chapter -1.pptx

PharmaceuticalAnalysis- II
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School of Pharmacy, CHS, AAU 2019/20 A.Y.

Spectroscopy
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Why we see different colors???

Outline
• Introduction
• Definition
• Electromagnetic radiation
• UV-visible spectra and its origin
• Wavelength determination
• Instrumentation
• Application
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Objectives
• To introduce to the students the principle and types of spectroscopic techniques
• To introduce to the students the principle and instrumentation of UV-visible
spectroscopy
• To introduce to the students the pharmaceutical applications of UV-visible
spectroscopy
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Learning outcomes
• At the end of this session students will be able to :
 define spectroscopy
 describe the basic principles of UV-visible spectroscopy
 describe the main components of UV-visible instrumentation
 describe the different applications of UV-visible spectroscopy
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Introduction to Spectroscopy
• Spectroscopy is the study of interaction between electromagnetic radiation
(EMR) and matter.
 The matter can be atoms, molecules or ions.
• Electromagnetic radiation is a form of energy whose behavior is described by
the properties of both waves and particles.
• Electromagnetic radiation consists of oscillating electric and magnetic fields that
propagate through space along a linear path and with a constant velocity
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 Electromagnetic radiation (EM):
 EM: the energy that radiates from all things
 in nature and from man-made electronic systems.
 EM is a form of energy and has both electrical and magnetic characteristics
 It includes cosmic rays, gamma rays, x-rays, ultraviolet light, visible light, infrared
light, radar, microwaves, TV, radio, cell phones and all electronic transmission
systems.
 It is made up of electric and magnetic fields that move at right angles to each
other at the speed of light. 7

(http://en.wikipedia.org)
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• An electromagnetic wave, therefore, is characterized by several fundamental
properties.
A. Wavelength (λ, lambda)
• Is defined as the distance between successive maxima, or successive minima.
• Different units of length are used to express wavelengths
 E.g. Angstrom, centimeter, micro and nanometer
 10 = 10-1 nm = 10-4  = 10-7 mm = 10-8 cm = 10-10 m
• For ultraviolet and visible electromagnetic radiation the wavelength is usually expressed in
nanometers (nm, 10–9 m), and the wavelength for infrared radiation is given in microns (µm, 10–6 m)
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B. Amplitude (A)
 Is the vertical distance from midline of a wave to the peak or trough.
 Measured by units of distance
C. Frequency (v, nu)
• is the number of waves that pass through a particular point in 1 second (Hz = 1 cycle/s)
• Frequency is expressed as ʋ (nu) in Hertz (Hz) or
Cycles per second (cps)
D. Wavenumber
• Number waves of per Centimeter
• as the total number of waves which can pass through a space of one cm. ῡ = 1/λ
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 Electromagnetic radiation consists of a beam of energetic particles called
photons.
 The energy of a photon, in joules, is related to its frequency, wavelength, or
wavenumber by the following equations. E = hv = hc/ λ = hc ῡ
 Where h is Planck’s constant, which has a value of 6.626 *10–34 J · s.
 Energy of the light or electromagnetic radiation is measured in
joules or Kcal/mole (1 Kcal = 4.184 KJ)
 Example-- What is the energy per photon of the sodium D line ( λ= 589 nm)? (Answer;
ῡ=5.09*1014/S, E= 3.37 *10-19J)
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The Electromagnetic Spectrum
 The frequency and wavelength of electromagnetic radiation vary over many
orders of magnitude.
 For convenience, electromagnetic radiation is divided into different regions based
on the type of atomic or molecular transition that gives rise to the absorption or
emission of photons (Figure below).
 The boundaries describing the electromagnetic spectrum are not rigid, and an
overlap between spectral regions is possible.
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The electromagnetic Spectrum
Type of
transition
Nuclear Core
level
electro
ns
Valence
electrons
Molecular
vibration
Molecular
rotations,
electron
spin
Nuclear
Spin
Spectral
region
ˠ-ray X-ray UV Visible IR Microwave Radio wave
Violet Blue Green Yellow Orange Red
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λ/E

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With matter
Interaction of electromagnetic radiation
School of Pharmacy, CHS, AAU 2019/20 A.Y. 19

• If beam of white light is passed through a beaker of water, it remains white
• If KMnO4 is added to the water---purple
– KMnO4 allows blue and red color to pass and absorbs the other colors
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• This is an example of the interaction b/n radiant energy and matter.
– In this case, the radiant energy is visible and we can see the effect of absorption with our eyes.
— However, absorption of radiation can take place over a wide range of radiant energy, most of
which can not be seen
– Such absorption effects can be measured using suitable instruments
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• The nature of the interaction between radiation and matter may include;
• Absorption: Absorption is a process whereby EM radiation is absorbed by particles or
molecules and converted to another form of energy
• Emission: The emission spectrum of a chemical element or chemical
compound is the spectrum of frequencies of EM radiation emitted due to an atoms making a
transition from a high energy state to a lower energy state. The energy of the emitted photon is
equal to the energy difference between the two states
• Scattering: Scattering is a random process whereby EM radiation is
absorbed and immediately re-emitted by particles or molecules
• Refraction, Reflection, Transmission???
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Matter is in a continuous motion
Motion could be rotational, vibration or transitional motion or combination of
these.
Each motion is associated with different level of energy.
Each motion can be made to occur at a faster rate (at higher energy level) by
applying an external energy.
This can be achieved by applying one of the regions of the EMR , since each
consists energetic particles called photons.
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• When an atom or molecule is exposed to electromagnetic radiation, the energy
can be absorbed in one of three ways:
1. The energy can promote an electron from a bonding orbital to a higher-energy
anti bonding orbital, a so-called electronic transition.
2. The energy can act to increase the vibration, or oscillation, of atoms about a
chemical bond. This is termed a vibrational transition.
3. The energy can bring about an increase in the rotation of atoms about a chemical
bond, which is a rotational transition.
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• After absorbing energy, each type of motion are promoted from the
lower energy level (Ground state, E0) to higher energy level
(Excited Level, E1 & E 2).
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• The effect of electromagnetic radiation on interaction with matter depends
on energy associated with the radiation
• Very energetic radiations (x-ray) may cause an electron to be ejected from
the molecules.
• Radiation in the infrared region of the spectrum have much less energy they
can cause vibrations in molecules.
• Microwave radiation is even less energetic than infrared radiation it can
neither induce electronic transition in molecules nor can it cause vibrations it
can only cause molecules to rotate.
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• Ultraviolet, visible
• Infrared
• Microwave
- electronic transitions
- molecular vibrations
- molecular rotation

