Analytical Technologies in Biotechnology

Prof. Dr. Ashwani K. Sharma

Department of Biotechnology
Indian Institute of Technology, Roorkee

Module - 6
Spectroscopic Techniques
Lecture - 1
Introduction and Basic Concepts

In previous lectures, we have covered 5 topics and these include microscopy, radio
isotope technique, chromatographic technique, electrophoreses and centrifugation
techniques. From this lecture, we are going to start a new topic and that is spectroscopic
techniques.

(Refer Slide Time: 00:48)

Now, spectroscopy can be defined as the study of interaction of electromagnetic

radiation with metal and it is analysis. So, in spectroscopy the electromagnetic radiation
say light, interacts with the metal in different ways, as we will discuss and this particular
property of the metal to interact with the light, and then formation is derived is used for
the analysis and characterization of small and large molecules. The interaction of
electromagnetic radiation with metal or as we say spectroscopy is a quantum
phenomenon or we can say observation of quantum mechanics.
And it depends on both the properties of the radiation and the structural composition of
materials, which are involved in here interacting with the electromagnetic radiation.
Now, different types of spectra obtained or the information, which is derived due to the
interaction of the radiation with a metal, will lead to the understanding of these
interactions and development of spectroscopic techniques, which will help in solving
many biological problems, where one can characterize these molecules and know about
them and likewise solve many biological problems.

(Refer Slide Time: 02:32)

Little bit into electromagnetic radiation, in very simple terms, electromagnetic radiation
can be defined as a form of energy, whose origin is the emission and absorption from
charged particle, it is like coming out of the metal. So, in terms of classic physics, if we
say electromagnetic radiation is mainly produced, when charged particles are accelerated
by forces acting on them.

So, it comprises of both, the electrical as well as magnetic components, which are
oscillating in directions perpendicular to each other, at a fixed intensity ratio and
mutually, they are at right angles to the direction of propagation. The energy of wave or
electromagnetic wave is given by Planck’s constant into the velocity of the light divided
by the wave length that is or you can say h nu.
(Refer Slide Time: 03:48)

And let me show, you on this screen the typical electromagnetic radiation in a wave
form, so like I said, it has 2 components, electromagnetic wave has 2 components,
electrical and magnetic component. So, if I can draw very specific, so there will be 2
components, one is electrical electric, you can say electric vector and another is magnetic
vector and if you consider a wave here. So, this is a magnetic vector at 90 degrees
electric vector and this is the propagation of the light actually that is direction of
propagation.

So, as we depict the wave nature of the light here, what we will have is if I just draw,
supposing this is your one vector or one direction and if I say this direction as say
electric vector here. Then there will be another direction, which will be perpendicular to
this and which can be made in a different manner, likewise actually and this one here,
this is you can say, you can give different term x or whatever and this is these are the 2
components of this wave, electric and magnetic components.

So, these are perpendicular to each other and then, to the direction of propagation here,
so that is a typical electromagnetic wave, which has one component, which is
perpendicular to 2 components, which are perpendicular to each other, one is electric
component or electric vector another is magnetic component. And they are perpendicular
to the direction of propagation, so that is a typical electromagnetic wave actually. Now,
as we said, the energy of this wave, in terms of like energy of the, this wave could be
expressed in term and in this equation here.

(Refer Slide Time: 06:25)

Where you have h is Planck’s constant c is the velocity of light and lambda is the
wavelength of the radiation. So, here it has particular like, this particular could be also
like, it could be written in this way that is h nu, where nu is the frequency and frequency
is expressed in terms of hertz actually that is one oscillation per second alright. So, this is
typical representation of a electromagnetic wave here, let us come back, so what you
have is your Planck’s constant like, we have shown you h c upon lambda, c is the
velocity of the light in this equation, which I have shown you and nu is the frequency of
the radiation. If the frequency of the radiation is the is in reciprocal seconds and it is
expressed in terms of hertz, which is one oscillation, per second is called one hertz.

Now, electromagnetic wave or you can say light, can interact with metal, where it can be
either absorbed or emitted or scattered. So, a molecule or atoms, they will either absorb
an electromagnetic radiation or they might emit, as they go into a higher state and when
come back to the ground state, they can emit a particular radiation or it is could be that
electromagnetic radiations can be scattered also by the metal.

