that should be kept in mind. If you keep one in mind, the other one will be
automatically remembered, but if you try to remember both of them together, there is a
possibility of mixing it up.

Now, you have already understood what are chromophores. But, what about
understanding another additive, which is called oxochrome? Oxochrome on their own do
not impart any color contribution. However, they help in the conjugation process. If a
functional group that does not absorb radiation by itself in the UV range, but has a
shifting effect on the main chromophore peak to longer wavelength as well as increasing
their intensities; for example, like hydroxy group – it is not absorbing by itself, but when
it is in conjugation with a double bond or other such conjugated system or NH2 or
benzene chromophore, then on the benzene chromophore, they will act as oxochrome.

Inorganic ions – a number of inorganic ions exhibit UV absorption as a consequence of

pi to pi star transition. For example, nitrate will absorb at 313 nanometers; whereas,
nitrite absorbs at 360 and 280 nanometers. Carbonate absorbs at 217 nanometers;
whereas, azide absorbs at 230 nanometers; whereas, trithiocarbonate absorbs at 500
nanometers. So, you see that different types of inorganic ions have different
wavelengths, because they absorb differently, and because they have different types of
oxochromes attached to the main chromophore.

(Refer Slide Time: 23:42)

Quantification for spectroscopy – now, this is the most important part of the
spectroscopy. Why, because this is the crux. What happens in a spectroscopic
transformation or data collection? Either we consider transmittance; that means, the
fraction of light that is incident and the fraction of light that is transmitted are compared;
that is called transmittance. Or, it can be a light as incident; it absorbs and then it
transmits the light, which is slightly less in its energy. Now, a part that the amount of
incident radiation, which has been absorbed by the medium is expressed as log 1 by T;
or, is equal to minus log T; or, is equal to log P0 by P.

Beer’s law (Beer-Bourguer-Lambert’s law) – radiation energy decays exponentially and

T, that is, the transmittance is always proportional to P over P 0. And therefore, it is to be
understood that it is proportional means, whatever be the molecular species
concentration, only that much will absorb, and therefore, it is a method of quantifying
these substances, because the number of species, the concentration is related directly to
its absorption energy.

(Refer Slide Time: 25:26)

Quantification – if we present in logarithm form, log T is equal to log P by P 0 is equal to

minus k b. The same stands for concentration, that is, log T is equal to log P by P 0 is
equal to minus k prime c; or, A is equal to k prime c; or, A is equal to abc. Now, what
are these a and b and c? The small a is the proportionality constant of absorption; and, b
is the path length of the method or the medium. Now, path length – that means how
much light is being shown, is also very important. And therefore, it is expressed in moles
per liter; C is expressed in moles per liter. And, path length or cell length is expressed in
centimeter. Then, the small a are the molar absorptivity has the special symbol, and the
units are liters per mole per centimeter. The molar absorptivity range for UV visible is 0
to 10 to the power of 5. So, it is as low as 0 and it can be as high as 10 to the power of 5.
(Refer Slide Time: 26:48)

When we try to see, understand Beer’s-Lambert’s law, we also have to remember that
these molecular species are not interactive among each other. Each species is an
individual, and that individual species absorbing light and the absorption directly
proportional to the concentration of the material. But, sometimes, when these molecules
are in aggregates, the Beer’s-Lambert’s law will not hold good. There is deviation from
its direct proportionality, because it is only valid at concentrations usually below 0.01
molar. If the solution is very concentrated, it may not have that complete effect of its
reaction and each entity will not be just one entity, but will be an aggregation of more
than one entity.

Molecules interference – distance between molecules affects charge distribution of

molecules particularly in the case of ions. Effect of relative index – concentrations effect
relative index, and therefore, molar absorptivity is affected. So, either it will be low or
too less; or, linear graph is no more linear. It then takes a turn and that is not considered
as one of the best methods of analysis, because for any law to be valid, it is important
that the calibration graph should be a straight line passing through the axis; and, the axis
is the point where the y-axis and the z-axis actually collide. So, this 0 should be passing
through the 0. And, one axis gives the absorbance, the other axis gives the concentration,
and that is what shows the proportionality between the two. But, if the solution is very
concentrated, if there are molecular aggregates, then this proportionality will not hold
good.
(Refer Slide Time: 29:13)

Beer-Lambert’s law also faces challenges when there are chemical deviations – either the
molecule is associated or dissociated, and the reaction takes place with the solvent and
other molecules like in buffered solution or unbuffered solution. Then, the instrument
will not show correct data; why, because the Beer’-Lambert’s law is not holding good.
So, one has to keep in mind, first thing, solutions should be very dilute, so they are fairly
apart. There should be no association, no dissociation or reaction with any kind of
solvents or other molecules when the analysis is being carried out.

Instrumented deviation – monochromatic radiation – quality of monochromator and

control of bandwidth and slit are other parameters that need to be considered.
Instrumental noise – accuracy of measurement of transmittance – quality of detector also
should be very good. If these have little bit of discrepancies, or if the instruments shows
any kind of deviation, or if the instrument is not regularly calibrated, or if the quality of
monochromatic light is not good, and the bandwidth and the split are not maintained at
the right parameters, then also, the Beer’s-Lambert’s law will not hold good.
(Refer Slide Time: 30:49)

Atomic and molecular spectrometry – this is what we are going to study in this whole
chapter of spectroscopy. What happens to a molecule when it is subjected to
electromagnetic radiation or what happens to molecules when they are subjected to
electromagnetic radiation? Atoms behave slightly different way – we just saw when we
were doing atomic absorption spectroscopy that came under the category of atomic
spectroscopy; whereas, molecular spectroscopy is where we are looking at the entire
picture of the molecule. Transitions between energy levels may occur in several ways
providing different types of spectra for analysis. So, molecular spectrometry is much
complicated as compared to atomic.