• Types of spectroscopy
• When radiation meets matter, the radiation is either scattered, emitted or
absorbed
• So they are of three types
1.Absorption spectroscopy
2.Scattering spectroscopy
3.Emission spectroscopy
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1. In absorption spectroscopy an electromagnetic radiation is absorbed by an atom or molecule which undergoes
transition from a lower energy state to a higher energy or excited state
• Absorption occurs only when the energy of radiation matches the difference in energy between two energy levels
2. Scattering spectroscopy measures certain physical properties by measuring the amount of light that a substance scatters
at certain wavelengths .
• One of the most useful applications of light scattering spectroscopy is RAMANSPECTROSCOPY
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3. Emissionspectroscopy
• Atoms or molecules that are excited to high energy levels can decay to lower levels by emitting radiation
• The substance first absorbs energy and then emits this energy as light
• Emission can be induced by sources of energy such as flame or electromagnetic radiation
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Quiz #1
• Q1. What are electromagnetic radiation? How that is classified?
• What is electromagnetic spectrum? Name the various electromagnetic regions?
• Q2. Describe the dual nature of EMR
• Q5. Describe the relationship of wavelength , wavenumber and frequency to energy of a
EMR.
• Q6. Why is absorption and not emission spectroscopy used to study the spectra of organic
compounds? (Reading Assignment)
• Calculate the wavenumber of a beam of infrared radiation with a wavelength
of 5.00 µm. (2000 cm-1 )
• Calculate the energy in joules of one photon of radiation (3.98 3 10220 J )
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Home study for next classes
• Atoms, elements and compounds
• Atomic structure: orbitals and electronic
configurations
• Chemical bonding theories: formation
of chemical bonds, Valence Bond Theory and Molecular orbital theory (sigma bonds, pi
bonds
• Various types of chemical bonding
• Electronegativity and chemical bonding, Bond polarity and intermolecular forces
• Resonance, Induction, conjugation
• Recommended reading : Organic Chemistry, John McMurry (Cornell
University) Chapter 1, and 2.
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Uv –Vis Spectroscopy
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UV–Vis Spectroscopy
 A spectroscopic technique which utilizes the UV/Visible region of the EMR is known as UV/visible
spectroscopy/spectrophotometer/.
 Near UV region-200 nm-400 nm
 Visible region-400-800 nm
 Absorption of light in these region mainly causes electronic transition.
1. The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals
ranges from 125-650 kJ/mole
2. This energy corresponds to EM radiation in the ultraviolet (UV) region, 150-350 nm, and
visible (VIS) regions 350-800 nm of the spectrum
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UV–Vis Spectroscopy
• The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation
2. If a particular electronic transition matches the energy of a certain band of UV, it will be
absorbed (energy is quantized)
3. The remaining UV light passes through the sample and is observed
4. From this residual radiation a spectrum is obtained with “gaps” at these discrete energies –
this is called an absorption spectrum
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Uv –Vis Spectrophy
 The outer electrons in an organic molecule may occupy one of three different energy levels
(- , - or n- energy level).
 Accordingly there are three types of electrons.
a) σ-electrons; They are bonding electrons, represent valence bonds and possess the lowest
energy level ( the most stable)
b) π-electrons; They are bonding electrons, forming the pi-bonds (double bounds), and
possess higher energy than sigma-electrons.
c) n-electrons; They are nonbonding electrons, present in atomic orbitals of hetero atoms (N, O,
S or halogens). They usually occupy the highest energy level of the ground state.
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• Observed electronic transitions
• Electrons reside in orbitals. A molecule also possesses normally unoccupied orbitals called
antibonding orbitals; these corresponds to excited state energy levels and are either s* or p*.
1. The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest
Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO)
2. For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the two contributing
atomic orbitals; for every bonding orbital “created” from this mixing (, ), there is a corresponding anti-
bonding orbital of symmetrically higher energy (*, *)
3. The lowest energy occupied orbitals are typically the ; likewise, the corresponding anti-bonding * orbital is of
the highest energy (the transition is termed s - s * transition.)
4. -orbitals occupy an anti-bonding energy level (p*) and the transition is termed p - p * transition.
They are somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy
than *.
5. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than  or 
(since no bond is formed, there is no benefit in energy). the n-electrons may occupy s * or p * levels to
give n-s* or n-p* transition
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Observed electronic transitions
Here is a graphical representation

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Observed electronic transitions
From the molecular orbital diagram, there are several
possible electronic transitions that can occur, each of a
different relative energy:

Organic compounds containing -Electrons:
Compounds contain -electrons only are the saturated hydrocarbons, which
absorb below 170 nm.
They are transparent in the near UV region (200 - 400 nm) and this make
them ideal solvents for other compounds studied in this range.
They are characterized by s--s* transition only.
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Organic compounds containing n-Electrons :
Saturated organic compounds containing hetero atoms, possess n-electrons in
addition to sigma-electrons.
They characterized by the s -s* and n – s* transitions.
n-electrons can also be transited to * when they exist in unsaturated
compounds
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Organic compounds containing -Electrons :
• Unsaturated compounds containing no hetero atoms are characterized by the -
* and -* transitions, such as ethylene (CH2=CH2).
• When these compounds containing hetero atoms, they can undergo -*, -*,
n-* and n-* transitions,
• Example acetone (CH3-COCH3).
• Many inorganic compounds in solution also show absorption in the visible
region.
• Increasing order in absorption wavelength
-* <n-* < -*< n-*
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• Of these transitions, the most important are the n-π* and π -
π *
• because they involve functional groups that are characteristic
of the analyte and wavelengths that are easily accessible
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Origin of UV-visible spectra
• Because light is a form of energy, absorption of light by matter causes the energy
content of the molecules (or atoms) to increase.
• The total potential energy of a molecule generally is represented as the sum of its
electronic, vibrational, rotational energies and other energies:
• The amount of energy a molecule possesses in each form is not a continuum but a
series of discrete levels or states.
• The differences in energy among the different states are in the order:
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Origin of UV-visible spectra…
• In some molecules and atoms, photons of UV and visible light have enough energy to cause
transitions between the different electronic energy levels.
• The wavelength of light absorbed is that having the energy required to move an electron from a
lower energy level to a higher energy level.
• These transitions should result in very narrow absorbance bands at wavelengths highly
characteristic of the difference in energy levels of the absorbing species.
• This is true for atoms
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Origin of UV-visible spectra…
• However, for molecules, vibrational and rotational energy levels are
superimposed on the electronic energy levels.
• Because many transitions with different energies can occur, the bands are
broadened.
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Spectral Changes
π to π * transitions, when occurring in isolated groups in a molecule, give rise to absorptions of
fairly low intensity.
However, conjugation of unsaturated groups in a molecule produces a remarkable effect upon the
absorption spectrum.
The wavelength of maximum absorption moves to a longer wavelength and the absorption
intensity may often increase.
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Uv –Vis Spectrosopy
The same effect occurs when groups containing n electrons are conjugated with a
π electron group; e.g.,
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• Conjugation raises the energy of the HOMO and lowers the energy of the LUMO, so less energy
is required for an electronic transition in a conjugated system than in a nonconjugated system
• The more conjugated double bonds there are in a compound, the less energy is required for the
electronic transition, and therefore the longer is the wavelength at which the electronic
transition occurs.
• Notice that both the λmax and the molar absorptivity increase as the number of conjugated
double bonds increases.
• Thus, the λmax of a compound can be used to predict the number of conjugated double bonds in
the compound.
• “The λmax increases as the number of conjugated double bonds increases.”
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Thus, the characteristic energy of a transition and hence the wavelength of absorption is a
property of a group of atoms rather than the electrons themselves.
 When such absorption occurs, two types of groups can influence the resulting absorption
spectrum of the molecule: chromophores and auxochromes.
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52
Ultraviolet excitation of 1,3-butadiene results in the promotion of an electron from the
highest occupied molecular orbital (HOMO), to the lowest unoccupied molecular
orbital (LUMO).

