This document discusses electronic spectroscopy and molecular spectroscopy. It begins by explaining that electronic spectroscopy relies on quantized energy states of electrons and involves electronic transitions between principal quantum states when electrons absorb enough energy. It then discusses different regions of the electromagnetic spectrum and how spectrometers are used to analyze absorption and emission spectra. The rest of the document discusses molecular spectroscopy, deviations from Beer's law, and applications of using spectrophotometry and Beer's law to determine equilibrium constants.

1. DISCOVER . LEARN . EMPOWER
Mr. Yunes Alsayadi
Assistant Professor
of Pharmaceutical Analysis
E 10695
UNIVERSITY INSTITUTE OF PHARMA
SCIENCES
Pharm.D
Spectroscopy
PST-392

2. Contents
Theory of electronic, atomic and molecular spectra.
Fundamental laws of photometry
Beer-lambert’s Law
Determining An Equilibrium Constant Using Spectrophotometry and Beer’s
Law
Chromophore
Auxochrome
Bathochramic Shift or Effect
Hypsochromic Shift or Effect
Hyperchromic Effect
Solvent effect on absorption spectra
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3. ELECTRONIC SPECTROSCOPY
All organic compounds are capable of absorbing electromagnetic radiation because all
contain valency electrons that can be excited to higher energy levels. The excitation
energies associated with electrons forming most single bonds are high.
Electronic Spectroscopy relies on the quantized nature of energy states. Given enough
energy, an electron can be excited from its initial ground state or initial excited state (hot
band) and briefly exist in a higher energy excited state.
Electronic transitions involve exciting an electron from one principle quantum state to
another. Without incentive, an electron will not transition to a higher level.
Only by absorbing energy, can an electron be excited. Once it is in the excited state, it will
relax back to it's original more energetically stable state, and in the process, release energy
as photons.
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4. The science of spectroscopy grew out of studies of the interaction of
electromagnetic energy with matter.
When light shines on an object, for example, we know that part of the light is
scattered (reflected) and part is absorbed. Of the initial part that is absorbed, some
is later emitted as light of a different color or wavelength.
Spectroscopy is that science which attempts to determine what specific energies
and amounts of incident light are absorbed by specific substances, and what
specific energies and amounts are later re-emitted.
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5. Regions of the electromagnetic spectrum

6. Electromagnetic radiation moves in waves

7. Optical instruments called
spectrometers reveal in photographic
or printed records—as a series of
specific wavelengths or
frequencies—the light energies
absorbed and emitted.
These records, in turn— referred to
as spectra—provide us with
important information pertaining to
the atomic and molecular structure of
the substances on which the
electromagnetic energy is focused.
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8. Often, during electronic spectroscopy, the electron is excited first from an
initial low energy state to a higher state by absorbing photon energy from
the spectrophotometer.
If the wavelength of the incident beam has enough energy to promote an
electron to a higher level, then we can detect this in the absorbance
spectrum. Once in the excited state, the electron has higher potential energy
and will relax back to a lower state by emitting photon energy. This is
called fluorescence and can be detected in the spectrum as well.
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9. 9

10.  Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not
transparent below 200 nm
 Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically
collected from 200-700 nm
 This limits the transitions that can be observed:
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11. 11

12.  An electron in a bonding s orbital is excited to the corresponding
antibonding orbital. The energy required is large. For example,
methane (which has only C-H bonds, and can only undergo   
transitions) shows an absorbance maximum at 125 nm. Absorption
maxima due to    transitions are not
 seen in typical UV-VIS spectra (200 - 700 nm)
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13. • Saturated compounds containing atoms with lone pairs (non-
bonding electrons) are capable of n   transitions. These
transitions usually need less energy than  
•   transitions. They can be initiated by light whose wavelength is in
the range 150 - 250 nm. The number of organic functional groups
with n   peaks in the UV region is small.
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14. • ▣ Most absorption spectroscopy of organic compounds is based
on transitions of n or electrons to the  excited state.
• ▣ These transitions fall in an experimentally convenient
region of the spectrum (200 - 700 nm). These transitions need an
unsaturated group in the molecule to provide the  electrons.
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15. 15
Chromophore Excitation max, nm Solvent
C=C →* 171 hexane
C=O
n→*
→*
290
180
hexane
hexane
N=O
n→*
→*
275
200
ethanol
ethanol
C-X
X=Br, I
n→*
n→*
205
255
hexane
hexane

