PHARMACEUTICAL ANALYSIS-II.ppt

Pharamacuetical
Analaysis II
By:Salahadin A.
1

2
UV- Visible
spectrophotometry

Application of instrument Technique in
Quality control Pharmaceuticals
 Pharmaceutical analysis- deals with methods for
determining the chemical composition of pharmaceutical
samples.
 Two types
 Classical (or so-called wet chemical methods) and
 Instrumental
 Instrumental methods involve studying the physical
properties of analytes.
 Conductivity, electrode potential, light absorption or emission, mass-
to-charge ratios are properties often probed.
 Qualitative - chromatography, electrophoresis and identification by measuring
physical property
 (e.g. spectroscopy, electrode potential)
 Quantitative - measuring property and determining relationship to conc.
3

Types of Instrumental Methods
Property Example
 Radiation emission Emission spectroscopy:
fluorescence,
 Radiation absorption Absorption spectroscopy -,
Spectrophotometry,
photometry
 Radiation scattering Turbidity, Raman
 Radiation refraction Refractometry,
interferometry
 Radiation diffraction X-ray
 Radiation rotation Polarimetry, circular
dichroism
 Electrical potential Potentiometry
 Electrical charge Coulometry
 Electrical current Voltammetry - polarography
 Electrical resistance Conductometry
4

Introduction
5
 UV-Visible spectrophotometry is the method of choice in
most laboratories in:
 pharmaceuticals
 nucleic acids
 proteins, foodstuffs and fertilisers
 in mineral oils and in paint.
• Modern spectrophotometers are:
quick
 accurate
reliable and
make only small demands on the time and skills of
the operator.

Intro…
6
Spectroscopy
• Is the study of interaction between electromagnetic
radiation and matter.
Spectrophotometry
 It is more specific than the general term Electromagnetic
spectroscopy in that spectrophotometry deals with visible
light, near-ultraviolet, and near-infrared.
 The color of a solid object is determined by what wavelengths are
reflected by the object (the other wavelengths being absorbed).
 The color of a solution is determined by those wavelengths that are
absorbed or transmitted by the molecules in that solution.

Electromagnetic radiation
7
 EMR is a form of energy whose behavior is described by
the properties of both waves and particles.
 The optical properties of EMR, such as diffraction, are
explained best by describing light as a wave.
 Many of the interactions between EMR and matter, such as
absorption and emission, however, are better described by
treating light as a particle, or photon.
 The exact nature of electromagnetic radiation remains
unclear

EMR..
9
 The human eye is only sensitive to a tiny proportion
of the total electromagnetic spectrum between
approximately 380 and 780 nm and within this area
we perceive the colors of the rainbow from violet
through to red.
 If, however, only a portion of the light is absorbed
and the balance is reflected, the color of the sample
is determined by the reflected light.
 Thus, if violet is absorbed, the sample appears
yellow-green and if yellow is absorbed, the sample
appears blue.
 The colors are described as complementary.

EMR…
10
 However, many substances which appear colorless do have
absorption spectra.
 In this instance, the absorption will take place in the infra-red or
ultraviolet and not in the visible region

Wave Properties of EMR
• EMR consists of oscillating electric and magnetic fields
that propagate
through space along a linear path and with a constant
velocity.
• In a vacuum, EMR travels at the speed of light, c, which is
3 x 108 m/s
EMR……
1
1

 An electromagnetic wave is characterized by several
fundamental properties, such as:
1-Wavelength (λ, lambda): which is the linear distance
measured along the line of propagation, between crest of
one wave to that of the next wave.
2-Amplitude: which is the vertical distance from midline of
a wave to the peak or trough.
12
EMR……

3- Frequency (v, nu) is the number of waves that pass through a
particular point in 1 second (Hz = 1 cycle/s)
4- Wavenumber ( , nu par): number of waves per centimeter
and which is expressed in cm-1.
 (  ) = 1/ , cm-1 .
 Relations between ,  and  : are given by the following equations:
C =  x , Since  = 1/
Then  = 1/ = /C Or C =  / 
Where C is the velocity of light in vacuum = 3 x 1010 cm/Sec.
EMR……
13