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n - π* transition.

Chromophores
A chromophore group is a functional group, not conjugated with another group, which exhibits a
characteristic absorption spectrum in the ultraviolet or visible region.
Some of the more important chromophoric groups are:
If any of the simple chromophores is conjugated with another (of the same type or different type) a
multiple chromophore is formed having a new absorption band
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Auxochromes
The absorption of a molecule may be intensified by groups called auxochromes which
generally do not absorb significantly in the 200-800nm region, but will affect the spectrum
of the chromophore when attached to it.
These include OH, NH2, CH3
, alkoxy and Halogens.
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increase
Other substituents may have any of four kinds of effects on the absorption:
I. Bathochromic shift (red shift) – a shift to longer l; lower energy
II.Hypsochromic shift (blue shift) – shift to shorter l; higher energy
III.Hyperchromic effect – an in increase in intensity
IV.Hypochromic effect – a decrease in intensity
200 nm 700 nm
e
Hypochromic
Hypsochromic
Hyperchromic
Bathochromic

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UV Spectroscopy
Absorption characteristics of chromophores
1. Alkanes – only posses s-bonds and no lone pairs of electrons, so only the
high energy s  s* transition is observed in the far UV
This transition is destructive to the molecule, causing cleavage of the s-
bond
*
 C C
C C

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UV Spectroscopy
2. Alcohols, ethers, amines and sulfur compounds – in the cases of
simple, aliphatic examples of these compounds the n  s* is the
most often observed transition; like the alkane s  s* it is most often
at shorter l than 200 nm
Note how this transition occurs from the HOMO to the LUMO
*CN
CN
nN sp3
C N
C N
C N
C N
anitbonding
orbital

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UV Spectroscopy
3. Alkenes and Alkynes – in the case of isolated examples of these compounds the p  p* is
observed at 175 and 170 nm, respectively
Even though this transition is of lower energy than s  s*, it is still in the far UV – however,
the transition energy is sensitive to substitution
*


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UV Spectroscopy
4. Carbonyls – unsaturated systems incorporating N or O can undergo n  p*
transitions (~285 nm) in addition to p  p*
Despite the fact this transition is forbidden by the selection rules (e = 15), it is
the most often observed and studied transition for carbonyls
This transition is also sensitive to substituents on the carbonyl
Similar to alkenes and alkynes, non-substituted carbonyls undergo the p  p*
transition in the vacuum UV (188 nm, e = 900); sensitive to substitution effects

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UV Spectroscopy
4. Carbonyls – n  * transitions (~285 nm);   * (188 nm)

*
n
CO transitions omitted for clarity
O
O
C O

Uv –Vis Spectrophotometer
Aromatic Systems
I) Benzene ring :
Benzene has three maxima at 184 nm ( the most intense), 204 nm and at 254 nm.
The first two bands have their origin in the ethylenic p-p* transition, while the longest B-band
is a specific feature of benzenoid compounds.
This band abbreviated B-band, is characterized by vibrational fine structures.
In structure elucidation both the B-band and the 204-nm ethylenic band, termed E-band are
useful while the far UV band (184 nm) is unsuitable for analytical purposes.
• In polycyclic aromatic compounds, the second primary band is often shifted to longer
wavelengths, in which case it can be observed under ordinary conditions
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Aromatic Compounds
1. General Features
One would expect there to be four possible HOMO-LUMO   * transitions at
observable wavelengths (conjugation)
Due to symmetry concerns and selection rules, the actual transition energy states
of benzene are illustrated at the right:
4* 5*
6*
2
1
3
1g
B2u
B1u
E1u
254 nm
(forbidden)
204 nm
(forbidden)
180 nm
(allowed)
Expected Actual

UV-Visible spectrophotometer…
[B] Aromatic Systems…
I) Benzene ring :
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184
nm 204
nm
254
nm

[B] Aromatic Systems…
II) Monosubstituted benzenes :
 Substitution on the benzene ring can cause bathochromic and hyperchromic shifts.
 Substituents that carry nonbonding electrons (n electrons) can cause shifts in the primary and
secondary absorption bands. The nonbonding electrons can increase the length of the p
system through resonance.
 The more available these n electrons are for interaction with the p system of the aromatic ring,
the
greater the shifts will be.
 Examples of groups with n electrons are the amino, hydroxyl, and methoxy groups, as well as the
halogens.
 In addition the B band loses most of its fine structure.
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Effect of pH on absorption spectra
The spectra of compounds containing acidic or basic groups are dependent on the pH of the
medium (e.g.) phenols and amines.
UV-spectrum of phenol in acid medium (where the molecular form predominates) is completely
different from its spectrum in alkaline medium (where the phenolate anion predominates).
Spectrum in alkaline medium exhibits bathochromic shift with hyperchromic effect.
The red shift is due to the participation of the pair of electrons in resonance with the  electrons
of the benzene ring, thus increasing the delocalization of the  electrons
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-
+
H
in acid medium in alkaline medium
O
O
OH
OH
(Phenol)l max = 270 nm (phenate anion) lmax= 290 nm

Effect of pH on absorption spectra…
On the other hand, UV spectrum of aniline in acid medium shows hypsochromic (blue)
shift with hypochromic effect (decrease in absorption intensity).
This blue shift is due to the protonation of the amino group, hence the pair of electrons is
no longer available and the spectrum in this case is similar to that of benzene (thus called
benzenoid spectrum).
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NH2 NH3
In alkaline medium in acid medium
Aniline, lmax= 280 nm Anilinium ion lmax= 254 nm
+
+ H+
- H+

Effect of Solvent on absorption spectra
The solvent in which the absorbing species is dissolved also has an effect on the spectrum of the
species.
Peaks resulting from n → π* transitions are shifted to shorter wavelengths (blue shift) with
increasing solvent polarity.
 The ground state is more polar than the excited state
 Hydrogen bonding solvents with unshared electron pairs in the ground state molecule
• Lowers the energy of the n-orbital
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Effect of Solvent on absorption spectra…
Often the reverse (i.e. red shift) is seen for π → π* transitions.
The ground state of the molecule is relatively non-polar, and the excited
state is often more polar than the ground state.
As a result, when a polar solvent is used, it interacts more strongly with the
excited state than with the ground state, and the transition is shifted to
longer wavelength.
69