So, the atoms and molecules mostly exists only in discrete energy levels, what does that
means, it means that electrons in atoms or a molecules may be distributed or they reside
in ground state or which say lower level of energy. And they can be excited to a higher
level or we call it excited state, where if exact amount of energy, which is equivalent to
the difference in energy levels or excited electrons in higher state.

So, if it is a absorption, then absorption will occur only if, there is a equivalent amount
that is the difference between the 2 energy states is absorbed and then, electron can be go
to higher or excited state. Likewise, when an excited electron might come back to a
lower level where again it will whichever level it comes, it will emit the equivalent
amount of energy that is difference in energy between the levels.

So, light could be absorbed or as well as emitted by a molecule by undergoing transition

and this particular phase, where a particular phenomenon, where the electron like
undergoes transition from one transition state to another is known as, what you call
transition here. And this transition is between discrete energy levels and these energy
levels and other characteristics are predicted by quantum mechanics, let me show you on
your screen these, what are these energy levels mean here alright. So, when we say
energy levels like, I said these atoms have a discrete energy levels and they reside in
these discrete energy levels and there is could be a ground state and higher excited states.

(Refer Slide Time: 10:38)

So, when we say these states, let us little bit understand them, if I, if we can just
understand this particular one alright, now if we see these 2 here and this is ground state
and this is the first excited state. Now, here in this one, so this is electronic state, you can
say and this going from this particular one is electronic energy level, another one is in
excited state that is the first excited state of that an electrode level. Now, within these
electronic energy levels, there are subdivisions, which are known as vibrational levels
actually.

So, these are called vibrational levels and these vibrational levels, will exist in the
excited states also, here we have shown you the first excited state and they will be in
here also. Now, when absorption or emission whatever occurs, say for example,
absorption occurs, say from the first or from very ground level, to the higher, if I say it is
going up to here. Then absorption has occurred the exact energy has been absorbed,
which leads to the electron from this state to the say 4th vibrational state of first excited a
state in electronic shell actually.

So, these are electronic shells and these are vibrational further division makes them into
or divides them into vibrational levels and further vibrational levels could be divided into
rotational levels, which are not shown in here. And this distance is this is distance
between, electrons and nucleus or we can say atoms or we can say between atoms of
molecule, of a molecule or between atoms in a particular molecule.

So, what you can see here is this is very clearly shown that, electronic the transition
occurs from one electronic state to another, this transition could be between the
vibrational levels of the 2 electronic states that is ground state. And first excited state and
rotational levels are not shown, which could be further division of vibration levels
actually. So, that is how likewise, if say this is transition from one level to another, but
there could be like transition in within the electronic level between say vibrational levels,
if I say here.

This is like changing vibrational level or in this particular excited state, it could be like
this could be returning into different vibrational levels. So, this is how a transition takes
place between different energy levels actually. So, I think, it should be very clear that
how an electromagnetic radiation interacts with metal by a absorption or emission and
electrons in atoms or molecules, which resides in the ground state or a lower level after
absorption of the equivalent energy that is the energy difference in the 2 levels, can get a
excited to a higher level.

So, and the reverse will happen emission actually, so a molecule that can be excited by
absorption is known as chromophore actually, the excitation energy is usually converted
to heat say by collision of excited molecule with another molecule or reemitted as
fluorescents. In both the cases intensity of light transmitted by chromophore is less than,
the intensity of the incident light. Now, will be discussing about a fluorescence
spectroscopy, in later lectures, now the major energy level, as we have were discussing
depends on spatial distribution of electrons.

And these are called electronic energy levels, so what I have shown you, these word
electronic energy levels and I have shown you, the ground state and first excited state.
Then there are vibrational levels within electronic levels or you can say in each
electronic states, indicating various modes of vibration of this of the molecule, now these
vibration this gives information about stretching and bending of various covalent bonds
within the same electronic state. And then, there could be further smaller subdivisions,
which were not shown in that diagram, these are called rational levels and they are not.

