Pharmaceutical Analysis-II

Amha Teklu (B.Pharm, MSc. Pharmaceutical Analysis & QA).

Email : (teklueshtu@gmail.com)

1
Course Description

◼ The course deals with the applications of important instrumental

analytical techniques such as spectro-chemical methods including
UV- Visible, atomic absorption, flame spectroscopy, mass
spectroscopy and nuclear magnetic resonance spectroscopy;
chromatographic methods including Gas Chromatography and High
Performance Liquid Chromatography in quality controls of
pharmaceutical products. The course also covers applications of
biological methods in pharmaceutical analysis

08-Mar-19 2
Course objectives
◼ After completing this course, you will be able to:

❑ Develop deep understanding on the principles, instrumentation

and applications of different modern instrumental analytical
techniques and their use in pharmaceutical and biochemical
analysis .

❑ Propose suitable analytical technique for a sample

❑ Perform analysis of drugs using instrumental techniques

❑ Develop deep understanding on the principles and applications of

microbiological methods in pharmaceutical analysis

08-Mar-19 3
 Course content
◼ Introduction
◼ Uv –Vis Spectroscopy
◼ Factors governing absorption of radiation in the UV/Visible
region
◼ The concept of Chromophore and Auxochrome
◼ Absorption intensity shifts
◼ Effect of pH on absorption
◼ Effect of Solvent on absorption spectra
◼ Conjugated dienes and Wood Ward and Fiesher’s rule
◼ Instrumentation
◼ Application in pharmaceutical analysis
◼ Analysis of binary mixtures
◼ Differential spectrophotometry
◼ Derivative spectra
◼ Colorimetry: principle and applications

4
Course content…
◼ Fluorescence spectrophotometry
❑ Introduction
❑ Structural requirements and factors affecting fluorescence spectra
❑ Instrumentation
❑ Applications in pharmaceutical analysis
◼ Infrared Spectrophotometry
❑ Introduction
❑ Fundamental vibrations and factors affecting vibrational frequency
❑ Interpretation of IR spectra
❑ Instrumentation
❑ Applications in pharmaceutical analysis

08-Mar-19 5
Course content…
◼ Atomic spectrophotometry

❑ Introduction

❑ Types of atomic spectrophotometric techniques

❑ Instrumentation

❑ Application

◼ Nephelometry and Turbidometry

❑ Introduction

❑ Instrumentation

❑ Pharmaceutical applications

◼ Mass spectrometry

❑ Introduction

❑ Fragmentation pattern

❑ Instrumentation
08-Mar-19
❑ Application 6
Course content…
◼ Nuclear magnetic resonance spectroscopy

❑ Principle, instrumentation and applications

◼ Introduction to chromatography

◼ High performance liquid chromatography

❑ Principle ,instrumentation and applications

◼ Gas chromatography

❑ Principle, instrumentation and application

◼ Biological methods of analysis

❑ Introduction

❑ Microbial assay

❑ Sterility test

❑ Preservative efficacy test

08-Mar-19
❑ Pyrogen test 7
References

◼ Connors, K.A. Textbook of Pharmaceutical Analysis, 3rd or latter

edition
◼ Beckett, A.H. and Stenlake, J.B. Practical Phamaceutical Chemistry,
Parts I & II, 4th or latter edition
◼ David G. Watson. Pharmaceutical Analysis, A Textbook for
Pharmacy Students and Pharmaceutical Chemists, 2nd or latter
Edition
◼ Gary D. Christian; Analytical chemistry, 6th edition, John Wiley
and Sons
◼ USP and BP: latter editions

08-Mar-19 8
Introduction to Spectroscopy
◼ If matter is exposed to electromagnetic radiation, e.g. visible
light, the radiation can be absorbed, transmitted, reflected,
scattered or undergo photoluminescence.
◼ If beam of white light is passed through a beaker of water, it
remains white.
❑ If KMnO4 is added to the water---purple
◼ 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

9
 Introduction to Spectroscopy
◼ Spectroscopy is the study of interaction between electromagnetic radiation
(EMR) and matter.
What is electromagnetic radiation?
◼ Electromagnetic radiation, or light, 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.

10
 Intro….
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 m = 10 cm = 10 mm = 10 m = 10 nm = 10
2 3 6 9 10 o.

▪ 1 = 10 nm = 10  = 10 mm = 10 cm = 10
0 -1 -4 -7 -8 -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).

11
 Intro….
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)
D. Wavenumber
➢ Number waves of per Centimeter

ῡ = 1/λ

12
 Intro…..
❖ 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.
Example: In 1817, Josef Fraunhofer (1787–1826) studied the spectrum
of solar radiation, observing a continuous spectrum with numerous dark
lines. Fraunhofer labeled the most prominent of the dark lines with
letters. In 1859, Gustav Kirchhoff (1824–1887) showed that the “D” line
in the solar spectrum was due to the absorption of solar radiation by
sodium atoms. The wavelength of the sodium D line is 589 nm. What are
the frequency, Energy and the wavenumber for this line?
13
 Intro…..
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.

14
 The electromagnetic Spectrum
Type of Nuclear Core Valence Molecular Molecular Nuclear
transition level electrons vibration rotations, Spin
electrons electron spin
Spectral ˠ-ray X-ray UV Visible IR Microwave Radio wave
region
Violet Blue Green Yellow Orange Red

λ/E

15
 Intro…..
How does EMR interact with Matter?
 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.

16
 Intro…..
◼ After absorbing energy, each type of motion are promoted
from the lower energy level (Ground state, E0) to higher
energy level (Excited Level, E1 & E2).