Effect of Solvent on absorption spectra…
For example, the figure below shows that the absorption maximum of acetone in
hexane appears at 279 nm which in water is shifted to 264 nm, with a blue shift
of 15 nm.
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Summary
• Spectroscopic techniques deal with the interaction of electromagnetic radiation with
matter.
• Electromagnetic radiation is a form of energy which has both wavelike and particle like
property.
• Based on the energy or wavelength, EMR is classified into different regions.
• EMR interacts with matter by promoting the different motions to higher energy level.
• Ultraviolet/visible spectroscopy involves the absorption of ultraviolet light by a molecule
causing the promotion of an electron from a ground electronic state to an excited
electronic state.
• The electronically excited states of organic molecules which absorb in the near-ultraviolet
and visible regions are created by the promotion of π –electrons to π *- and n-
electrons to π *-orbitals.
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Summary
• However, not all frequencies of light can be absorbed by a given molecule.
• A necessary condition for light of frequency ν to be absorbed by a molecule in its ground state is that
the energy gap between the ground state and the excited state to which excitation occurs is
exactly equal to h ν.
• If that is not true absorption will not occur and the molecule is said to be transparent to
light of frequency ν.
• The electronic absorption spectrum of a molecule is a graphical representation of the intensity of
light absorbed in producing electronic transitions in the molecule as a function of the wavelength of
the light.
• The relationship between the concentration of analyte and the intensity of light absorbed is the
basis of quantitative applications of spectrophotometer.
• In addition, features of absorption spectra such as spectral position, and shape and breadth of the
absorption band are related to molecular structure and environment and therefore can be used
for qualitative analysis.
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Calculation of λmax of an organic compound
I. Woodward's rules:
 Named after Robert Burns Woodward, are several sets of empirically derived rules
Which attempt to predict the wavelength of the absorption maximum ( λmax ) in an
ultraviolet-visible spectrum of a given compound.
A. Rules for conjugated dienes
 These rules specify a base value (214 nm) for the parent diene which is 1,3-
butadiene.
 The value is red shifted upon alkyl substitution or attachment of ring carbons or ring
residues or olefin
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R2C=CR-CR=CR2

A. Rules for conjugated dienes…
It is also affected by the presence of double bonds out side a ring (exocyclic),
extra double bonds in conjugation, and auxochromes.
74

Rules for diene and triene abspr[tion
• Value assigned for a parent heteroanular or open diene 214 nm
• Value assigned for a parent homoanular diene 253 nm
• Increment for
• Each alkyl substituent or ring residue 5 nm
• The exocyclic double bond 5 nm
• A double –bond extension 30 nm
• Auxochrome
• OCOCH3 0 nm
• OR 6 nm
• SR 30 nm
• Cl, - Br 5 nm
• NR2 60 nm
75

Rules for diene and triene abspr[tion
A. Rules for conjugated dienes… Examples
76

B. Rules for enones
77

B. Rules for enones…
• Examples
78

B. Rules for enones…
• Examples
79

α, β -unsaturated aldehydes, acids and esters follow the same general trends as enones,
but have different base values.
80

C. Rules for Benzoyl Derivatives
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C. Rules for Benzoyl Derivatives…
• Example
The Woodward’s rules work well only for conjugated polyenes having four double bonds or less.
For conjugated polyenes with more than four double bonds the Kuhn rules are used.
82

II. Simplified Kuhn and Hausser rule
• According to this rule
• λmax = 134(n)1/2 +31
• Where n is the number of conjugated double bonds
• Example
• λmax =476 nm
•
• λmax =476 nm
•
83

Quantitative
The attenuation of electromagnetic radiation as it passes through a sample is described
quantitatively by two separate, but related terms: transmittance and absorbance.
Transmittance is defined as the ratio of the electromagnetic radiation’s power exiting the
sample, PT, to that incident on the sample from the source, P0,
Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies
between 100% (no absorption) and 0% (complete absorption).
84

Quantitative
An alternative method for expressing the attenuation of electromagnetic radiation is
absorbance, A, which is defined as
A = -log T, = -log PT/Po = log Po/pt
Absorbance is the more common unit for expressing the attenuation of radiation because
it is a linear function of the analyte’s concentration.
Besides absorption by the analyte, several additional phenomena contribute to the net
attenuation of radiation, including reflection and absorption by the sample container,
absorption by components of the sample matrix other than the analyte, and the scattering
of radiation.
To compensate for this loss of the electromagnetic radiation’s power, we use a method
blank.
85

Quantitative
Absorbance and Concentration: Beer’s Law
Beer’s law states that, using a monochromatic wavelength, Absorbance is directly
proportional to concentration.
A= e bc, or A= abc, or A= A1% 1 cm b c
Where
A is absorbance
a is absorptivity where the concentration is expressed in gm/L
 ∈ is molar absorptivity where the concentration is expressed in
mol/L
C is concentration
 b is the path length of sample cell
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Example:
A 5.00x10–4 M solution of an analyte is placed in a sample cell that has a pathlength of 1.00
cm. When measured at a wavelength of 490 nm, the absorbance of the solution is found to be 0.338.
What is the analyte’s molar absorptivity at this wavelength? Ans(Molar A. = 676 cm-1 M-1
)
A sample has a percent transmittance of 50.0%. What is its absorbance? Ans (A= 0.301)
The molar absorptivity of a substance is 2.0 × 104 cm-1 mol-1 L. Calculate the transmittance through a
cuvette of path length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of the substance. Ans (T= 0.63)
87

Quantitative
Limitations to Beer’s Law
Deviations from the direct proportionality between the measured absorbance and concentration
when path length is constant may be encountered.
Assumptions of the absorption law:
The incident beam is monochromatic
The absorbers absorb independently of each other.
Incident radiation consists of parallel rays perpendicular to the surface of the absorbing
medium.
Path length traversed is uniform over the cross section of the beam.
Absorbing medium is homogenous and does not scatter the radiation.
88

Quantitative
Limitations to Beer’s Law…
Deviations from linearity are divided into three categories: fundamental,
chemical, and instrumental.
I. Fundamental Limitations:
• Beer’s law is valid only for low concentrations/diluted solutions/ of
analyte.
• At higher concentrations the individual particles of analyte no longer
behave independently of one another.
• There will be reflection, Refraction and scattering
89

Quantitative
II. Chemical Limitations
Deviations from Beer’s law also arise when an analyte associates,
dissociates or reacts with a solvent to produce a product having a different
absorption spectrum from the analyte.
III. Instrumental Limitations
• Using polychromatic radiation always gives a negative deviation from Beer’s
90