So, much used in biology in an energy level diagram, lowest electronic level is ground
state and others are excited state and as we have shown the first excited, state in here.
Now, absorption energy, absorption of energy can happen, only if the amount absorbed
corresponds exactly to the difference between energy levels.

(Refer Slide Time: 17:23)

So, light of a particular wavelength, can only be absorbed only when there is a like, so if
you consider this alright, so amount could be absorbed only, when like in terms of like
say energy change, for electronic transition, we can say. But, you should have is that,
change the difference in energy that is between the 2 states has to be equal to, so the
energy absorbed should be equal to the difference between the 2 energy states that is the
first ground state and then excited state.

And also between the vibrational levels, to which vibrational level that, particular
electron is excited or in terms of like we can say, that if particular, if tells for wavelength
of electromagnetic radiation then, we can say that it should be that, if it fulfills this
particular one that is again this component here. Then, the wavelength the energy is will
depend on the wavelength of electromagnetic radiation as well. And the wavelength, at
which the maximum absorption takes place is called lambda max, lambda max means,
the wavelength, at which a particular material or molecule absorbs the maximum in
terms like.

(Refer Slide Time: 18:48)

If I say in terms of absorption plot, if there is an absorption, which is large absorption,

there could be minor absorption around here, but if there is a particular wave length
gives maximum absorption, then this wavelength will be called lambda max. So, a plot
of the absorption versus wavelength is called an absorption spectrum and this absorption
spectrum comes from, the absorption of particular wavelengths actually, which is what,
which is the information that we obtained from absorption spectrum.

Likewise, if it is a emission spectrum then, the spectrum or it will be called emission

spectrum and there, we will be looking for the emitted wavelength, emitted radiations,
rather than, absorbed radiations. So, these are 2, complimentary techniques,
spectroscopic techniques, now E M radiation or electromagnetic radiations, they are
classified by the frequency of the radiation or you can say in reverse, it is a wavelength,
but on the basis of frequency, if we say.

And they will be categorized into different types of electromagnetic radiations, these
could be radio waves microwaves, infrared radiation, visible light, ultraviolet radiation x-
rays and gamma rays and the total radiations including all these frequencies form the
electromagnetic spectrum. Now, different radiations of the electromagnetic spectrum has
been utilized in the biological science, for variety of purposes, now if you can see on
your screen.

(Refer Slide Time: 20:37)

This is the particular diagram to depict various kinds of electromagnetic radiations and as
per their frequency and that is increasing frequency or increasing wavelength, as the
frequency increases, wavelength decreases and vice versa. So, if you can see in here,
there are gamma rays, at very high frequency then, x-rays and this frequency here is
depicted in terms of hertz actually and wavelength here is depicted in terms of meters.
So, if you see gamma rays with very high frequency then, x-rays then next in
electromagnetic spectrum is u v that is ultraviolet rays.

Then after ultraviolet rays, these are visible spectrum that is what we can see or the
colors, which we can perceive then, comes IR region infrared region in the spectrum,
microwave then, radio waves and then long radio waves which are present in here. Now,
if we consider like I said there are different radiations of the electromagnetic spectrum
have been utilized in the biological science, for a verity of purpose. If I compare in here
this one, if you can, if little bit, we can go into that then, u v and visible region.

These are utilized and little bit of IR region, these are utilized, for as a standard, what
you call this particular region from u v, till little bit of IR is for a standard absorption
spectroscopy. And here, in absorption spectroscopy mainly inner shell at the lower
electrons and outer shell electrons are considered here, for standard absorption
spectroscopy. Then second is in IR region, you have like, we have told you about,
vibrational and rotational thing.

Now in the IR region, there is a vibrational part is taken and this is utilized, for infrared
in vibrational region, it is utilized for infrared and raman spectroscopy. So, here in higher
regions, there will be nuclear magnetic resonance spectroscopy, which will be utilized.
So, these are like different like, another important thing is where, we are utilizing in u v
visible, where absorption spectroscopy is being used, this is in electronic level actually,
where inner shell and outer shell electrons are taken.