16. ANALYTICAL TECHNIQUES –SPECTROSCOPY
Spectroscopy is the branch of science dealing with the study of interaction of electromagnetic
radiation with matter.
It is a powerful tool available for the study of atomic and molecular structure and it is also used in
the analysis of most of the samples.
Types of Spectroscopy:-
The study of spectroscopy is divided into two types. They are,
1. Atomic spectroscopy
2. Molecular spectroscopy.
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17. Atomic spectroscopy is the determination of elemental composition by its
electromagnetic or mass spectrum. The study of the electromagnetic spectrum of
elements is called Optical Atomic Spectroscopy.
Electrons exist in energy levels within an atom. These levels have well defined
energies and electrons moving between them must absorb or emit energy equal to
the difference between them.
Since every element has a unique electronic structure, the wavelength of light
emitted is a unique property of each individual element.
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Atomic Spectroscopy

18. The science of atomic spectroscopy has yielded three techniques for analytical use:
1. Atomic Absorption
2. Atomic Emission
3. Atomic Fluorescence
The process of excitation and decay to the ground state is involved in all
three fields of atomic spectroscopy. Either the energy absorbed in the
excitation process, or the energy emitted in the decay process is
measured and used for analytical purposes.

19. If light of just the right wavelength impinges on a free, ground state atom, the atom may absorb the
light as it enters an excited state in a process known as atomic absorption. By measuring the
amount of light absorbed, a quantitative determination of the amount of analyte element present
can be made.
In atomic emission, a sample is subjected to a high energy, thermal environment in order to
produce excited state atoms, capable of emitting light. The energy source can be an electrical arc, a
flame, or more recently, a plasma.
Atomic emission using electrical arcs has been widely used in qualitative analysis. Emission
techniques can also be used to determine how much of an element is present in a sample. For a
"quantitative" analysis, the intensity of light emitted at the wavelength of the element to be
determined is measured.
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20. 20
The third field of atomic spectroscopy is atomic fluorescence. This technique
incorporates aspects of both atomic absorption and atomic emission. Like atomic
absorption, ground state atoms created in a flame are excited by focusing a beam
of light into the atomic vapor. Instead of looking at the amount of light absorbed in
the process, however, the emission resulting from the decay of the atoms excited
by the source light is measured. The intensity of this "fluorescence" increases with
increasing atom concentration, providing the basis for quantitative determination.
Most scorpions glow a blue-green color when illuminated by ultraviolet light or
natural moonlight.

21. Molecular spectroscopy
 A molecule is a collection of positively charged atomic nuclei surrounded by a
cloud of negatively charged electrons.
 Molecular spectra result from either the absorption or the emission
of electromagnetic radiation as molecules undergo changes from
one quantized energy state to another. The mechanisms involved are similar to
those observed for atoms but are more complicated. The additional complexities
are due to interactions of the various nuclei with each other and with the electrons,
phenomena which do not exist in single atoms.

22. There are two primary sets of interactions that contribute to observed molecular
spectra.
 The first involves the internal motions of the nuclear framework of the molecule
and the attractive and repulsive forces among the nuclei and electrons.
 The interaction of electromagnetic radiation with these molecular energy
levels constitutes the basis for electron spectroscopy, visible, infrared (IR) and
ultraviolet (UV) spectroscopies, Raman spectroscopy, and gas-phase microwave
spectroscopy.
 The first set of interactions can be divided into the three categories given here in
decreasing order of magnitude:
 Electronic,
 Vibrational,
 And rotational.