INSTRUMENTATION…
Today a wide range of instruments are available for making molecular
absorption measurements in the UV-visible range.
These vary from simple and inexpensive machines for routine work to highly
sophisticated devices.
However, the basic components of these instruments remain the same.
The five essential components of UV-VIS instruments are
• A stable radiation source
• Wavelength selector
• Sample holder
• Radiation detector or transducer , and
• Signal processing and output device
91

92
INSTRUMENTATION…
The general layout of the essential components in a
simple absorption instrument is

Radiation Sources
• A deuterium discharge lamp for UV region (160-375 nm)
• A tungsten filament lamp or tungsten-halogen lamp for
Visible and NIR regions (350 - 2500 nm)
• The instruments automatically swap lamps when scanning
between the UV and VIS-NIR regions
93

Wavelength Selectors
In spectrophotometric measurements we need to use a narrow band of wavelengths of light.
This enhances the selectivity and sensitivity of the instrument and give a more linear
relationship between the optical signal and concentration of the substance to be determined
• There are different types of wavelength selectors.
• These include Filters and moncochromators
94

A. Filters
 Either absorption or interference filters are used for wavelength selection:
• Absorption filters
Usually function via selective absorption of unwanted wavelengths and transmitting the
complementary color.
The most common type consists of colored glass or a dye suspended in gelatin and
sandwiched between two glass plates.
They have effective bandwidths from 30 to 50 nm. They are inexpensive and widely used
for band selection in the visible region.
95

A. Filters….
• Absorption filters…
• If a solution appears orange, this implies that orange light is not being
absorbed.
96

A. Filters….
Interference filters
As the name implies, an interference filter relies on optical interference to provide a
relatively narrow band of radiation.
It consists of a transparent material (calcium or magnesium fluoride) sandwiched between
two semitransparent metallic films coated on the inside surface of two glass plates.
When it is subjected to a perpendicular beam of light, a fraction passes through the first
metallic layer and the other is reflected.
97

A. Filters….
Interference filters…
Fraction that is passed undergoes a similar partitioning upon passing
through the second metallic film, thus narrower bandwidths are obtained.
98

B. Monochromators
One limitation of an absorption or interference filter is that they do
not allow for a continuous selection of wavelength.
If measurements need to be made at two wavelengths, then the filter
must be changed in between measurements.
An other limitation is that they do not give narrow band of
wavelength.
An alternative approach to wavelength selection, which provides for a
continuous variation of wavelength, is the monochromator.
These are of two types; the prism and grating monochromators.
99

INSTRUMENTATION…
B. Monochromators…
Prisms
The radiations of different colors having different wavelengths are
refracted to different extent due to the difference in the refractive
index of glass for different wavelengths.
100

Instrumentation…
In a prism monochromator, shown below fine beam of the light from the source
is obtained by passing through an entrance slit. This is then collimated on the
prism with the help of a lens.
The refracted beams are then focused on an exit slit. The prism is then rotated in
a predetermined way to provide the desired wavelength from the exit slit.
101

Instrumentation…
Gratings
A grating is made by cutting or etching a series of closely spaced
parallel grooves on the smooth reflective surface of a solid material as
shown below
The surface is made reflective by making a thin film of aluminium on it
and the etching is done with the help of a suitably shaped diamond
tool.
102

Instrumentation…
Gratings
In grating monochromator (Fig. above), a fine beam of the light from the source
falls on a concave mirror through an entrance slit.
This is then reflected on the grating which disperses it. The dispersed radiation is
then directed to an exit slit.
The range of wavelengths isolated by the monochromator is determined by the
extent of dispersion by the grating and the width of the exit slit.
Rotation of the grating in a predetermined way can be used to obtain the desired
wavelength from the exit slit.
103

Instrumentation…
 Sample cells
The UV-VIS absorption spectra are usually determined either in vapor phase or in
solution.
In order to take the absorption spectrum of the analyte it is taken in a cell called
a cuvette which is transparent to the wavelength of light passing through it.
A variety of quartz cuvettes are available for the spectral determination .These
are of varying path lengths and are equipped with inlet and outlets.
For measurements in the visible region the cuvettes made of glass can also be
used.
However, since glass absorbs the ultraviolet radiations, these cannot be used in
the UV region.
104

Instrumentation…
 Sample cells…
 Therefore, most of the spectrophotometers employ quartz cuvettes (Fig below),
as these can be used for both visible and UV region.
Usually square cuvettes having internal path length 1.0 cm are used, though
cuvettes of much smaller path lengths say of 0.1 mm or 0.05 mm are also
available.
105
The faces of these cells
through which the radiation
passes are highly polished
to keep reflection and
scatter losses to a minimum.

Instrumentation…
 Sample cells…
Now a days ‘spectral grade’ solvents are available which have high purity and
have negligible absorption in the region of absorption by the chromophore.
In a typical measurement of absorption spectrum, the solution of the sample is
taken in a suitable cuvette and the spectrum is run in the desired range of the
wavelengths.
The absorption by the solvent, if any, is compensated by running the spectrum
for the solvent alone in the same or identical cuvette and subtracting it from
the spectrum of the solution.
This gives the spectrum only due to the absorption species under investigation.
In double beam spectrometers, the sample and the solvent are scanned
simultaneously
106

Instrumentation…
 Detectors
The detectors are used to convert a light signal to an electrical signal which can
be suitably measured and transformed into an output.
The detectors used in most of the instruments generate a signal, which is linear
in transmittance i.e. they respond linearly to radiant power falling on them.
The transmittance values can be changed logarithmically into absorbance units
by an electrical or mechanical arrangement in the signal read out device.
There are three types of detectors which are used in modern
spectrophotometers.
107

Instrumentation…
A. phototube
• A phototube consists of a photoemissive cathode and an anode in an evacuated
tube with a quartz window.
• These two electrodes are subjected to high voltage (about 100 V) difference.
• When a photon enters the tube and strikes the cathode, an electron is ejected
and is attracted to the anode resulting in a flow of current which can be
amplified and measured.
108

B. Photomultiplier (PM) Tube
A photomultiplier tube consists of a series of electrodes, called dynodes.
 The voltage of successive electrodes is maintained 50 to 90 volt more positive
than the previous one.
When a radiation falls on the cathode an electron is emitted from it. This is
accelerated towards the next photoemissive electrode by the potential
difference between the two. Here, it releases many more secondary electrons.
109

Instrumentation…
B. Photomultiplier (PM) Tube…
These, in turn are accelerated to the next electrode where each
secondary electron releases more electrons.
The process continuous up to about 10 stages of amplification. The
final output of the photomultiplier tube gives a much larger signal than
the photocell.
110