Infrared spectroscopy is performed, as per vibrational shell here or vibrational level,

these are transition, I am talking about here, it is a electronic transition, these are
vibrational transition and as you go for say, nuclear magnetic resonance, it is a nuclear
transition, we can say. So, these are different parts of the spectrum or electromagnetic,
different types of electromagnetic radiations and the types of transitions, we were talking
about and to how to, what kind of techniques have been developed on that bases.

So, in summary we can say that, u v based and even particular x-rays are taken as
standard absorption spectroscopy with electronic transition, from x-rays till u v based
little bit of infrared. Then, infrared is mostly vibrational transition and it is utilized for
raman spectroscopy and infrared spectroscopy and then finally, you have nuclear
magnetic resonance, which is in say microwave region here, which is based on nuclear,
this resonance actually. So, these are different types of transitions, which we were which
have been utilized for obtaining information regarding the metal that is interaction of the
metal, with the electromagnetic radiation.
(Refer Slide Time: 25:10)

At so as we have seen that is spectroscopy is the study of interaction of energy with the
metal and it is analysis and this has originated, this is spectroscopy originated with the
study of visible light dispersed by the prism according to it is wavelength. So, like as we
know that, visible light is a combination of different wavelengths and when they pass
through the prism, they are dispersed actually, according to their wavelengths, as we
have talked about this in microscopy also.

So, in daily life, also the colors of things can be taken, as an example of spectroscopy,
because objects absorb light of a particular frequency and emit a particular frequency.
And failing in the visible, which are falling in the visible range and giving it a particular
characteristic color actually, so that, we can see it. So, this is like in daily life we are
observing different colors, because of like absorption and emission of a particular
frequency wavelengths.

Now, the principle concept of the spectroscopy is resonance and the corresponding
resonant frequency that is, it is resonating between the 2 different energy states. So, there
is a major or principle concept is resonance and resonant frequencies, in quantum
mechanical terms resonance can be defined as the energy equivalence between 2
stationery states of a system, which is provided by the external energy source.

Now, spectroscopy is a very wide field here and where multiple subdivisions exist and it
is and it is classified in a verity of ways in like, nature of radiation, types of interaction
and types of materials being studied. Traditionally, spectroscopy has been restricted to
where, particular absorption or emission takes place that is a particular molecule or
atoms absorbed, a particular wavelength or electromagnetic radiation of a particular
energy.

And gets excited that information or where, emission takes place, because of emission of
a particular radiation and those information are taken and that is the our characteristic or
you can say traditional spectroscopy. And many techniques, which might not have fallen
in spectroscopy earlier, have been regarded or considered in terms of the spectroscopy,
like for example, scattering actually, where or interaction in terms of not absorption or
emission.

But, say in terms scattering of light that could be one form, where that could also be
called as a particular kind of spectroscopy, as we will discuss, as we go along here. So, it
could be divided like a spectroscopy has become a very wide field and lot of multiple
subdivisions. And it could be divided into various ways for example, what are the nature
of radiations are being used, what are the types of interactions, which are happening and
what are the types of materials, which are being studied.

Let us take each one of them separately here, in terms of nature of radiative energy or
radiations, which will include radiations belongs to different parts of the electromagnetic
spectrum. Particles it could be like electromagnetic spectrum, which as we have seen
from right from x-rays till microwaves and then, ultraviolet and visible rays infrared part
of the spectrum all these are utilized, where interactions with metal takes place.

Also other than, electromagnetic spectrum, there could be particles like electrons or
neutrons, which could be taken like there is electrons when they, pass through metal,
they could be scattered or there could be other interactions likewise, neutrons can
interact in various ways. And at times could be also say waves like, say pressure waves
or others, used as energy source for a spectroscopic study.

So, it is not a typical only electromagnetic spectrum or electromagnetic radiations, which

are interacting, it could be in the form of particles like, electrons and neutrons as well,
where scattering or other interactions might occur will also be covered under
spectroscopy. Then, there are types of interactions actually, now there could be different
types of interactions that will form the bases of spectroscopic technique, actually and as
we were, talking the traditionally absorption and emission were, the main constituent or
main types of interactions, which were considered or covered under the spectroscopy.