Example:
if we have a visible radiation of 500 nm, then:
 in cm = 500 x 10-7 = 5 x 10-5 cm.
 = 1/ = 1/5 x 10-5 = 0.2 x 105 = 2 x 104 cm-1.
and  = C X  = 3 X 1010 . 2 X 104 = 6 X 1014 Hz
14

EMR…
15
Light as energy
 Light like any other matter consists of energy packets called
photons.
 The absorption and emission of light by compounds occur
in these packets (photons).
 The energy (E) of a photon is directly proportional to the
frequency and inversely proportional to the wavelength.
 It can be related to C,  and  by the following equation:
E = h = h C/
Where h is a constant called Planck’s constant , which equal to
6.625 x 10-27 erg. sec.

Example: What is the energy of a 500 nm
photon?
 = c/  = (3 x 1010 cm s-1)/(5.0 x 10-5 cm)
 = 6 x 1014 s-1
E = h =(6.626 x 10-27 erg.s)(6 x 1014 s-1) = 4.0 x 10-
12 erg
16

How Light Interacts With Matter
As radiation passes from a
vacuum through the
surface of a
portion of matter, the
electrical
vector of the radiation
interacts
with the atoms and
molecules
17

How Light Interacts….
18
 The nature of the interaction depends upon the
properties of the matter.
 Each interaction can disclose certain properties of the
matter.
refraction
transmission
absorption
reflection scattering

 There are two types of absorption
 Atomic
 Molecular
19

20
Atomic absorption
 In an atom there are a number of shells and of
subshells where e-’s can be found.
 The energy level of each shell & subshell are
different and quantised.
 The exact energy level of each shell and sub-shell
varies with sub.
 Under normal situation an e- stays at the lowest
possible shell - the e- is said to be at its ground
state.
 Upon absorbing energy (excited), an e- can
change its orbital to a higher one - we say the e- is
at its excited state.

21
 The excitation can occur at different degrees
 Low E tends to excite the outmost e-’s first
 An e- at its excited state is not stable and tends to return its
ground state.

22
ΔE transition = E1 -
E0 = hv = hc/
Absorption and emission for the sodium atom in the gas phase, Illustrates discrete energy
transfer
ΔE transition =
E2 - E0 = hv = hc/
590 nm
330 nm

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
23

24
Molecular spectrum

25
 When a molecule interacts with photons in UV-Vis
region, the absorption of energy results in displacing
an outer electron in the molecule is given by the
equation:
E = Es - Eg = h = h C/
 The energy E associated with the absorption bands
of a molecule is given by:
E = E electronic + E vibrational + E rotational
E electronic > E vibrational > E rotational
 The number of possible energy levels for a molecule is
much greater than for an atomic particles

26
 The energy levels of a molecule at each state / sub-state
are quantised.
 To excite a molecule from its ground state (S 0) to a higher
E state (S 1, S 2, T 1 etc.), the exact amount of energy
equal to the difference between the two states has to be
absorbed.
 Ordinarily, the lifetime of an atom or molecule excited
by absorption of radiation is brief because several
relaxation processes exist which permit its return to
1
,
0
2
,
2
' v
v S
S E
E
hcv 


S0
T1
S2
S1
v1
v2
v3
v4
v1
v2
v3
v4
v1
v2
v3
v4
v1
v2
v3
v4
Inter- system
crossing
Internal
transition
B
B
E1
E2
C
F
A
B
Fluorescence
D
Phosphorescence
27

Spectrum
28
A


Line
spec.(atoms)
max
 It is the display of the energy level
of
EMR as a function of wave
number or
wavelength of EMR energy.
The energy level of EMR is
usually
expressed in terms of
absorbance,
transmission, Intensity.
 It may be:
a) line spectrum: occur with atomic

Spectrum…
29
b) band spectrum: occurs with molecules due to the
presence of different vibrational and rotational sub-
levels which the molecules may occupy on
transition to excited state.
 What an spectrum tells
 A peak (a valley) represents the A (T) of EMR at that
specific wavenumber or wavelength.
 The wavenumber or λ at the tip of peak is the most
important, especially when a peak is broad.