 The source of the energetic state depends on the photon’s

energy.
❖ Absorption of UV/Visible affects all types of motions, the
major effect being transition of valence electrons
❖ Absorption of IR radiation results in Vibrational and
rotational energy
17
Intro…
Absorption vs Emission

◼ When applying EMR to a substance energy is transferred between a

photon of EMR and that substance i.e. the substance absorbs radiation

❑ Absorption: A transition from a lower level to a higher level with

transfer of energy from the radiation field to an absorber substance .

❑ Emission: A transition from a higher level to a lower level with

transfer of energy from the emitter to the radiation field. If no
radiation is emitted, the transition from higher to lower energy
levels is called non-radiative decay.

18
Introduction…

◼ When an atom or molecule is exposed to electromagnetic radiation, the

energy can be absorbed in three ways: (depending up on the energy of
EMR)

◼ The energy can promote an electron from a bonding orbital to a

higher-energy antibonding orbital, a so-called electronic transition.

◼ The energy can act to increase the vibration, or oscillation, of atoms

about a chemical bond. This is termed a vibrational transition.

◼ The energy can bring about an increase in the rotation of atoms

about a chemical bond, which is a rotational transition.

19
Intro….

 For absorption to occur, the energy of the photon must exactly

match an energy level in the atom (or molecule) it contacts
Ephoton = Eelectronic transition

20
Intro…

Atomic VS molecular absorption

Atomic absorption
◼ Passage of radiation through a medium that consists of mono-atomic
particles results in absorption of a few frequencies

◼ Simplicity is due to small number of possible energy states for the

absorbing particles.

◼ The energy of photon that can interact with a transition jump

depends on the energy d/c b/n the electronic levels.

21
Intro…
Molecular absorption

◼ More complex than atomic absorption because many more

potential transitions exist.

A molecule may absorb light energy in three ways:

◼ By raising an electron to a higher energy level (electronic).

◼ By increasing the vibration of constituent nuclei (vibrational).

◼ By increasing the rotation of molecule about its axis

(rotational)

E total = E electr + E vibrat + E rotat

22
Intro…

Energy levels of molecules

23
Introduction…
Analytical spectroscopic techniques

◼ Analytical spectroscopy is the science of determining how much of a

substance is present in a sample by accurately measuring how much
light is absorbed or emitted by atoms or molecules within it.

◼ Different types of spectroscopy are available, depending on the type or

wavelength of EMR absorbed or emitted by the atom or molecule (refer
to the tables in the succeeding slides).

◼ In absorption spectroscopy the energy carried by a photon is absorbed

by the analyte, promoting the analyte from a lower-energy state to a
higher-energy, or excited, state.

24
Introduction…

◼ The intensity of photons passing through a sample containing the analyte is

attenuated because of absorption. The measurement of this attenuation,
which we call absorbance, serves as analytical signal.
◼ Emission of a photon occurs when an analyte in a higher-energy state
returns to a lower-energy state .
✓ Note:The higher-energy state can be achieved in several ways, including
thermal energy, radiant energy from a photon, or by a chemical reaction.
Emission following the absorption of a photon is also called
photoluminescence, and that following a chemical reaction is called
chemiluminescence.

◼ Analytical spectroscopic techniques based on photoluminescence:

fluorescence and phosphorescence

25
Introduction…
Table: Representative Spectroscopies Involving an Exchange of Energy
Type of Region of Spectroscopic techniques
energy EMR
transfer spectrum
Absorption ɣ-ray Mossbauer spectroscopy
X-ray X-ray absorption spectroscopy
UV/Visible UV/Vis spectroscopy
atomic absorption spectroscopy
Infrared Infrared spectroscopy
Microwave Microwave spectroscopy
Radio nuclear magnetic resonance
wave spectroscopy
Emission X-ray X-ray fluorescence
UV/Visible Fluorescence spectroscopy
Phosphorescence spectroscopy
Atomic emission spectroscopy
Atomic fluorescence spectroscopy
26
Introduction…

Table: Representative Spectroscopies That Do Not Involve an Exchange of

Energy

Region of EMR Type of Spectroscopic techinques

spectrum interaction

X- ray Diffraction X-ray diffraction

UV/visible Refraction refractometry
Scattering Nephelometry and turibidimetry

27
Introduction…
Spectra

◼ Absorption spectrum is a graph of a sample’s absorbance of

electromagnetic radiation versus wavelength (or frequency or
wavenumber).

◼ It has characteristic shape with the  of maximum absorbance ( max).

◼ It is characteristic for each molecule according to its structure and the

type of transitional energy.

◼ Therefore it is used for identification of a chemical substance

(qualitative analysis). Also  max is used for quantitative measurement,
in order to increase sensitivity and to minimize error of the analytical
method.
28
Introduction…

Absorption spectrum for molecules

max

29
Uv –Vis Spectroscopy

30
 Uv –Vis Spec…..
▪ 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.

▪ 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.

31
 Uv –Vis Spec…..
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.

32
 Uv –Vis Spec…..
▪ Electrons reside in orbitals. A molecule also possess normally
unoccupied orbitals called antibonding orbitals; these corresponds
to excited state energy levels and are either s* or p*.

◼ In excited state the s -electrons occupy an anti-bonding energy

level (s *) and the transition is termed s - s * transition. p -
electrons occupy an anti-bonding energy level (p*) and the
transition is termed p - p * transition, While the n-electrons may
occupy s * or p * levels to give n-s* or n-p* transition.

33
 Types of Electronic Transitions
  Anti-bonding
 -   Anti-bonding

n-
-

 - 

n - 
-

n Non-bonding
 Bonding

 Bonding

150 200 250 300

Wavelength, nm
 Uv –Vis Spec…..
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.