But, there could be lot of other interactions like, scattering, which could be elastic
scattering and reflection or we call it as diffraction, in many comes like a x-rays and
electron scattering and crystallography. Like x-ray crystallography, where electrons like,
x-rays are diffracted by the molecule, which are arranged in a crystal in a particular
regular array. So, that could be elastic scattering could be one form of interaction, which
is also comes under spectroscopy then, they could be inelastic scattering, where it could
be like, it might involve change of energy between the radiation.

And the metal and that will shift the wavelength of this cathode radiation and it will
include like, raman and compton scattering. So, there could be both, elastic scattering
like in x-ray crystallography or x-rays in electronics scattering in crystallography or it
could be inelastic scattering, where change in or shifting of the wavelength of the
scattered radiation could occur and that includes raman and compton scattering.

There could also be coherent or resonance spectroscopy in coherent or resonance

spectroscopy like, radiative energy, couples the 2 quantum states of the material in
coherent interaction. And that is sustained by the radiating field one example is N M R
spectroscopy is widely used method in this typical example of resonance method. So,
there could be different types of interactions right from absorption emission, which are
traditionally considered and then, could be scattering that is elastic scattering or inelastic
scattering and then, there could be a resonance spectroscopy or interactions as well.

Apart from types of radiation or types of interaction also one other thing should be
considered in here, when we are talking about a spectroscopy that is types of materials,
which are involved in here. So, this spectroscopic techniques are designed to interact
with specific kinds of materials, which could be like, it could be atoms only, in terms of
atomic absorption and atomic emission spectroscopy, like we will be talking about
atomic absorption and emission spectroscopy, where absorption and emissions often
referred to as atomic spectral lines.

And these atomic spectral lines are due to the an electronic transition of an outer shell
electron to an excited state and atoms of different elements have distinct spectra or we
can say, spectral lines. On the bases of these spectral lines, there it could be spectral
lines, in terms of absorption of particular lines or it could be in terms of emission lines
emission spectra, where a particular wave length have been emitted actually.

Both ways these have these are characteristics of a particular atom and these are very
helpful in recognizing or identifying the particular types of elements or atoms, in these
sort of spectroscopy. So, one is interaction could be with like materials like atoms,
another could be interaction with the molecules, molecular spectra can be obtained, due
to electron spins states, like where could be, one is electron spin states could be there.
Another could be molecular rotations actually, which could also utilized for deriving
information then, molecular vibrations, which occur all the time and electronic states.

So, spin states could also be utilized and electronic state, which we have discussed
earlier could be utilized. Then, there are vibrations are which are also happening and
these are relative motions of the atomic nuclei and are studied by both infrared and
Raman spectroscopy. Electronic excitations as we have discussed are studied using
visible, ultraviolet and fluorescence spectroscopy, whereas for example, there could be,
so we have discussed about atoms molecules.

There could be crystals as well actually like, I said these are regular arrangement or
regular array of atoms and the regular arrangement of atoms in a particular array in
crystals. And these regular lattice structure of crystals will scatter x-rays electrons on
neutrons and these are utilized for crystallographic studies, where the structure, 3
dimensional structure of molecules, could be obtained by generating the electron density
maps.

So, this is like particular way that is scattering method, could be utilized for crystals for
deriving information that is a structural and chemical compositions, then nuclei or metal
could be interacting like, nuclei also have a distinct energy states and distinct nuclear
spin states. And these can have their energy separated by a magnetic field and which,
forms the bases for N M R spectroscopy. So, in n m r spectroscopy, it is the nuclear
resonance which is utilized or magnetic nuclear magnetic resonance, which is utilized for
deriving information.

So, these are different ways like, you can say a different types of spectroscopic
techniques, which are on the bases, on which these spectroscopic technique could be put
in and different ways like of interaction or types of radiation or different types of matters
are discussed in here. So, alright this was the basics, certain basic concepts in terms of
electromagnetic radiation, in terms of a types of interactions, which are happening and
how this information could be derived.

What are the different spectroscopic techniques, which have been developed like on the
bases or electronic transition or on the bases of say vibrational transitions or say nuclear
transitions. The techniques like absorption techniques or say infrared technique,
spectroscopy or say nuclear magnetic resonance spectroscopy have been developed. Also
other techniques, which will be included in here like, x-ray diffraction or x-ray scattering
and mass spectrometry, which is not typical spectroscopy technique, as we have
explained. In traditional it is not typical spectroscopy technique, but very useful very
powerful technique and is important, so we have included it in here.