Spectrum
30
A broad peak may sometimes consist of several peaks
partially overlapped each other.
The height of a peak corresponds the amount
absorption/emission thus can be used as a
quantitative information (e.g. conc).
 There are two parameters which define an absorption
band :
1. Its position (max) on wavelength scale
2. Its intensity on the absorbance scale.

UV-Visible Spectrophotometry
31
 What occur to a molecule when absorbing UV-visible
photon?
 A UV-visible photon (200-700nm) promotes a bonding or non-bonding
electron into antibonding orbital - the so called electronic transition
 Bonding e-’s appear in s & p molecular orbitals; non-bonding in n
 Antibonding orbital's correspond to the bonding ones
 Molecules which can be analyzed by UV-visible
absorption are:
 Chromophores -functional groups each of which absorbs a
characteristic UV or visible radiation.

Types of electronic transitions
 Absorption of radiation in the UV-VIS region depends upon the
number and arrangement of electrons in absorbing molecules.
 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) δ-electrons: possess the lowest energy level ( the most stable)
b) П -electrons; forming the П -bond and possess higher energy
than δ-electrons.
c) n-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

Types of electronic…
33
 In excited state:
The δ -electrons occupy an anti-bonding energy level
(δ *) and the transition is termed δ - δ * transition.
П -electrons occupy an anti-bonding energy level (П
*) and the transition is termed П - П * transition
 While the n electrons may occupy δ* or П * levels to
give n- δ * or n- П * transition.

34
 electronic transitions of formaldhyde

 Bonding
 Bonding
n- Nonbonding
*- Antibonding
*- Antibonding
150 200 250 300 350
Wavelength, nm
Energy
*
Transtion
n-*
Transtion
*
Transtion
n-*
Transtion
35

36
Organic compounds containing -Electrons:
 Compounds contain -electrons only are the
saturated HC, which absorb below 170 nm (in the
far UV region).
 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 characterized by  -  * transition only.

37
Organic compounds containing n-Electrons :
 Characterized by the  -  * & n – * transitions.
 The majority of these compounds show no
absorption in near UV region.
 They are useful as common solvents in near UV
region.
 However, their intense absorption usually extends to
the edge of near UV producing what is called end
absorption (cut off wavelength) mostly in the 200

Organic compounds containing -Electrons :
 Unsaturated compounds containing no hetero atoms are
characterized by the -*
and -* transitions, such as (CH2=CH2).
 When these compounds containing hetero atoms, they can undergo
-*, -*, n
* and n-* transitions, example acetone (CH3-COCH3).
Solvent , nm Solvent  , nm
Water 190 Chloroform 247
Ether 205 Carbon tetrachloride 257
Ethanol 207 Benzene 280
Methanol 210 Acetone 331
Cut-off wavelengths of some common solvents:
38

Some important terms
39
Chromophores: (Chrome = color, phore = carrier).
 They are functional groups, which confer color on substances
capable of absorbing UV and/or visible light (200 - 700 nm).
 They have unsaturated bonds (double or triple bonds) such as
C=C, C=O, N=N and C=N, ……….etc (-electrons).
Auxochromes:
 They are functional groups which can not confer colors on
substances but have the ability to increase the coloring power of
Chromophores.
 They does not absorb radiations longer than 200(absorbed far)
nm, but when attached to a given chromophore, causes a shift
to a longer wavelength with increase in absorption intensity.

Some important…
40
Bathochromic (Red) shift:
 Shift of absorption to longer wavelength due to substitution
and solvent effects.
 Multiple conjugations -Reduce transition energy-high
sensitivity
-Enhance probability of transition.
 CH2=CHCH2CH2CH=CH2 λ max =185 nm &
 CH2=CHCH=CH2 λmax =217
Hypsochromic (Blue) shift:
 It is shift of absorption to shorter wavelength.
Hyperchromic & hypochromic effects:
 It is the increase and decrease in absorption intensity
respectively.

Absorption characteristics of
Chromophores
41
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.