The intensity of the spectral emission or the reduction of intensity by absorption is

related to the concentration of the species producing the spectrum. So, when we try to
look at the intensities of the spectrum emission or reduction of intensity by absorption, it
will be either emitted or absorbed. These are the two things that happened when
electromagnetic radiation is shown. And, that will produce a different type of spectrum.
Certain transitions are favored by quantum theory rules; why, because energies are
quantized. It is not that by putting some energy, some excitation will take place. The
energies when they are matching with the molecular part or chromophore, will only
show excitation, not otherwise.
(Refer Slide Time: 32:44)

Transitions – for a given set of energy levels defined as quantum numbers, there are
several possible types of transition, but only several possible types. If energy is supplied
by passing electromagnetic radiation, the molecule may absorb energy and may get
promoted to higher energy level. So, it is possible that if the energy levels are matching,
then only these absorptions will take place. And therefore, if energy supplied by passing
electromagnetic radiation, the molecule may absorb only when it is compatible and may
be promoted to higher level, because it is rightly matching with the kind of energy that is
required for pi to pi star transition. If the molecule is activated thermally and then it gets
promoted, it would emit energy in falling back to the lower level. So, obviously, if a
molecule is heated and then it is thermally excited, then it will give out that heat by
emitting that energy and comes back to its natural position.

Let me tell you one thing that molecules always want to be at a peaceful situation. So,
whenever they are excited, either they have to emit or they have to absorb. And so, they
cannot keep this energy within themselves. So, this absorption has to be emitted in some
form or the other; they may revert back to their original space by emitting or they may
show that they are excited, and that absorption will be studied.
(Refer Slide Time: 34:34)

Quantitative spectrometry – the word quantitative means it is an absolute value; a

quantity is decided. In emission spectrometry, the intensity of the spectral line is related
to the number of emitting species present in the emitting medium and to probability of
the transition. So, in emission spectroscopy, it is the intensity of spectral line, which
shows that it is going to emit species that have been absorbed or excited or elevated to a
higher energy level.

In absorption spectrometry, the intensity of the incident radiation is reduced, which is

because it has interacted with the atoms or the molecules, raising them to higher energy.
So, here what happens that incident light has some energy and the light which is coming
out has some other energy. This is at a higher energy; this is at a lower energy. So, it is
assumed that this loss of energy is what has been taken up by the atom. So, it is very
quantitative; as much it will absorb as the concentration of these molecules or atoms are
present in the medium. It cannot get lost; it cannot get added up; it has to be in sign of
proportionality. And, that is what helps us to understand the spectroscopic method.
(Refer Slide Time: 36:10)

Coming to infrared spectroscopy – particularly, which is used in organic chemistry.

(Refer Slide Time: 36:15)

Infrared spectroscopy – because here the light source will be infrared – it is the subset of
spectroscopy that deals with the infrared region of the electromagnetic radiation or
electromagnetic spectrum. It covers a range of techniques, the most common being a
form of absorption spectroscopy meant to identify compounds. So, it should be
understood, that part of electromagnetic radiation or spectrum, which deals with infrared
region is what the study of the present lecture is. It covers a range of techniques, the most
common being the form of absorption spectroscopy meant to identify the compounds
only from the point of view of their functional group – whether an OH moiety is present
or not; whether NH 2 is present or not; whether carbonyl is present or not; if the carbonyl
present, then whether it is aldehyde or ketone; or, does it belong to acyl carbnyl; or, does
it belong to anhydride carbonyl – is the study that will be understood through the use of
infrared radiation.

(Refer Slide Time: 37:48)

The infrared portion of the electromagnetic spectrum is divided into three regions; as
what we saw also in one of the earlier slides, the near-, the mid- and the far-infrared,
named for their relation to the visible spectrum. The far-infrared, approximately in the
region of 400 to 10 centimeter inverse, lying adjacent to the microwave region, has low
energy and may be used for rotational spectroscopy. Second, the mid-infrared,
approximately ranging from 4000 to 400 c m - ( centimeter inverse) – this is the one
which is most properly used – may be used to study the fundamental vibrations and
associated rotational and vibrational structure. And therefore, these are regions, which
are of very great importance for knowing the functional group and its presence.
(Refer Slide Time: 38:53)

And, the third region that is important is the higher energy near-IR, approximately
ranging from 14000 to 400 centimeter inverse, can excite overtone or harmonic
vibrations. The names and the classifications of these sub-regions are merely for
conventions, because they belong to different regions and they do different things in a
molecule. That is why the information imparted by these groups is also very clear. They
are neither strict divisions nor based on exact molecular or electromagnetic properties.

(Refer Slide Time: 39:39)

Near infrared spectroscopy – near infrared spectroscopy, which we will refer as NIRS is
a spectroscopic method utilizing the near infrared region of the electromagnetic spectrum
from about 800 nanometers to 2500 nanometers. Typical applications include
pharmaceutical, medical diagnostics including blood sugar and oximetry, food and
agrochemical quality control, as well as combustion research. So, you see, it has a
versatile area in which it can be used. And, the information imparted by the near infrared
spectroscopy can be so important that it can serve in the areas of pharmaceutical, medical
diagnostics including blood sugar, in the area of food and food chemistry, in the area of
agrochemical quality control, and in the area of combustion research.