(Refer Slide Time: 38:40)

So, the techniques, which are little bit into the techniques, which we are going to discuss
in this section here, one is ultraviolet visible absorption spectroscopy, which we are
going to discuss here. In here, when electromagnetic radiation passes, through a
transparent material, a portion of radiation may be absorbed and as a result of energy
absorption atoms or molecules will pass from a state of low energy that is ground state to
state of higher energy that is excited state.

And ultraviolet visible spectroscopy is based on the transitions of electrons from one
atomic orbital or molecular orbital to another and due to the absorption of the
electromagnetic radiations. So, we are going to discuss in detail about, this technique, in
coming lecture, then another based on vibration transition is infrared spectroscopy,
which deals with the infrared region of the electromagnetic spectrum, having higher
wavelength and lower frequency then visible light.

It consist of 3 regions that is near 0.8 to 2.5 micrometer midpoint 2.5 to 25 micrometer
and far infrared that is 25 to 1000 micrometer and based on the facts that are molecules
absorb specific frequencies in this range that are characteristic of the structure and absorb
frequency resonance resonates within the frequency of the vibration of the group. So, IR
active molecule require the change in the dipole moment the IR spectrum is recorded by
passing a beam of infrared light, through the sample and when, the frequency of the
infrared beam is equal to the vibrational frequency of the bond IR absorption occurs.

(Refer Slide Time: 40:33)

Then, next technique, we are going to discuss about, it fluorescence spectroscopy,

fluorescence spectroscopy is a type of spectroscopy, where fluorescence from a sample is
analyzed. The phenomenon involves the absorption of light usually in ultraviolet region,
that will excite the electrons in molecules of the certain compound and will cause them
to emit light at a longer wavelength, when they come back to the ground state, usually in
visible region.
So, that we are going to discuss in coming lectures, then also we going to discuss, we
have included certain other techniques, like which are typically, spectroscopy techniques
of course.

(Refer Slide Time: 41:17)

Like circular dichroism, which are very useful in where is biophysics applications,
circular dichroism here the electromagnetic radiation consist of an electric and magnetic
field that oscillates perpendicular to each other and to the direction of the propagation.
So, while linearly polarize light occurs, when the electric field vector oscillates, only in
one plane and circularly polarized light occurs, when the direction of the electric field
vector rotate about, it is propagation direction, while the vector retain the constant
magnitude.

At single point in space the circularly polarized, vector will trace out circular over the
one period of the wave frequency, hence the name has been given. So, the circular
dichroism refers to the differential absorption of the left and right circularly polarized
light. The phenomenon of circular dichroism is exhibited by kairali molecules and hence
biological molecules are excepted to exhibit the same, due their dextrorotatory
levorotatory components.

More over a distinct secondary structure in part the characteristics c d spectrum and like
alpha helices and double helices in d n a have characteristics c d signatures, the far u v c
d spectrum of proteins can reveal important characteristics of their secondary structure.
And c d spectrum can be rear used to estimates the fraction of molecule that is the in
alpha helix confirmation. So, the beta sheet confirmation the and also some other, like
say random coil confirmation could be obtained, these fractional assignments place
important constraints on the possible secondary confirmation that protein can be in.

Then, we are going to discuss about nuclear magnetic resonance spectroscopy, which
explode the magnetic properties of certain atomic nuclei to determine physical and
chemical properties of the atoms or molecules, in which they are present. The method is
based on the nuclear magnetic resonance and provides a detailed information about, the
chemical environment reaction state and structure of the molecule. Basically, when
sample contains n m r active nuclei for example, 1 h or 30 c or 19 f or 31 p, these have
fractionally spin of half.