2. Carbon-hetero atom chromophores: -C=O, -
C=N, -C=S…
 They exhibit some common characteristics; n-  *
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 H
bonding
 Alkyl substitution; Cause red shift due to hyper-
Absorption Characteristics…
42

3. Conjugated Chromophores
43
CH2 = CH2
CH2 = CH – CH2 – CH = CH2
170-180 nm
170-180 205-215 nm
CH2 = CH – CH = CH2
have additive effect only
because there is little or
no electronic interaction
b/n separated
Chromophores.
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 Chromophores

44
4. 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
* transition, while the longest λ band is a specific
feature of benzenoid compounds.
 This band abbreviated B-band, which is
characterized by vibrational fine structures

 In structure elucidation both the B-band and the 204-
nm ethylenic
band, (E-band) are useful while the far UV band (184
nm) is
unsuitable for analytical purposes.
184 nm
204 nm 254 nm
45

(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.
D D
W X W X
h
h
46

 Which compound in each of the following pairs is likely
to absorb radiation at longer wavelength (Give reasons) :
CH3-CH2-CH3 or CH3-CH=CH2
CH3-CH2-CH=CH2 or CH3-CH2-CH=O
CH3-CH2COOH or CH3-CH2CH=O
CH2=CH-CH=CH2 or CH2=CH-CH2-
CH=CH2
4
7

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 is completely different
from its spectrum in alkaline medium
 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 
48

Effect of pH on….
49
-
+
H
in acid medium in alkaline medium
O
O
OH
OH
(Phenol)max = 270 nm (phenate anion) max= 290 nm

 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
In alkaline medium in acid medium
Aniline, max= 280 nm Anilinium ion max= 254 nm
+
+ H+
- H+
Effect Of pH On….
50

Effect of Solvents on absorption
spectra
 The solvents may have a strong effect on the position of
max due to its effect on the energy of transition.
-* Transitions: Two cases arise;
1- -* bands of dienes:
 Not shifted by any change of solvent polarity due to
absence of charge separation in either ground or
excited states.
2- -* bands of enones:
 Are red shifted on increasing solvent polarity due to
stabilization of excited state by dipole-dipole solvent
interaction.
51

n-* Transition:
 Blue shift with increasing solvent polarity due to stabilization
of the
ground state by hydrogen bonding: R-C=O…….HOR
 Hydrogen bonding lowers the energy of the ground state
(i.e.) increase
energy of transition and hence decrease wavelength, .
G2
G1
G
S2
S1
 - * bands of enones n - * bands of enones
Effect of Solvents…
52

Calculation of max of an organic
compound
 These rules specify a base value ( 214 nm ) for the parent diene
which is 1,3-butadiene.
R – CH = CH – CH = CH – R’
 The value of 214 nm is red-shifted upon:
 alkyl substitution or attachement of ring carbons (Ring residues)
 by the presence of double bonds outside (exocyclic) a ring.
CH2
endocyclic exocyclic
R
CH3
CH3
R
Counted Ring residues and alkyl substitutions
I- According to Woodward Feiser Rules:
(A) Rules For conjugated dienes:
53

homoannular heteroannular
 Also the presence of the two double bonds within the same ring
(homoannular)
gives a different value from that of heteroannular systems where the
two double
bonds occur in two different rings (and still in conjugation).
 Addition of EXTRA double bonds in conjugation.
 Attachment of AUXOCHROMES to the conjugated system.
OCH3
SH
Auxochrome attachements
Extra double bond
Calculation of .max….
5
4

OCH3
OAc
Cl
SH
OH
Check that this chemical compound containing
2-E xtra double bonds
5- Auxochromes attachements
5- Ring residues
No alkyl substitutions
3- E xocyclic double bounds
One homoannular nature
5
5

Woodward Rules for Conjugated Dienes can be summarized as :
Component nm
Base value for heteroannular or opened-chain dienes 214
Base value for homoannular dienes 253
Add the following Values to the base value:
(a) Each extra double bond in conjugation 30
(b) Each Alkyl Substituent or ring residue 5
(c) Each exocyclic nature 5
(d) Each auxochrome has its corresponding value:
- OAc 0
- OR (including OH) 6
- SR (including SH) 30
- Cl or Br 5
- NR2 (including NH2 & NHR) 60
(e) Solvent Correction 0
5
6