23.  The other set encompasses the interactions of nuclear magnetic and
electrostatic moments with the electrons and with each other.
 The second set of molecular interactions form the basis for
 Nuclear magnetic resonance (NMR) spectroscopy,
 Electron spin resonance (ESR) spectroscopy,
 And nuclear quadrupole resonance (NQR) spectroscopy.
 The first two arise, respectively, from the interaction of the magnetic moment
of a nucleus or an electron with an external magnetic field. The nature of this
interaction is highly dependent on the molecular environment in which the
nucleus or electron is located.

24. Differences between atomic spectra and
molecular spectra
Atomic Spectroscopy
It deals with the interactions of electromagnetic radiation with atoms.
Molecular Spectroscopy
It deals with the interaction of electromagnetic radiation with molecules.

25. Photometry is the science of the measurement of light, in terms of its
perceived brightness to the human eye
 It is distinct from radiometry, which is the science of
measurement of radiant energy (including light) in terms of
absolute power.
 In modern photometry, the radiant power at each wavelength is
weighted by a luminosity function that models human
brightness sensitivity.
Fundamental Laws of photometry

26. There are two principal laws utilized to produce photometric measurements. They are known as the
inverse square law and the cosine law.
 The inverse square law determines the correlation between illumination from a constant-intensity
source and its distance from the surface.
 It states that ““The intensity of illumination of surface (E) or illumination of surface (E) is
inversely proportional to the square of the distance between the surface and source”.
 In other words, the it states that the illuminance decreases with the square of the distance
between the light source and the illuminated surface. This means: if the distance between
the light source and illuminated surface is doubled, four times the luminous intensity is
needed for the same illuminance.
Fundamental Laws of photometry

27. Lambert’s Cosine Law
Lambert’s cosine law states that the strength of light on a surface of a fixed
area varies with incident angle.
When light hits a surface obliquely, the illumination of the surface is
directly proportional to the cosine of the angle θ between the direction of
the incident light and the surface normal.
It means that the radiant intensity from the ideal diffusely reflecting surface
and cosine of the angle θ between the direction of incident light and surface
normal are directly proportional. Mathematically,
E (illumination from a surface) ∝ Cos Ɵ
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28. 28

29. ⚫ Lambert’s Law : It states the relationship between the radiant
power of absorbed light with the thickness of the medium.

30. When a beam of monochromatic light is allowed to pass
through a transparent medium, the rate of decrease of
radiant power with the thickness of the medium is directly
proportional to the radiant power of the incident light.
dP is change in radiant power, db is small thickness of sample, k1 is proportionality
constant and minus sign indicate radiant power decrease
-dP P
db
-dP = k1P
db
Lambert’s Law

31.  Beer’s Law : When monochromatic light passes through a ‘transparent medium’,
the rate of decrease of transmitted radiation with the increase in the concentration
of the medium is directly proportional to the intensity of the incident light.
 Beer's Law states that the concentration of a chemical solution is directly
proportional to its absorption of light.
 Combined formed is called Lambert-Beer’s law.

32. ⚫ The law deals with the relationship between the radiant power of
the incident light and transmitted light as a function of both the
thickness of the medium and the concentration of the absorbing
species.
⚫ Lambert’s law
Absorbance = Constant x Thickness of the medium
x Concentration of the
⚫ Beer’s Law
Absorbance = Constant
medium
Lambert-Beer’s law

33. Lambert-Beer’s Law
The combined law may be given by the relation –
x
Absorbance = Constant x [Thickness of the medium]
[Concentration of the medium]
For a given material, the sample path length and concentration of the sample are
directly proportional to the absorbance of the light.