❖ For example. C-H bond in methane absorbs at 125 nm Ethane has

additional absorption at 135 nm, which is due to C-C bond, C-H
bond energy is > than C-C bond energy
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
❖ Most transitions occurs at <200 nm

35
 Uv –Vis Spec…..
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-*

36
 Uv –Vis Spec…..

◼ 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

37
 Uv –Vis Spec…..
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:
38
 Uv –Vis Spec…..
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

39
 Uv –Vis Spec…..
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.

40
 Uv –Vis Spec…..
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.

41
 Uv –Vis Spec…..
❖ The same effect occurs when groups containing n electrons are
conjugated with a π electron group; e.g.,
O

CH3CCH3
200nm

❖ 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.

42
Uv –Vis Spec…..
Chromophore

◼ A chromophore (Chrome = color, phore = carrier) group is a functional

group, not conjugated with another group, which exhibits a characteristic
absorption spectrum in the ultraviolet or visible region.

❑ i.e. it is part of an organic molecule responsible to absorb light in UV-

visible region.

◼ They have unsaturated bonds (double or triple bonds) such as C=C, C=O,
N=N and C=N, ……….etc. (-electrons).

◼ 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.
43
44
Uv –Vis Spec…..
Auxochromes
◼ Auxochromes are groups which generally do not absorb significantly in the
UV-visible (200-800nm) region, but will affect the spectrum of the
chromophore to when attached to it.

◼ This is a functional group attached to the chromophore which does not

absorb light energy itself but which influences the wavelengths of light
absorbed by the chromophore.

◼ They may increase the absorption intensity of the molecule.

◼ Most often auxochromes extends the conjugation of a chromophore by

sharing of nonbonding electrons.

◼ Examples: OH, NH2, alkoxy and Halogens.

45
Uv –Vis Spec…..
Absorption and intensity shifts
Bathochromic (Red) shift
◼ Shift of absorption to longer wavelength due to substitution
and solvent effects.

◼ Alkyl substitution; Cause red shift due to hyper-conjugation

and stabilization of excited state.

◼ Attachment to auxochromes: Cause red shift and increased

absorption intensity due to extension of conjugation.

46
Absorption of radiation in the UV-visible region…
Hypsochromic (Blue) shift

◼ It is shift of absorption to shorter wavelength.

◼ Attachment of hetero atom or group to -C=O . e.g. Cl, -NH2 or –OH causes blue
shift due to resonance elevation of the p* level hence increase energy of n-p*
transition
Hyperchromic and hypochromic shift
 It is the increase and decrease in absorption intensity respectively

08-Mar-19 47
 Uv –Vis Spec…..

Hyperchromic
Hypsochromic Bathochromic
Absorbance

APEX

Hyporchromic
Wavelength, nm
Absorption characteristics of chromophores
1- Ethylenic chromophores:
❖ Their bands are difficult to observe in near UV region, so they are not
Useful analytically.
❖ However, substitution and certain structural features may cause red shift
rendering the band observable in the near UV region.
Examples:
✓ Alkyl substitution: cause red shift due to hyper-conjugation and
stabilization of excited state
✓ Exocyclic nature: cause red shift due to relaxation of strain upon
excitation.
✓ Attachement to auxochromes: cause red shift and increased absorption
intensity due to extension of conjugation.
49
 Uv –Vis Spec…..
2- Carbon-hetero atom chromophores:
❖ Such as -C=O, -C=N, -C=S, -N=O, ….etc.

❖ They exhibit some common characteristics; n-p* band in the range

of 275-300 nm., which is the most apparent band, has low
intensity and long wavelength.
❖ This band undergoes a blue shift on increasing the solvent polarity
due to increasing the energy of transition as a result of hydrogen
bonding
❖ Alkyl substitution; Cause red shift due to hyper-conjugation.

50
3-Conjugated chromophores
[A] 1,3-Butadienes and conjugated enones
CH2 = CH – CH2 – CH = CH2
Separated chromophores (by two or more
single bonds)
CH2 = CH – CH2 – CH = CH2 CH2 = CH2
have additive effect only because there is little or
no electronic interaction between separated
chromophores.
If the two chromophoric groups are present in a 170-180
molecule and they are separated by only one nm
single bond (a conjugated system), CH2 = CH – CH = CH2

CH2 = CH – CH = CH2 or CH2 = CH – CH = O

a large effect on the spectrum results, more than
found by mere addition.

170-180 205-215 nm 51
 Uv –Vis Spec…..
3- Conjugated chromophores
[A] 1,3-Butadienes and conjugated enones…
❖ The reason is that the -orbitals overlap there by decreasing the
energy gap b/n adjacent orbitals.

❖ When two chromophoric groups are conjugated such as in dienes,

the high intensity →* transition is generally red shifted by
about 15 - 45 nm with respect to the single unconjugated
chromophore.

52
 Uv –Vis Spec…..
[B] 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.

53
 Uv –Vis Spec…..
[B] Aromatic Systems…
I) Benzene ring :

184 254
nm 204
nm nm

54
 Uv –Vis Spec…..
[B] Aromatic Systems…
II) Monosubstituted benzenes :
❖ When the benzene ring is substituted with a single functional
group a Red shift occurs for both the E- and B-bands with
increase in the absorption intensity.
❖ This occurs whether the substituent is an electron donating or
electron withdrawing group.
◼ In addition the B band loses most of its fine structure.

hn
D D

hn
W X W X

55
 Uv –Vis Spec…..
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.

OH O O
-
OH
H+

in acid medium in alkaline medium

(Phenol) max = 270 nm (phenate anion)  max= 290 nm
56
 Uv –Vis Spec……
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).

NH2 +
NH3

+ H+
- H+

In alkaline medium in acid medium

Aniline,  max= 280 nm Anilinium ion  max= 254 nm

57
 Uv –Vis Spec……
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

58
 Uv –Vis Spec……
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.

59
Uv –Vis Spec……
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.

60
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

R2C=CR-CR=CR2

61
Calculation λmax of an organic compound…

Some terminologies

◼ Homoannular: Homo means 'same', annular means 'ring', so

homoannular means same ring. If a conjugated diene is contained
within one ring, it is homoannular.