The following examples illustrate the use of these rules:
Basic Value 214 253 253
Extra D.B. --- --- 30
Exocyclic D.B. 5 --- 5
Ring residue 15 10 15
Alkyl Substituent 5 5 10
Auxochromes
OR 6 6 6
SR --- --- 30
Cl & Br --- 5 5
NR2 60 60 ---
Calculated max 305 339 354
OR
NH2
OH
Cl
NH2
OH
Cl
Cl
SH
OCH3
5
7

Calculate the max of the following compounds :
Cl
OH
Cl
OH
Cl
OR
OCH3
NH2
OR
Cl SH
OH
Br
OR
OH
Cl
Cl
Br
OAc
SH
OH
OR
Cl Br
OR
NH2
Cl
NH2
CH2
Br
5
8

II- Simplified Kuhn and Hausser rules:
These rules can be used for calculating max for conjugated polyenes as follows:
max (nm) = 134 n + 31
where n is the number of conjugated double bonds.
Example : Calculate the max of the following compound :
max = 134 5 + 31 = 330.6 or 331 nm
This rule is also useful for calculating number of double bonds from the observed max as n
= (max - 31/134)2
Example : If max of a compound is 433 nm calculate the approximate number of double
bonds :
The number of double bonds (n) = [(433 –31) / 134]2
= 9
CH2OH
5
9

 Using the simplified Kuhn and Hausser rules, Calculate the approximate
λmax for the following compounds :
Calculate the approximate number of double bonds present in each compound , if
you gave the following λmax for each:
1- 420 nm , 2- 530 nm , 3- 485 nm
4- 565 nm 5- 612 nm 6- 710 nm
CHOH
OH
C CH
CH
OH
6
0

Instrumental design
Components
61
 A spectrophotometer is an instrument for measuring the
T or A
of a sample as a function of the wavelength of EMR.
 The key components of a spectrophotometer are:
1. Source that generates a broad band of EMR
2. Wavelength selectors
3. Sample holder
4. One or more detectors to measure the intensity
of radiation
5. Signal Processor

i- Light Sources. Two types:
1- Continuous Sources: which produce spectra over a broad range(e.g.):
 Tungsten lamp (provides visible spectrum; 400-1200 nm)
 Deuterium lamp (provides ultra-violet spectrum; 190-400 nm)
2- Discontinuous or Discrete Sources: which produce only specific
(discrete) wavelengths .
 Hollow cathode lamp (HCL)
 Electrodeless discharge lamp (EDL)
Tungsten Lamp Deuterium Lamp Hollow cathode lamp
Instrum…
6
2

Instrum…
 The ideal light source would yield a
constant intensity over all
wavelengths with low noise and
long-term stability.
 Two sources are commonly used in
UV-visible spectrophotometers.
a) Deuterium arc lamp: yields a good
intensity continuum in the UV region
 Although modern deuterium arc
lamps have low noise.
 Over time, the intensity of light
from a deuterium arc lamp
decreases steadily.
 Such a lamp typically has a half-
life of approximately 1,000 h.
63
deuterium arc lamp

Instrum…
b) Tungsten-filament: consists of a
tungsten filament contained in a
glass envelope.
 The life of the lamp is limited by
the evaporation of tungsten.
c) Tungsten-halogen lamp:
• The halogen gas prevents the
evaporation of tungsten and increases
the lifetime of the lamp to more than
double that of the ordinary tungsten
lamp.
• yields good intensity over part of the UV
spectrum and over the entire visible
range.
 This type of lamp has very low noise
and low drift and typically has a useful
life of 10,000 h.
64
tungsten-halogen lamp