34. • the number of molecules capable of absorbing
light of a given wavelength, the the extent of light
absorption.
• The more effectively a molecule absorbs light of a
given wavelength, the greater the extent of light
absorption.
• From these guiding ideas, BEER –LAMBERT LAW
may be formulated:
▣ Where,
▣ A = absorbance
▣ I° = intensity of light incident upon sample cell
▣
▣
▣
▣
I = intensity of light leaving sample cell
c = molar concentration of solute
l = length of sample cell
Ɛ = molar absorptivity

35. Deviations to the law
The Beer-Lambert law maintains linearity under specific conditions
only.
The law will make inaccurate measurements at high concentrations
because the molecules of the analyte exhibit stronger intermolecular
and electrostatics interactions which is due to the lesser amount of
space between molecules.
This can change the molar absorptivity of the analyte. Not only does
high concentrations change molar absorptivity, but it also changes the
refractive index of the solution causing departures from the Beer-
Lambert law.
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36. Applications
The important use of the Beer Lambert law is found in electromagnetic spectroscopy.
 To analyze the drugs, for that, let’s take an example of a tablet:
 Let’s suppose we have a tablet and we don’t know which drug is present in it. Though we may know the
drug, then the question arises about what its molar concentration is.
 In electromagnetic spectroscopy, we use electromagnetic radiation (we may take UV rays), which scans
the tablet and determines the qualitative (drug present) and the quantitative (concentration) property of the
tablet.
 The same method can be used in determining the molar absorbance of bilirubin in blood plasma samples.
 We use Beer Lambert Law to conduct a qualitative and quantitative analysis of biological and dosimetric
materials that may contain organic or inorganic materials.
 We can determine the concentration of various substances in cell structures by measuring their absorbing
spectra in the cell.
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37. Limitations of the Beer-Lambert law
The linearity of the Beer-Lambert law is limited by chemical and instrumental
factors. Causes of nonlinearity include:
Deviations in absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity.
Scattering of light due to particulates in the sample.
Fluorescence or phosphorescence of the sample
Changes in refractive index at high analyte concentration
Shifts in chemical equilibria as a function of concentration
Non-monochromatic radiation, deviations can be minimized by using a relatively
flat part of the absorption spectrum such as the maximum of an absorption band
stray light.
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38. Determining An Equilibrium Constant Using
Spectrophotometry and Beer Lambert’s Law
One of the fundamental problems in chemistry is how to determine the extent of a
reaction. While not every chemical reaction goes to completion, they usually
approach an equilibrium state. When the system reaches equilibrium, the
concentrations of the reactants and products no longer change over time. This does
not mean that the reaction has ceased. In fact, the reaction continues to progress
forward, as well as backward.
It is said to be in a dynamic state of equilibrium where the rate of the products
being formed from the reactants is exactly the same as the rate of the products
being decomposed to form the reactants.
For the general equilibrium reaction
 aA + bB < = > cC + dD (1)
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39. When studying the equilibrium of chemical systems, one of the most
important quantities to determine is the equilibrium constant, Keq. At
equilibrium at a given temperature, the mass action expression is a
constant, known as the equilibrium constant, Keq. The equilibrium
expression for the reaction in Equation 1 is given as:
The value of the equilibrium constant may be determined from
experimental data if the concentrations of both the reactants and the
products are known. Additionally, all equilibrium concentrations can
be calculated if a single equilibrium concentration is known along with
all other “initial” concentrations.
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40. It may be recalled that in spectrophotometric studies, the Beer-Lambert law,
or Beer’s Law, can be used to determine the concentration of highly colored
species. Mathematically, Beer’s Law can be stated as:
A = abc
where “a” is the molar absorptivity, “b” is the path length, “c” is the
concentration, and “A” is absorbance. Molar absorptivity, a, is a
proportionality constant that has a specific value for each absorbing species
at a given wavelength. The path length, b, is the distance across the solution
in centimeters and is dependent upon the size of the cuvette. In this case, the
path length will be kept constant at 1.00 cm. The concentration, c, of the