◼ Heteroannular: Hetero means ‘different’. Therefore a

heteroannular diene (or polyene) is contained in two or more rings

◼ Exocyclic : Exo means 'outside', so an exocyclic alkene must be

outside of a ring, but still part of a ring. One of the alkene carbons
is part of a ring and the other is not part of the same ring.

08-Mar-19 62
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.

63
Calculation λmax of an organic compound…
A. Rules for conjugated dienes…
◼ Base value
❑ Acyclic conjugated diene 217nm
❑ Heteroannular diene 214 nm
❑ Homoannular diene 253 nm
◼ Increments

❑ Double bond extending conjugation + 30 nm

❑ Exocyclic double bond + 5 nm

❑ Alkyl substituent or ring residue + 5 nm

❑ Acetate group + 0 nm

❑ Ether group + 6 nm

❑ Thioether group + 30 nm

❑ Bromine, chlorine + 5 nm

❑ Secondary amine group + 60 nm

08-Mar-19 64
Calculation λmax of an organic compound…

Examples

08-Mar-19 65
Calculation λmax of an organic compound…
Examples…

08-Mar-19 66
Calculation λmax of an organic compound…

B. Rules for enones (conjugated carbonyl compounds)

08-Mar-19 67
Calculation λmax of an organic compound…

Example

08-Mar-19 68
Calculation λmax of an organic compound…

◼ α, β -unsaturated aldehydes, acids and esters follow the same general

trends as enones, but have different base values.

08-Mar-19 69
Calculation λmax of an organic compound…
C. rules for benzoyl derivatives

08-Mar-19 70
Calculation λmax of an organic compound…

◼ Example

08-Mar-19 71
Calculation λmax of an organic compound…
Kuhn and Häusser rule
◼ According to this rule

max (nm) = 134  n + 31

Where n is the number of conjugated double bonds

◼ 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.
Example
CH 2 OH

max (nm) = 134  n + 31

max (nm) = 134  5 + 31 = 330.6  331 nm
08-Mar-19 72
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

73
❖The general layout of the essential components in a simple
absorption instrument is

74
❑ Radiation Sources
◼ Two sources are required to scan the entire UV-VIS
band:
❑ Deuterium lamp – covers the UV part – 150-350 nm
❑ Tungsten lamp – covers visible portion - 350-800 nm

75
❑ 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 monchromators

76
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.

77
A. Filters….
Absorption filters…
◼ If a solution appears orange, this implies that orange
light is not being absorbed.

78
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.

79
A. Filters….

Interference filters…

❖ Fraction that is passed undergoes a similar partitioning upon

passing through the second metallic film, thus narrower
bandwidths are obtained.

80
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.

81
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.

82
❖ 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 collected 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.

83
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.

84
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.

85
❑ 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.
86
 ❑ 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.

❑The faces of these cells through which the

radiation passes are highly polished to keep
reflection and scatter losses to a minimum.

87
❑ 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
88
 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.

89
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.

90
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.

91
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.

92
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.

93
◼ 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.

94
◼ 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.
95
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.

96
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.

97
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.

◼ The grating disperses all the wavelengths simultaneously.

08-Mar-19 98
◼ 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.

08-Mar-19 99
Instrument calibration
◼ The calibration of an instrument involves comparison of the value or
values of a particular parameter measured by the instrument under
strictly defined conditions with pre-set standard values.
◼ Pharmacopoeial monographs usually rely on standard A(1%, 1 cm) values
in order to calculate the concentration of drugs in extracts from
formulations.

◼ In order to use a standard value the instrument used to make the

measurements must be properly calibrated with respect to its
wavelength and absorption scales.
◼ The UV-visible spectrophotometer is calibrated for absorbance scale,
wavelength scale , stray light and for its resolution power.

08-Mar-19 100
Instrument calibration…
Calibration of absorbance scale (photometric accuracy)

◼ The BP uses potassium dichromate solution to calibrate the absorbance

scale of a UV spectrophotometer, the A (1%, 1cm) values at specified
wavelengths have to lie within the ranges specified by the BP.

◼ The absorbance scale calibration wavelengths with corresponding

A(1%,1cm) values for 0.0065% w/v potassium dichromate solution which
are specified by the BP, are as follows : 235 nm (122.9-126.2),257 nm
(142.4-145.7), 313 nm (47.0-50.3), 350 nm (104.9-108.2)

08-Mar-19 101
Instrument calibration…
The UV spectrum of 0.0065% w/v potassium dichromate solution between
220 and 350 nm.

08-Mar-19 102
Instrument calibration…
Calibration of wavelength scale

◼ Wavelength accuracy is defined as the deviation of the wavelength reading

at an absorption band or emission band from the known wavelength of the
band.

◼ The wavelength deviation can cause errors in the qualitative and

quantitative results of the instrumental measurements.

◼ The wavelength scale of a UV-visible spectrophotometer is cheeked by

determining the specified wavelength maxima of a 5 % w/v solution of
holmium perchlorate.

◼ The tolerances for calibration wavelengths specified by the BP are: 214.15

± 1 nm, 287.15 ± 1 nm and 361.5 ± 1 nm (see the spectra on next slide).

08-Mar-19 103
Instrument calibration…

The absorbance maxima of 5 % w/v solution of holmium perchlorate

between 200 and 400 nm.

08-Mar-19 104
Instrument calibration…

Determination of instrumental resolution

◼ The resolving power of an instrument is controlled by its slit

width settings.

◼ For some pharmacopoeial tests a certain resolution is

specified.