Instrum…
 Either a source selector is used to
switch between the lamps as
appropriate, or the light from the two
sources is mixed to yield a single
broadband source.
 An alternate light source is the
xenon lamp which yields a good
continuum over the entire UV and
visible regions.
 The noise from currently available
xenon lamps is significantly worse
than that from deuterium or tungsten
lamps
 Xenon lamps are used only for
applications in which high intensity
is the primary concern.
65
xenon lamps

ii. Wavelength selectors
 Narrower bandwidth tend to enhance the sensitivity and
selectivity of the absorbance measurements and give a more
linear r/ship between the optical signal and concentration of
the substance to be determined
 i.e. narrower bandwidth representing better performance.
 Ideally, the output from a wavelength selector would be
radiation of a single wavelength.
 Two types of wavelength selectors are used:
 Filters and
 Monochromators.
Instrum…
6
6

A. Filters:
 Either absorption or interference filters are used for wavelength
selection:
1. 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.
Instrum…
67

Instrum…
2. 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.
 The thickness of the dielectric layer is carefully controlled and
determines the wavelength of the transmitted radiation.
 When it is subjected to a perpendicular beam of light, a
fraction passes through the first metallic layer and the other
is reflected.
68

Instrum…
Figure 16; Interference filter
White
radiation
Narrow
band
radiation
Glass plates
Dielectric layer
Mealic films
Interference Filters
69
Fraction that is passed undergoes a similar partitioning
upon passing
through the second metallic film, thus narrower
bandwidths are
obtained.

B) Monochromators:
 All monochromators contain
 an entrance slit,
 a collimating lens or mirror to produce a parallel beam of
light
 a prism or grating to disperse the radiation into its
component wavelengths
 a focusing element and exit slit
Instrum…
70

Dispersion devices
 It cause different wavelengths of light to be dispersed at
different angles.
 When combined with an appropriate exit slit, these
devices can be used to select a a narrow waveband
 Two types of dispersion devices, prisms and
holographic gratings, are commonly used in UV-visible
spectrophotometers.
 A prism generates a rainbow from sunlight.
 This same principle is used in spectrophotometers.
 Prisms are simple and inexpensive, but the resulting
dispersion is angularly nonlinear (see Figure).
 Moreover, the angle of dispersion is temperature
sensitive.
Instrum…
72

 For these reasons, most modern
spectrophotometers contain
holographic gratings instead of prisms.
 These devices are made from glass
blanks, onto which very narrow
grooves are ruled.
 The dimensions of the grooves are of
the same order as the wavelength of
light to be dispersed.
 Finally, an aluminum coating is applied
to create a reflecting source.
 Light falling on the grating is reflected
at different angles, depending on the
wavelength.
 Holographic gratings yield a linear
angular dispersion with wavelength
and are temperature insensitive.
73
Instrum…

Instrum…
 However, they reflect light in different orders, which overlap.
 As a result, filters must be used to ensure that only the light
from the desired reflection order reaches the detector.
 A concave grating disperses and focuses light
simultaneously.
 A monochromator consists of an entrance slit, a dispersion
device, focusing mirror and an exit slit.
 Ideally, the output from a monochromator is monochromatic
light.
 In practice, however, the output is always a band(group),
optimally symmetrical in shape.
 The width of the band at half its height is the instrumental
bandwidth (SBW).
74

Instrum…
Optics
 Either lenses or concave mirrors are used to relay
and focus light through the instrument.
 Simple lenses are inexpensive but suffer from
chromatic aberration(devation from what is normal or
desirable), that is, light of different wavelengths is not
focused at exactly the same point in space.
 Achromatic lenses combine multiple lenses of
different glass with different refractive indices in a
compound lens that is largely free of chromatic
aberration.
 Such lenses are used in cameras.
75

Instrum…
 They offer good performance but at relatively
high cost.
 Concave mirrors- are less expensive to
manufacture than achromatic lenses and are
completely free of chromatic aberration.
 However, the aluminum surface is easily
corroded, resulting in a loss of efficiency.
76

Instrum…
iii- Sample cells (sample holders):
 For UV/Vis instrument, this is a light tight box in w/c the
container
holding the sample so/n is placed.
 The container is called cuvette.
 For the UV region sample compartment is made of quartz
since quartz
will not absorbed in the UV region except at vacuum UV.
 For the Visible region, compartment composed of simple
glass or
plastic cells since they absorb in the UV but not absorb in the