absorbing species is in moles of solute per liter of solution.
For more details, refer this link:
https://adamcap.com/schoolwork/1470/#:~:text=The%20reaction%20is%20
represented%20by,and%20ultimately%20the%20equilibrium%20constant.
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41. Chromophores
It is Greek word
 Chroma means colour, phoros means bearer.
The term chromophore was previously used to denote a functional group of some
other structural feature of which gives a color to compound. For example- Nitro
group is a chromophore because its presence in a compound gives yellow color to
the compound.
But these days the term chromophore is used in a much broader sense which may
be defined as “any group which exhibit absorption of electromagnetic radiation in
a visible or ultra-visible region “It may or may not impart any color to the
compound.
Some of the important chromophores are: ethylene, acetylene, carbonyls, acids,
esters and nitrile groups etc.
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42. Auxochrome
An auxochrome (from Ancient Greek αὐξάνω auxanō "increase" and
χρῶμα chrōma "colour") is a group of atoms attached to a
chromophore which modifies the ability of that chromophore to
absorb light.
It is a group which itself does not act as a chromophore but when
attached to a chromophore, it shifts the absorption towards longer
wavelength along with an increase in the intensity of absorption.
Some commonly known auxochromic groups are: -OH, -NH2, -OR, -
NHR, and –NR2.
For example: When the auxochrome –NH2 group is attached to
benzene ring. Its absorption change from λ max 225 to λmax 280.
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43. Bathochromic Shift or Red Effect
The shift of an absorption maximum to a
Ionger wavelength due to the presence of
an auxochrome, or solvent effect is called
a bathochromic shift or red shift.
For example, benzene shows λmax 256
nm and aniline shows λmax 280 nm. Thus,
there is a bathochromic shift of 24 nm in
the λmax of benzene due to the presence
of the auxochrome NH2.
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44. Hypsochromic Shift or Blue Effect
The shift of an absorption
maximum to a shorter
wavelength is called
hypsochromic or blue shift.
This is caused by the removal
of conjugation or change in
the solvent polarity.
Hyperchromic Effect.
An effect which leads to an increase in
absorption intensity εmax is called
hyperchromic effect .
The introduction of an auxochrome usually
causes hyperchromic shift. For example,
benzene shows B-band at 256 nm, εmax
200, whereas aniline shows B-band at 280
nm,due to the hyperchromic effect of the
auxochrome NH2.
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45. Solvent effect on absorption spectra
Solvents play an important role in
UV spectra.
 Compound peak could be obscured
by the solvent peak. So, a most
suitable solvent is one that does not
itself get absorbed in the region
under investigation.
A solvent should be transparent in a
particular region. A dilute solution of
sample is always prepared for
analysis.
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Most commonly used
solvents are as follows.in
another solvent.
Solvent Absorption Maxima
(nm)
Water 191
Ether 215
Methanol 203
Ethanol 204
Chloroform 237
Carbon Tetrachloride 265
Benzene 280

46. Two broad categories of shift phenomena have been defined:
(1) Many, especially the larger, shifts are attributed to specific
chemical effects of the solvent on one or both electronic states of the
chromaphore. Some important specific effects are: hydrogen-bond
formation; proton or charge transfer between solvent and solute; and
solvent-dependent aggregation, ionization, and isomerization
equilibria. Such specific effects are outside the scope of this
discussion.
(2) The second broad category of shifts are attributed to physical
interactions between the solute and solvent molecules. These
interactions must be considered even when the chromaphore is not
involved in a solvent-dependent reaction.
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47. The presence of specific and nonspecific interaction between the solvent
and the solute molecules are responsible for the change in the molecular
geometry, electronic structure and dipolar moment of the solute.
These solute-solvent interactions affect the solute’s electronic absorption
spectrum and this phenomenon is referred to as solvatochromism.
Moreover, the behaviour of a solute in a neat solvent is very different from
the behaviour in mixed binary solvent systems.
In these kinds of systems, the solute may induce a change in the
composition of the solvents in the cybotactic region compared to that in the
bulk leading to preferential solvation. This situation commonly results from
specific (hydrogen bonding) and non-specific (dielectric effects)
interactions.
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48. THANK YOU
For queries
Email: yunes20171@gmail.com
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