◼ The resolving power of an instrument can be assessed by using

a 0.02% w/v solution of toluene in hexane. The BP specifies
that the ratio of the absorbance for this solution at 269 nm to
that at 266 nm should be at least 1.5.

08-Mar-19 105
Instrument calibration…
Determination of stray light
◼ Stray light is light which falls on the detector within a UV instrument
without having passed through the sample.
◼ It can arise either from light scattering within the instrument or by entry of
light into the instrument from outside.
◼ It gives a false low absorbance reading for the sample since it appears as
though the sample is absorbing less light than it actually is.
◼ Stray light is cheeked by measuring the absorbance of a 1.2% solution of
KCl in water against a water blank at a wavelength of 200 nm. If the
absorbance of the sample is less than 2 then stray light is present and the
instrument needs be serviced.

08-Mar-19 106
Applications in pharmaceutical analysis
◼ UV- visible spectroscopy has wide applications in pharmaceutical analysis,
these include:

❑ Quantification (assay) of drugs in formulations

❑ The UV spectrum of a drug is often used as one of a number of

pharmacopoeial identity cheeks.

❑ Determination of pka values of some drugs.

❑ Determination of partition coefficient and solubility of drugs.

❑ Used to determine the release of drugs from formulations with time

e. g. in dissolution testing.

❑ Can be used to monitor the reaction kinetics of drug degradation

08-Mar-19 107
 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.

◼ Example 1: an absorption band at 254 nm with characteristic

vibrational fine structures may be an evidence for existence of
aromatic structure.
08-Mar-19 108
Qualitative applications …

◼ Example 2: An absorption band at about 280-290 nm, which is displaced

toward shorter wavelength with increasing solvent polarity strongly, indicates
the presence of aromatic carbonyl group.
◼ Example 3: Confirmation of presence of aromatic amine or phenolic structure
may be obtained by testing the pH effect on their spectra.
NH2 +
NH3

+ H+
- H+

In alkaline medium in acid medium

Aniline,  max= 280 nm Anilinium ion  max= 254 nm

OH O O
-
OH
H+

in acid medium in alkaline medium

(Phenol) max = 270 nm (phenate anion)  max= 290 nm
08-Mar-19 109
Qualitative (Pharmacopoeial) identification of drugs
 To ascertain the presence of a specific compound in formulation or
pharmaceutical active ingredient, test and standard solutions are prepared and
the spectra‘s are recorded concomitantly for both test solution and standard
solution.

◼ There are about three ways for qualitative analysis of drugs in pharmaceutical
preparations.
1 . comparison of spectra
◼ The spectra of the test and standard drug are compared

◼ If the UV absorption spectra of the test solution and standard solution exhibit
maxima and minima at the same λ, the UV spectrum is used as a
pharmacopoeial identification checks.

08-Mar-19 110
Qualitative applications …

2. Absorptivitie ratio

◼ Absorptive values of the test and standard drug are compared

and their ratio are with in a specified limit.

❑ Example. in the USP for methyldopa the A measured at λ

max of about 280 nm of a 0.05mg/ml solution should agree
within 3.0% of USPRS Methyldopa.

08-Mar-19 111
Qualitative applications …
Example: Official limits of UV absorptivity used as identification tests ( USP )
Substance Wavelength,nm Absorptivity tolerance
Solvent

Dexamethasone Methanol 239 Do not differ by more than 3 %

Dextromethorphan HBr 0.1N HCl 278 Do not differ by more than 3 %
Dienestrol Alcohol 228 Do not differ by more than 3 %
Estradiol Alcohol 280 Do not differ by more than 3 %
Famotidine Buffer 265 Do not differ by more than 3 %
Fluorometholone Methanol 239 Do not differ by more than 3 %
Fluophenazine HCl Methanol 259 Do not differ by more than 3 %
Hydralazine HCl Water 260 Do not differ by more than 3 %
Hydrocortisone Methanol 242 Do not differ by more than 3 %
Indomethacin Cl in H2O 318 Do not differ by more than 3 %
Levodopa 0.1N HCl 280 Do not differ by more than 3 %
Nalidixic acid 0.01N NaOH 258 Do not differ by more than 3 %
Testosterone propionate Alcohol 241 Do not differ by more than 3 %

08-Mar-19 112
Qualitative applications …

3. Absorbance ratio

◼ Absorbance ration at two different wavelengths is within

specified limit and it can be calculated as follows.

Q= Absorbance (A at λ1)/Absorbance (A at λ2) = constant within

a certain limit
◼ 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

08-Mar-19 113
Quantitative applications

◼ The attenuation of electromagnetic radiation as it passes through a

sample is described quantitatively by two separate, but related
terms: transmittance and absorbance.

◼ Transmittance (T) is defined as the ratio of the electromagnetic

radiation’s power exiting the sample, It, to that incident on the
sample from the source, I0
T = It / Io

◼ Multiplying the transmittance by 100 gives the percent transmittance

(%T), which varies between 100% (no absorption) and 0%
(complete absorption).

08-Mar-19 114
Quantitative applications…
◼ An alternative method for expressing the attenuation of
electromagnetic radiation is absorbance, A, which is defined as

A = -Log T = Log Io / It

◼ 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.

08-Mar-19 115
Quantitative applications…
Beer’s and Lambert’s laws
◼ The quantitative aspects of spectrophotometry are based on two very similar laws.

◼ The first is Beer’s law , which states that ‘the intensity of a beam of parallel,
monochromatic light decreases exponentially with the concentration (c) of the

absorbing molecules at constant path length (b/l)’.

◼ Beer’s law can be expressed mathematically as:

◼ where I0 is intensity of light incident on the sample, It is intensity of light

transmitted by the sample, k is a constant and c is the concentration of the
sample.