Instrumentation…
C. Diode Array Detector
• These detectors employ a large number of silicon diodes arranged side by side
on a single chip.
• When a UV-VIS radiation falls on the diode, its conductivity increases
significantly. This increase in conductivity Is proportional to the intensity of the
radiation and can be readily measured.
• Since a large number of diodes can be arranged together, the intensity at a
number of wavelengths can be measure simultaneously.
111

Instrumentation…
Signal Processing and Output Devices
The electrical signal from the transducer is suitably amplified or processed
before it is sent to the recorder to give an output.
The subtraction of the solvent spectrum from that of the solution is done
electronically.
The output plot between the wavelength and the intensity of absorption is the
resultant of the subtraction process and is characteristic of the absorbing
species.
112

Types of Uv-visible spectrometers
Broadly speaking there are three types of spectrometers.
1. Single Beam Spectrometers
As the name suggests, these instruments contain a single beam of light. The
same beam is used for reading the absorption of the sample as well as the
reference.
The radiation from the source is passed through a filter or a suitable
monochromator to get a band on monochromatic radiation.
It is then passed through the sample (or the reference) and the transmitted
radiation is detected by the photodetector.
The signal so obtained is sent as a read out or is recorded.
113

Types of Uv-visible spectrometers…
1. Single Beam Spectrometers
Typically, two operations have to be performed – first, the cuvette is filled with
the reference solution and the absorbance reading at a given wavelength or the
spectrum over the desired range is recorded.
Second, the cuvette is taken out and rinsed and filled with sample solution and
the process is repeated.
The spectrum of the sample is obtained by subtracting the spectrum of the
reference from that of the sample solution.
114

2. Double Beam Spectrometers
In a double beam spectrometer, the radiation coming from the
monochromator is split into two beams with the help of a beam splitter.
These are passed simultaneously through the reference and the sample cell.
The transmitted radiations are detected by the detectors and the difference in
the signal at all the wavelengths is suitably amplified and sent for the output.
115

3. Photodiode Array Spectrometer
In a photodiode array instrument, also called a multi-channel instrument,
the radiation output from the source is focused directly on the sample.
This allows the radiations of all the wavelengths to simultaneously fall on the
sample.
The radiation coming out of the sample after absorption (if any) is then made
to fall on a reflection grating.
116

117
3. Photodiode Array Spectrometer
The grating disperses all the wavelengths simultaneously.
These then fall on the array of the photodiodes arranged side by side.
In this way the intensities of all the radiations in the range of the spectrum
are measured in one go.
The advantage of such instruments is that a scan of the whole range can be
accomplished in a short time.

School of Pharmacy, CHS, AAU 2019/20 A.Y. 118

APPLICATIONS OF UV-VISIBLE
SPECTROPHOTOMETER
119

Application #1
Principles: radiation in the wavelength range 200-800nm is passed through a
solution of a compound.
 The electrons in the molecule become excited so that they occupy a higher
quantum state and in process absorb some of the energy passing through
the solution.
The wavelength at which the solution (analyte) absorbs and the Intensity of
absorption is determined by the structure and the concentration of the
analyte respectively. Can be used for qualitative and quantitative analysis if
appropriate Instrument is used
120

Application #2
• Important advantages of spectrophotometric methods
include:
• 1- Wide applicability; large number of organic and inorganic species absorb light in
the UV-Visible ranges.
• 2- High sensitivity; analysis for concentrations in the range from 10-4 to 10-6 M are
ordinary in the Spectrophotometric determinations.
• 3- Moderate to high selectivity; Due to selective reactions, selective measurements
and different mathematical treatments.
• 4- Good accuracy; Relative errors in concentration measurement lie in the range of
0.1 to 2 %.
• 5- Ease and convenience; Easily and rapidly performed with modern instruments.
121

Application #3
Qualitative Applications
In terms of qualitative analysis of the analyte, the UV-VIS spectrometry is of a
secondary importance for the identification and the determination of structural
details.
The information obtained from it needs to be supplemented by that from IR,
NMR and mass spectrometry.
Nonetheless, it can still provide information about the presence or absence and
the nature of the chromophore in the molecule.
122

1- Identification of Chromophores
• Example, the presence of an absorbance band at a particular wavelength often
is a good indicator of the presence of a chromophore.
• Useful information about substance can be obtained via examination of
its lmax and εmax, which could be correlated with the structural features
(See the following table).
123

1. Identification of chromophores…
124
Absorption characteristics of some common
organic chromophores:

2-Confirmation of identity
• The spectrum is a physical constant, which along with melting & boiling
points, refractive index and other properties may be used for
characterization of compounds
• Although UV-visible spectra do not enable absolute identification of an
unknown, they frequently are used to confirm the identity of a substance:
125

2-Confirmation of identity…
2.1 Through comparison of the
measured spectrum with a
reference spectrum.
a) An absorption band at 254 nm
with characteristic vibrational
fine structures may be an
evidence for existence of
aromatic structure.
b) Three characteristic bands at
278, 361 &550 nm with
absorbance ratio of 2:3:1 is very
characteristic for
cyanocobalamin.
126

2.2 Identification by using Absorbance ratio
• Absorbance ratio of a given drug at two different wavelength is constant,
provided that
• beer’s Law is obeyed at the selected wavelengths
• The same concentration of the sample is used for both
wavelengths
• Absorption ratio or molar absorptivity ratio determination
• Q value
• e.g. ASA λmax 265 &299, USP tolerance Q is 265/299 be 1.5-1.56
127
2-Confirmation of identity…

3- Approximate determination of the number of double
bonds:
By using Simplified Kuhn and Hausser rule :
lmax (nm) = 134 n + 31
where n is the number of conjugated double bonds.
4- Identification of the position and/or conformation of certain functional
groups:
d g b a
C = C – C = C – C = O enones
• a-Alkyl cause red shift about 10 nm & a-OH about 35 nm
• b-Alkyl cause red shift about 12 nm & b-OH about 30 nm
• g-Alkyl cause red shift about 18 nm & g-OH about 50 nm
128

II. Quantitative Analysis ...
Scope
- Applications of spectrophotometric methods are so numerous and touch
every field in which quantitative chemical information are required.
- In general, about 90% of all the quantitative determinations are performed
by spectroscopic techniques.
- In the field of health alone, 95 % of all quantitative determinations are
performed by UV-Visible spectrophotometer and similar techniques.
129

Quantitative Analysis#1
Assay of single component
• The assay of absorbing substance may be
quickly carried out by preparing a
solution in a transparent solvent and
measuring its absorbance at a suitable
wavelength
• wavelength should be maximum
• Small errors in setting the wavelength
have a little effect on the measured
absorbance
• Higher sensitivity (high molecular
absorptivity)
130

Quantitative Analysis#2
The concentration of the absorbing substance is then calculated
from the measured absorbance using one of the three principal
methods
1. Use of standard absorptivity value
• Use of A1%
1cm or e values
• Avoids preparation of standard solution
• Reference std are expensive and difficult to obtain
• E.g. calculate the concentration of methytestosterone in an
ethanolic solution of w/c the absorbance is a 1 cm cell at its lmax
, 241nm was found to be 0.890. ( A1%
1cm =540 )
• Ans: 0.00165g/100 ml
131