And when, they have placed in magnetic field, they exist in 2 spin states pulse half,
which is aligned magnetic field and minus half, which opposes the magnetic field and
difference in energy between the 2 spin states will depend on the external field strength.
When these are radiated sample is radiated with the radio frequency energy, which
corresponds to the except spin state separation of the specific sort of nuclei that will
cause excitation of nuclei, from plus half state, spin state 2 higher state, and resonant
frequency absorption and the energy absorption of the energy and intensity of the signal
proportional to the magnetic field. And there could be lot of different kinds of n m r
technique, which could be one dimensional technique, for smaller compounds to 3 or 4
dimensions techniques for large molecules, like protein or nucleic acids.
(Refer Slide Time: 44:57)

Another technique, we are going to talk about is x-ray spectroscopy and will be more
talking about x-ray scattering or x-ray diffraction methods, now x-ray spectroscopy
refers to the spectroscopic technique, for characterization of different material using, x-
ray excitation. Now, when an electron from inner shell of atom is lost, due to some sort
of excitation, then vacancies field with an electron from the outer shell, and the
difference in energy emitted as x-ray of appropriate wavelength, characteristic for the
element.

So, the analysis of spectrum of x-rays provides a qualitative information about, chemical
compositions and comparison of spectrum with spectra of standards of known
compositions provides quantitative results. Now, x-rays can be absorbed biometry also,
x-ray absorption spectra then, there could be x-ray fluorescence analysis also, using x-
ray fluorescence.

Apart from this, there are x-rays scattering diffraction technique, which reveal
information about, the crystallites structure or structure of the molecule and these
techniques are based on scattering or we can say diffraction of x-ray beam by crystals at
different angles and intensities. And from these angles and intensities of the diffracted
beam 3 dimensional distribution of density of electron within crystals is obtained and
therefore, the mean positions of atoms in the crystal and the structural information can be
derived.
(Refer Slide Time: 46:36)

So, dealing about, this in later chapters then, also we are going to discuss about atomic
absorption and flame emission spectroscopy atomic absorption and flame emission
spectroscopy methods are very powerful tools, for both qualitative and quantitative
analysis. In atomic absorption technique absorption of radiant energy by free atoms in
gas state occurs and that is the bases for obtaining the information, where as in emission
of flame emission technique, there is intensity of the light of a flame or a plasma, at
particular wavelength is emitted and this gives information. Wavelength of atomic
spectral lines helps in identification of elements by the intensity is proportional to the
number of atom present.
(Refer Slide Time: 47:25)

Finally, we are going to discuss about, mass spectroscopy or mass spectrometry to be a

to say mass spectrometry is an analytical tool used for measuring, the mass to charge
ratio of the sample that are charged. The instrument that do for same are called, which
does, it which calls mass spectrometers and which has ionizer analyzers and detectors
here the sample as to be introduced into the ionization source of the instrument.

Once in the side the ionization source the sample molecules are ionized and because ions
are easier to manipulate the neutral molecules, these ions are extract into the analyzed
region of the mass spectrometer. Where are there separated according to the mass to
charge ratio and the separated ions are detected and thus signals send to the data system,
where the these ratio and the z are ratio are stored, together with relative abidance for
presentation in the format of mass to charge ratio spectrum.

The analyzer and detector of the mass spectrometer and often the ionization source to are
maintain under high vacuum to give the ions are reasonable chance from 1 ion to another
of the instrument to the other, without endurance from air molecules. Entire operation of
the mass spectrometer and often the sample introduction process also is enter complete
data system controlled on modern mass spectrometers.

And this technique is utilized for various things like are determining masses of particles
for determining the alignmental compositions, for illustrated in the chemical structure
and etcetera, and this also we are going to discuss in coming chapters or coming lectures.
So, in this lecture to summarized, we discussed about, basic concepts in terms of
electrometric radiation and spectroscopy, which is the interaction of the metal with
electromagnetic radiation.

We have discussed about different types of spectroscopy and the basis for, those
spectroscopic techniques like, electronic transitions or vibrational or say nuclear
resonance and the lot of differences spectroscopic techniques have been developed. The
area of the spectroscopy also be widen to include more spectroscopic techniques, like on
the basis of scattering and other phenomenons. So, in the will be starting with specific
techniques, in the next lecture and in the next lecture, we are going to discuss about, the
first technique that is u v visible spectroscopy.

Thank you.