08-Mar-19 116
Quantitative applications…

◼ When expressing the relationship in logs we have:

log Io / It = K C
log Io/It  C (concentration)

It Log Io/It

b
C b
C

08-Mar-19 117
Quantitative applications…

◼ The second relationship is Lambert’s law, which states that the

‘intensity of a beam of parallel, monochromatic light decreases
exponentially as the light travels through a thickness of
homogeneous medium’, expressed mathematically as :

◼ where I and I0 are as before, l/b is the thickness of the medium

(or path length) through which the light passes and k is (another)
constant.

08-Mar-19 118
Quantitative applications…
◼ Again expressing the relation ship in logs and graphically we have:

log Io/It  b (thickness ) log Io / It = K b

It Log Io/It

C
C
b
b

08-Mar-19 119
Quantitative applications…

◼ These two fundamental equations are so similar that they can be combined
into one relationship, the Beer–Lambert law or equation, which can be
expressed as

◼ Here k is yet another constant, the value of which depends on the units used
for the concentration term, c, and on the path length, although this is
usually 1 cm.

08-Mar-19 120
 Quantitative applications…

◼ If the units of concentration are molarity (i.e. number of moles per

litre), then the constant is  (the Greek letter ‘epsilon’) and is
known as the molar absorptivity, with units of L mol–1 cm–1,. e is
equal to the absorbance of a 1 M solution in a cell of path length 1
cm and is usually a large number, approximately 10, 000–20, 000.

◼ In this case the Beer–Lambert equation is written as

08-Mar-19 121
Quantitative applications…

◼ When the concentration of the sample is expressed in percentage weight in

volume(%w/v) or g/100 mL, the constant used is A1%,1cm, usually written
as A1 1, and is called the specific absorbance, with units of dL g–1 cm–1.

◼ The A1 1 value is very useful in pharmacy and pharmaceutical analyses

where the molecular weight of the sample may be unknown (e.g.
macromolecules such as a protein) or where a mixture of several
components is being analysed in the same sample. This gives the most
useful form of the Beer–Lambert equation:

08-Mar-19 122
Quantitative applications…

◼ If the units of concentration are in grams per liter then the constant is a
used and is known as the absorptivity

A = abC

◼ Notice that  and a can be related with the following relationship

 = a x molecular weight

08-Mar-19 123
Quantitative applications…
◼ Example 1:A 5.00x10–4 M solution of an analyte is placed in a sample cell that
has a path length 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)
◼ Example 2: A sample has a percent transmittance of 50.0%. What is its
absorbance? Ans (A= 0.301)
◼ Example 3: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)
◼ Ex 4

◼ 0.001g/100ml &1mg/100ml
08-Mar-19 124
Quantitative applications…
Limitations to Beer’s Law
◼ According to Beer’s law, a calibration curve of absorbance versus the
concentration of analyte in a series of standard solutions should be a straight
line with an intercept of 0 and a slope of ab or eb.
◼ In many cases, however, Deviations from the direct proportionality between
the measured absorbance and concentration may be encountered.

08-Mar-19 125
Quantitative applications…

◼ Assumptions of the Beer’s 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.

08-Mar-19 126
Quantitative applications…
◼ Deviations from linearity are divided into three categories: fundamental,
chemical, and instrumental.
Fundamental Limitations to Beers Law
◼ Beer’s law is valid only for low concentrations of analyte. There are two
contributions to this fundamental limitation to Beer’s law.
❑ At higher concentrations the individual particles of analyte no longer behave
independently of one another. The resulting interaction between particles of

analyte may change the value of .

❑ A second contribution is that the absorptivity, a, and molar absorptivity, ,

depend on the sample’s refractive index. Since the refractive index varies with the
analyte’s concentration, the values of a and e will change.

◼ For sufficiently low concentrations of analyte, the refractive index remains

essentially constant, and the calibration curve is linear.
08-Mar-19 127
Quantitative applications…

Chemical limitations
◼ Chemical deviations from Beer’s law can occur when the
absorbing species is involved in an equilibrium reaction.
◼ 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.
◼ Depending on the resulting products, it may result in positive
or negative deviations.

08-Mar-19 128
Quantitative applications…
Instrumental Limitations to Beer’s Law
◼ There are two principal instrumental limitations to Beer’s law. The
first limitation is that Beer’s law is strictly valid for purely
monochromatic radiation; that is, for radiation consisting of only one
wavelength.
◼ Using polychromatic radiation always gives a negative deviation from
Beer’s law, but is minimized if the value of  is essentially constant
over the wavelength range passed by the wavelength selector.
◼ Stray radiation is the second contribution to instrumental deviations
from Beer’s law. Stray radiation arises from imperfections within the
wavelength selector that allows extraneous light to “leak” into the
instrument
08-Mar-19 129
Quantitative applications…
Methods of drug assay
◼ There are two methods of using spectroscopic measurements in drug
analysis, the absolute and the comparative methods of assay.
Absolute method
◼ Most common in the British Pharmacopoeia and European
Pharmacopoeia
◼ In this procedure the absorbance is measured experimentally and the
Beer–Lambert equation is solved for c, the drug concentration. For
this reason, the British Pharmacopoeia and European
Pharmacopoeia quote A11 values in drug monographs.

08-Mar-19 130
Quantitative applications…
Comparative method of assay

◼ the comparative method of assay can be single point standardization or

calibration curve/graph method and common in USP.

◼ In this type of assay ( single point standardization) a standard solution of

the drug to be analysed is prepared, the absorbance of the sample and the
standard are measured under identical conditions, and the concentration of
the sample is calculated from the relationship

◼ where [test] is the concentration of the sample and [std] is the

concentration of the prepared standard

08-Mar-19 131
Quantitative applications…

◼ The comparative method of assay has the advantage that it can be used
even if the drug undergoes a chemical reaction during the assay (e.g.
formation of a coloured derivative to allow measurement in the visible
region of the spectrum), but suffers from the disadvantage that an authentic
sample of the drug in question must be available for comparison.