Problems
• 1. The BP assay for orciprenaline tablets
• Weigh and powder20 tablets. Shake a quantity of the powder containing80 mg of
orciprenaline sulphate with 50 ml of 0.01M hydrochloric acid, filter and add sufficient 0.01M
hydrochloric acid to the filtrate to produce 100 ml. Dilute 10 ml to 100 ml with 0.01 M
hydrochloric acid and measure the absorbance of the resulting solution at the maximum at
276 nm. Calculate the content of orciprenaline sulphate taking 72.3 as the value of A (1%, 1
cm) at 276 nm.
• The following information was obtained during the assay:
Weight of 20 tablets = 2.5534 g
Weight of tablet powder assayed = 0.5266 g
Absorbance reading = 0.5878
Stated content per tablet = 20 mg.
Calculate the % of the stated content of the orciprenaline sulphate in the tablets (Answer: 98.56%)
132

• Assay in the analysis of furosemide tablet;
• Tablet powder containing ca 0.25 g of furosemide is shaken with 300 ml of 0.1 M NaOH to
extract the acidic furosemide.
• The extract is then made up to 500 ml with 0.1 M NaOH.
• A portion of the extract is filtered and 5 ml of the filtrate is made up to 250 ml
with 0.1 M NaOH.
• The absorbance of the diluted extract is measured at 271 nm.
• The A (1%, 1 cm) value at 271 is 580 in basic solution
• From the data below calculate the % of stated content in a sample of furosemide tablets:
• Stated content per tablet: 40 mg of furosemide
• Weight of 20 tablets: 1.656 g
• Weight of tablet powder taken for assay: 0.5195 g
• Absorbance reading: 0.596
• Answer: 102.4%
133

Problem
Calculate the concentration of in μg/ ml of a solution of trypthophan (M.wt.=204.2)
in 0.1 M HCl, giving an absorbance at its lmax , 277nm of 0.613 in a 4 cm cell.
(e=5432).
134

Assay of Methyldopa BP 2004
Weigh and powder 20 tablets. Dissolve a quantity of the powder containing of 0.1g
of anhydrous methyldopa as completely as possible in sufficient 0.05M Sulphuric
acid to produce 100ml and filter. To 5ml of filtrate add 2 ml of iron (II) sulphate-
citrate solution, 8ml of glycine buffer solution and suffiecient water to produce 100
ml. Measure the absorbace of the resulting solution at the maximum of 550nm.
Calculate the content of methyldopa taking 89 as the value of A (1%, 1cm) at the
maximum wavelength. (A=0.529)
135

2. Use of a calibration graphs
• Use of calibration graph Y = ax + b
• Example: the absorbance values at 250 nm
of 5 standard solutions, and sample solution
of a drug are given below:
• Conc. (μg/ml) A 250 nm
• 10 0.168
• 20 0.329
• 30 0.508
• 40 0.660
• 50 0.846
• Sample
0.661
 Calculate the concentration of the sample.
• (Y= 0.01679X-0.0008, C= 36.5 ug/ml)
136

3. Single point
standardization
Involves the measurement of the absorbance of a
sample solution and of a standard solution of the
reference substance
• By proportionality
• C test= (A sample * C std)/ A std
137

Simultaneous analysis of a two component mixture.
• When a solution of two light-absorbing substances is to be analyzed
spectophotometrically, the presence of one often affects the light
absorbing property of the other.
• If they do not interact or react light absorption will be additive.
• The analysis of such components will wholly depend on the nature of
their individual absorption spectrum.
• A two-component mixture may be analyzed by making absorbance
measurements at two characteristic maxima (one for each component)
and solving the following pair of simultaneous equations:
138

Simultaneous analysis of a two component mixture.
• At lmax(1) : A = A1 + A2 or A = e1 b C1 + e2 b C2
• At lmax(2) : A’ = A’1 + A’2 or A’ = e’1 b C1 + e’2 b C2
A and A’ are experimentally measured absorbances and e1 , e2 , e’1 and e’2
can be evaluated from individual std solutions of cpds 1 and 2.
• Thus, from these equations C1 and C2 can be calculated.
• Accuracy of this method could be increased by proper selection of lmax at which
d/ce in absorptivities are large.
139

E.g. Mixture of Co+2 and Cr3+
140

Simultaneous analysis …
Binary mixtures cannot be analyzed unless:
 Spectral data for the pure substances are available.
 The absorptivity values for the components can be easily and accurately determined
 The absorptivity values for the components are sufficiently d/t at the chosen wavelength to
permit an accurate solution of the simultaneous equations.
 The absorbance values for the mixture are accurately determined.
141

Example
• The lmax of ephedrine HCl and Chlocresol are 257 nm and 279 nm respectively and A1%1cm
values in 0.1 M HCl solution are
• Ephedrine at 257=9
• Ephedrine at 279=0
• Chlorocresol at 257=20
• Chlorocresol at 279=105
• Calculate the concentration of ephedrine HCl and Chlorocresol in a
batch of ephedrine HCl injection, diluted 1 to 25 with water, giving
the following absorbance values in 1 cm cell. (A279=0.424, and A
257=0.97)
142

• Difference Spectrophotometer- the selectivity and accuracy of
Spectrophotometric analysis of samples containing absorbing interferents may
be markedly improved by the technique of difference spectrophotometer.
• Principle: a component in a mixture is analysed by carrying out a reaction
which is selective for the analyte.
• This could be simply bringing about a shift in wavelength through adjustment
of pH of the solution in which the analyte is dissolved or a chemical reaction
such as oxidation or reduction.
• The measured value is the difference absorbance (∆A) b/n two equimolar
solutions of the analyte in different chemical forms which exhibit different
spectral characteristics.
143

• Difference Spectrophotometer…
• The criteria for applying difference spectrophotometery to the assay of a
substance in the presence of other absorbing substances are that:
 Reproducible changes may be induced in the spectrum of the analyte by the addition of
one or more reagents
 The absorbance of the interfering substance is not altered by the reagents.
 The simplest and most commonly employed technique for altering the
spectral characteristics of the analyte is the adjustment of the pH by means
of aqueous solutions of acid, alkali or buffers.
144

• Difference Spectrophotometer…
• ∆A = Aalk(total)- Aacid (total)
• = Aalk+Aint- (Aacid + Aint)
• = Aalk-Aacid
• ∆A = ∆ε .b. C
• If the substance is not affected by pH, it can be quantitatively converted by
means of a suitable reagent to a chemical species that has d/t spectral
properties to its unreacted parent species.
145