◼ In the calibration curve method it is often necessary to prepare a range of

concentrations of a standard sample of the analyte and measure the
absorbance of each solution. When these data are plotted, a straight line of
positive slope should be obtained that passes through the origin.

08-Mar-19 132
Quantitative applications…

◼ Constructing graphs of this type not only confirms that the

Beer–Lambert law applies to the assay at the wavelength of
measurement but also allows the graph to be used for
calibration purposes.

◼ A solution of unknown concentration is prepared in exactly the

same way as the standards and its absorbance is measured at the
same wavelength as the standards.

◼ This absorbance is then read off the calibration graph and the
concentration is calculated.
08-Mar-19 133
Quantitative applications…

Notice

◼ Dilution is an important aspect in preparing solutions for quantitative

analysis. Thus, the accurate preparation of the test and standard
solutions with desired stock and diluted concentrations is crucial.

◼ The wavelength selected for measuring absorbance should be

maximum

◼ Small errors in setting the wavelength have a little effect on the

measured absorbance

◼ Higher sensitivity (high molecular absorptivity)

08-Mar-19 134
Quantitative applications…
Worked examples

◼ Example 1: The A11 of cocaine at its λmax is 430. In an experiment, 11.20

mg of cocaine was weighed and made up to 1 litre with 0.1 M HCl. If the
measured absorbance in a 1 cm quartz cell was 0.470, calculate the purity
of the sample of cocaine.

08-Mar-19 135
Quantitative applications…
◼ Example 1…

◼ Example 2

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Quantitative applications…
◼ Example 2…

solution

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Quantitative applications…

Example 2…

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Quantitative applications…
◼ Example 3

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Quantitative applications…

◼ Example 3…

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Quantitative applications…
◼ Example 3 : solution

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Quantitative applications…
◼ Exercise

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Quantitative applications…
Assay by Derivatization
◼ Sometimes assay of drugs can be carried out by chemical Derivatization in
UV –visible region.

Example: assay of pencillins

◼ The pharmacopeias (usually :BP) utilize formation of a derivative in order

to quantify pencillins in formulations. This is mainly for two reasons
❑ Some penicillins do not have distinct chromophore

❑ A further problem with these molecules is that when they are in

suspensions they are not readily extracted away from excipients; since
they are quite insoluble in organic solvents which are miscible with
water.

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Quantitative applications…
assay of pencillins by Derivatization…
◼ Using the formation of a complex with the mercuric ion in the presence of
imidazole as a catalyst, a derivative of the penicillin structure is produced,
which has an absorption maximum between 325 and 345 nm.

Fig. reaction of penicillins with mercury imidazole reagent

◼ In the assay comparison with pure standard for the particular penicillin is
carried out rather than relying on a standard A1 1 value.
◼ This assay is used by the BP for analysis of preparations containing
amoxicillin, ampicillin, cloxacillin ,fluocloxacillin and others.
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Quantitative applications…
Worked example: assay of cloxacillin injection

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Quantitative applications…
Analysis of binary mixtures

◼ Beer’s law can be extended to samples containing several absorbing

components provided that there are no interactions between the
components.

◼ Individual absorbances, Ai, are additive. For a two-component mixture of X

and Y, the total absorbance, Atot, is

◼ Generalizing, the absorbance for a mixture of n components, Am, is given as

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Quantitative applications…
Analysis of binary mixtures…
◼ 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:
At  max(1)
A = A1 + A2 or A = e1 b C1 + e2 b C2
At  max(2)
◼ A’ = A’1 + A’2 or A’ = e’1 b C1 + e’2 b C2

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Quantitative applications…
◼ 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 max at

which difference in absorptivities are large.

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Quantitative applications…

◼ 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.

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Quantitative applications…

Worked examples
◼ Example 1

◼ The max 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)

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Quantitative applications…
◼ Solution
◼ A at 279nm

❑ A= A(1%1cm)b C ephe + A(1%1cm)b C chlocresol

0.424=105* C chlocresol ,because at 279 nm ephedrine doesn’t absorb

C=0.00404 g/100ml

A at 257 nm

A= A(1%1cm)*b Cephed + A(1%1cm)*b*C chlor

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Quantitative applications…
Example 2
◼ A pharmaceutical mixture contains two drugs, sulfanilamide and
sulfathiazole. When the UV spectrum of the mixture was obtained, it was
found that the two spectra overlapped as shown in the next slide . Pure
samples of each drug were available and the spectrum for each drug was
Obtained under identical conditions. Using the data tabulated below,
calculate the concentrations of each drug in the mixture.

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Quantitative applications…
Spectra of the two component mixtures

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Quantitative applications…
solution

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 Quantitative applications…
◼ Solution..

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Difference Spectrophotometry
◼ In difference spectroscopy a component in a mixture is analyzed 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.
◼ The selectivity and accuracy of Spectrophotometric analysis of samples
containing absorbing interferents may be markedly improved by the
technique of difference spectrophotometer.

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Difference Spectrophotometry…

◼ The criteria for applying difference spectrophotometry 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.

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Difference Spectrophotometry…

∆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.

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Difference Spectrophotometry…

Worked example : analysis of aspirin in dextropropxyphene.

◼ Selective alkaline shift of aspirin is used to determine it in a preparation
containing dextropropoxyphene, naphthalene sulphonic acid and caffeine.
◼ Dextropropoxyphene, naphthalene sulphonic acid anion and caffeine do not
undergo applicable alkaline shifts where as aspirin does .