Derivative spectroscopy
• Derivative spectroscopy uses first or higher derivatives of absorbance with
respect to wavelength for qualitative analysis and for quantification.
• If a spectrum is expressed as
absorbance, A, as a function of
wavelength,, the derivative
spectra are:
146

Derivative spectroscopy…
• A first-order derivative is the rate of change of
absorbance with respect to wavelength.
• It passes through zero at the same wavelength as
λmax of the absorbance band. This is characteristic
of all odd-order derivatives.
• The most characteristic feature of a second-order
derivative is a
negative band with minimum at the same
wavelength as the maximum on the zero-order
band.
• A fourth-order derivative shows a positive band.
• A strong negative or positive band with minimum
or maximum at the same wavelength as λ max of
the absorbance band is characteristic of the even-
order derivatives.
147

• Note that the number of bands observed is equal to the derivative order
plus one.
Advantages
Derivative spectrum shows better resolution of overlapping bands the
fundamental spectrum and may permit the accurate determination of the λ
max of the individual bands.
It permits discrimination against broad band interferences, arising from
turbidity or non-specific matrix absorption.
Thus, the information content of a spectrum is presented in a potentially
more useful form, offering a convenient solution to a number of analytical
problems, such as resolution of multi-component systems, removal of
sample turbidity, matrix background and enhancement of spectral details.
148

Derivative spectroscopy…
Background elimination Resolution
149

Other Applications
A. Monitoring drug degradation kinetics
Can be simply done when the product has a different absorption spectrum than that
of un-degraded drug.
The rate of disappearance of the spectrum or appearance of other spectrum (as a
function of time ) may be used to determine rate constant for hydrolysis or
degradation.
Oxidation reactions and any other type of reactions that yield products whose
spectra are different from the reactants , may be followed and their rate constant
estimated.
150

Other Applications
B. Detection in Chromatography
 Mainly used in HPLC and HPTLC.
They are the most widely used detectors, because:
Most drugs absorb UV-Visible radiation.
More sensitive and more selective than the bulk property detectors (e.g. R.I.
detectors).
Some absorbance detectors have one or two fixed wavelengths (280 and/or
254 nm).
More modern HPLC instruments have variable wavelength detectors using the
photodiodes
151

Other Applications
C. Determination of Equilibrium Constants (Laboratory)
 Acid dissociation constants and metal ion-ligand stability constants can be determined
spectrophotometrically if the species involved have absorptivities which differ from one
another.
Example : Determination of the pKa of Methyl red indicator ;
Acidic (HMR) and basic (MR-) forms of methyl red are shown below
152
CO2-
(CH3)2N N NH
+
CO2-
(CH3)2N N=N
HO-
H+
Acid form, pH= 4, (HMR) Red, 520 nm Basic form, pH= 6, (MR-) Yellow 430 nm

Other Applications
C. Determination of Equilibrium Constants
 The pKa of methyl red indicator is given by the equation:
pKa = pH - log [MR-]/[HMR]
 Both HMR and MR- have strong absorption peaks in the visible portion of
the spectrum.
153
A
430 nm 520 nm pH
l 5.0
Measured at
520 nm
Measured at
430 nm

Other Applications
The color change interval from pH 4 to pH 6 can be obtained with acetate
buffer system.
At pH = 4, the acid is completely unionized and at pH = 6, the acid is
completely ionized
At intermediate pH values, the two species are present.
Plotting absorbance (A) against pH values at l1 and l2 gives two curves.
The pH at the point of intersection represents the pKa of the indicator.
154

Other Applications
D. Determination of complex stoichiometry
The stoichiometry for a metal–ligand complexation reaction of the following
general form.
Can be determined by one of three methods: the method of continuous
variations, the mole-ratio method, and the slope-ratio method.
i. Method of continuous variations (CVM)
Also called Job’s method, is the most popular.
In this method a series of solutions is prepared such that the total moles of metal
and ligand, ntot, in each solution is the same.
 Thus, if (nM)iand (nL)i are, respectively, the moles of metal and ligand in the i-th solution,
then
155

Other Applications
i. Method of continuous variations…
 The relative amount of ligand and metal in each solution is expressed as the mole fraction of ligand, (XL)i, and the mole fraction of
metal, (XM)i,
 Absorbance versus the mole fraction of ligand will be plotted.
CVM
156
A
L/M ratio
0.0 1.0
A
[L]/[L]+[M]

Other Applications
i. Method of continuous variations…
The intersection of the two lines drawn from both sides occurs when
stoichiometric mixing of metal and ligand is reached.
Mole fraction of ligand at this intersection is used to determine the value of y
for the metal–ligand complex, MLy.
157

Other Applications
ii. Mole-ratio method
In the mole-ratio method the moles of one reactant, usually the metal, are held
constant, while the moles of the other reactant are varied.
The absorbance is monitored at a wavelength at which the metal–ligand
complex absorbs.
A plot of absorbance as a function of the ligand-to-metal mole ratio (nL/nM)
has two linear branches that intersect at a mole ratio corresponding to the
formula of the complex.
158

Other Applications
iii. slope-ratio method
In the slope-ratio method two sets of solutions are prepared.
The first set consists of a constant amount of metal and a variable amount of
ligand, chosen such that the total concentration of metal, CM, is much greater
than the total concentration of ligand, CL.
Under these conditions we may assume that essentially all the ligand is
complexed. The concentration of a metal–ligand complex of the general form
MxLy is
159

Other Applications
iii. slope-ratio method …
 If absorbance is monitored at a wavelength where only MxLy absorbs, then
and a plot of absorbance versus CL will be linear with a slope, sL, of
 A second set of solutions is prepared with a fixed concentration of ligand that is much
greater than the variable concentration of metal; thus
 The ratio of the slopes of the two straight lines gives the combining ratio between M and
L:
160

Other Applications
E. Spectrophotometeric
titrations
 One or more of the reactants or products
absorb radiation.
 They are carried out in a vessel for which
the light path is constant.
 The absorbance is directly proportional to
concentration.
Titration Curves
• Plot of absorbance as a function of titrant
volume and consists of two straight-line
regions with different slopes
161

Other Applications
E. Spectrophotometric titrations…
Advantages
 More accurate results than direct titrimetric analysis are obtained.
 Can be used for the titration of very dilute solutions (Sensitive)
 Do not need favorable equilibrium constants as those required for titration
that depends upon observations near the end point.
 Can be used for all types of reactions (Redox, acid-base, complexometric ,
pptmetry…etc).
162

Colorimetry
 Is a technique which involves measurement of absorbance in the visible region is known as
colorimetry.
 Involves measurement of color intensity of compounds.
Requirements for colorimetry
 the substance should be colored or
 The substance should be able to be derivatized in to colored product.
While derivatizing
 The reagent should be specific
 The color produced should be stable enough until the analysis is completed
 Color intensity should be directly proportional to the concentration of the analyte.
Application- colored drugs and those drugs which can be derivatized.
163

• Thank you!!!
164

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