Fig. UV difference spectrum used in the quantification of aspirin

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Difference Spectrophotometry…

Analysis of aspirin in dextropropxyphene compound tablet…

◼ the analysis is carried out by using standard additions which

involves adding known amount of aspirin standard to the
sample and comparing the absorbance of the original sample
extract with the absorbance of the spiked sample.

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Difference Spectrophotometry…

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Difference Spectrophotometry…

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Derivative spectra

◼ Derivative Spectrophotometry is an analytical technique which consists

in the differentiating of normal spectrum by mathematical
transformation of spectral curve into a derivative (first- or higher
derivatives).

◼ In calculus, the derivative of a function is the instantaneous rate of

change of that function with respect to a variable. This can also be
thought of as the slope of the graph or function at any point on the graph.

◼ Derivative spectroscopy uses first or higher derivatives of absorbance with

respect to wavelength for qualitative analysis and for quantification.

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Derivative spectra…

◼ If a spectrum is expressed as absorbance, A, as a function of wavelength,,

the derivative spectra are:

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Derivative spectra…

◼ 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.

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Derivative spectra…

Advantages
✓ Derivative spectrum shows better resolution of overlapping bands of
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.

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Derivative spectra…

i. Enhancement of spectral details- resolution of overlapping peaks

◼ the derivative centroid bandwidth of the even order derivatives decreases

with increasing order (look at the derivative spectra on the previous slide).

◼ Relative to the zero-order spectrum the derivative centroid bandwidth for a

Gaussian band is observed to decrease to 53 %, 41 %, and 34 % of the
original bandwidth in the second, fourth, and sixth orders respectively.

◼ This feature may be used in qualitative analysis to identify the presence of

two analytes with very similar λmax values that are not resolved in the
absorbance spectrum .

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Derivative spectra…
◼ In absorbance mode, when two Gaussian bands with 40 nm natural band
width (NBW) and separated by 30 nm, are added the result is a single band
with a maximum midway between the two component bands.
◼ The two components are not resolved. In the fourth derivative the presence
of these two bands is clearly visible with maxima centered close to the max
of the component bands.

Fig resolution enhancement

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Derivative spectra…
◼ Although the bands have been resolved (spectra in the above slide) there is
no indication of whether these arise from two chromophores in a single
compound or in two different compounds.

◼ It is often claimed that this increased resolution and the increased

differentiation between spectra in the derivative mode allows
multicomponent analysis of mixtures of components with similar spectra
that cannot be resolved in the absorbance mode.

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Derivative spectra…
ii. Elimination of baseline shift
◼ A common, unwanted effect in spectroscopy is baseline shift. This may arise
either from instrument (lamp or detector instabilities) or sample handling
(cuvette repositioning) effects.
◼ Because the first derivative of a constant absorbance offset is zero, using the
first derivative spectra always eliminates such baseline shifts and improves the
accuracy of quantification.

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Derivative spectra…
iii. Discrimination
◼ The other important effect of the derivative process is that broad bands are
suppressed relative to sharp bands and this suppression increases with
increasing derivative order.

◼ This arises from the fact that the amplitude of a Gaussian band in the nth
derivative is inversely proportional to the original bandwidth,, raised to the
nth degree:

◼ This property is used to improve the accuracy of quantification of a narrow

band component in the presence of a broad band component and to reduce
error caused by scattering.
◼ The broad band component may arise from matrix effect.

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Derivative spectra…
◼ The matrix effect often occurs in liquid formulations such as syrups and
linctuses where a small amount of a highly coloured dye is used to colour the
mixture i.e. the broad absorption bands usually may arise from these excipients.

Fig. broadband discrimination

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 Derivative spectra…
Example : assay of Ephedrine Elixir (BP)

◼ This preparation is an oral solution containing 0.3% w/v of ephedrine

hydrochloride in a suitably flavoured vehicle. The elixir is pink in colour and
the presence of the pink dye interferes with the simple UV assay, but the
preparation may be analyzed by derivative spectroscopy as follows:
❑ A number of standard dilutions of pure ephedrine hydrochloride are prepared and
their D2 spectra are measured. A calibration curve of D2 peak height vs
concentration is drawn.

❑ The elixir is diluted and the D2 spectrum of the elixir is obtained. then the
concentration of ephedrine is obtained form the curve.

◼ Ephedrine has a simple benzenoid absorbance (max 263 nm;) which appears
sharp and clear against the background absorbance when the D2 spectrum is
obtained.
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Derivative spectra …

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Other applications of UV-Visible spectrophotometry

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.

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Other applications…
Determination of equilibrium constants
◼ 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

CO2- CO2-
+ HO-
(CH3)2N N NH (CH3)2N N=N
H+
Acid form, pH = 4, (HMR) Red, 520 nm Basic form, pH = 6, (MR-) Yellow 430 nm
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Other applications…

◼ 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.

A
Measured at
520 nm

Measured at
430 nm

 430 nm 520 nm pH 5.0

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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 1 and 2 gives two

curves.

◼ The pH at the point of intersection represents the pKa of the

indicator.

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Other applications…

◼ A general equation for determination of pKa from absorbance measurement at

a particular wavelength is given below. The following equation can be used
for an acid (for a base the log term is subtracted)where increasing pH producers
a bathochromic/hyperchromic shift:

Where A is the measured absorbance in a buffer of known pH at the wavelength

selected for analysis ,Ai is the absorbance of the fully ionized species and Au is the
absorbance of the un-ionized species.
◼ The wavelength used for analysis is one where there is the greatest difference
between the ionized and unionized species

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Other applications…

◼ Example

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Other applications…

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

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Other applications…
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

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Other applications…

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 ,

precipitation …etc.).

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 Colorimetry
➢ 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.

185