Instrum…
 The standard path-length of
cells for measurements of
absorption in the uv-visible
range is 1 or ½ cm path-length,
although cells of path length
from 0.1 to 10 cm can also be
used.
78

iV) Detectors:
Two Types of detectors are used in this respect:
1- Heat Sensitive Detectors
2- Photoelectric Detectors
 Photoelectric detectors are the most frequently used for this
purpose.
 They give electrical signal, which is directly proportional to the
intensity of
the transmitted light.
The following types of photoelectric detectors are used:
1- Photovoltaic cells 2- Phototubes
3- Photomultiplier Tubes (PMT’s) (The most widely used)
4- Photoconductivity tubes and Silicon photodiodes
Instrum…
7
9

(A) Photocells
(Phototubes):
 Light (radiant energy) falls on
the
cathode surface which excites
electrons and generates an
electric
current which is proportional to
light intensity
 (In other words) Converts the
energy of an incoming photon
into a
current pulse. Conversion is
Instrum…
8
0

B) Photomultiplier Tubes
 The PMT (see Figure below) combines signal conversion
with several
stages of amplification within the body of the tube.
 The nature of the cathode material determines spectral
sensitivity.
A single photomultiplier yields good sensitivity over the
entire UV-
visible range.
Instrum…
81

This type of detector yields high
sensitivity at low light levels.
However, in analytical
spectroscopy
applications, high sensitivity is
associated with low
concentrations,
which result in low
absorbances,
which in turn result in high
intensity
levels.
To detect accurately small
differences
between blank and sample
measurements, the detector
must have
low noise at high intensity levels.
Instrum…
82

Radiation enters over the grill and
strikes
the cathode photo-emissive
surface
 Radiation striking the cathode is
converted
into photo-electrons
 The photo-electrons are attracted to
the
first (+) dynode which produces a
cascade
of electrons which travel to dynode
2 due
to its higher potential.
Each electron strikes the second
dynode
releases a cascade of new
electrons which
Instrum…
83

Instrum…
C) Photodiodes
 Photodiode detectors have a wider dynamic range
and, as solid-state devices, are more robust
(stronger) than photomultiplier tube detectors
 In a photodiode, light falling on the semiconductor
material allows e- s to flow through it, thereby
depleting the charge in a capacitor connected across
the material.
 The amount of charge needed to recharge the
capacitor at regular intervals is proportional to the
intensity of the light.
 Earlier photodiodes had low sensitivity in the low UV
range, but this problem has been corrected in modern
detectors.
84

Instrum…
 Some modern spectrophotometers contain an array
of photodiode detectors instead of a single detector.
 A diode array consists of a series of photodiode
detectors positioned side by side on a silicon crystal.
 Each diode has a dedicated capacitor and is
connected by a solid-state switch to a common
output line.
 The amount of charge needed to recharge the
capacitors is proportional to the number of photons
detected by each diode, which in turn is proportional
to the light intensity.
85

Instrum…
 The absorption spectrum is obtained by measuring
the variation in light intensity over the entire
wavelength range.
 The array typically comprises between 200 and
1000 elements, depending on the instrument and
its intended application.
 Photodiode arrays are complex devices but,
because they are solid state, have high reliability.
86

Instrum…
a)The conventional spectrophotometer
87

Instrum…
 The absorbance of a sample is determined by measuring
the d/c b/n intensity of light reaching the detector without
the sample (the blank) and with the sample.
 This design is well-suited for measuring absorbance at a
single point in the spectrum.
 It is less appropriate, however, for measuring different
compounds at different wavelengths or for obtaining
spectra of samples.
 To perform such tasks with a conventional
spectrophotometer, parts of the monochromator must be
rotated
 This introduces the problem of mechanical
irreproducibility into the measurements.
 Moreover, serial data acquisition is an inherently slow
88

Instrum…
b)The diode array spectrophotometer
 Polychromatic light from a source is passed through the
sample area and focused on the entrance slit of the
polychromator.
89

Instrum…
 The bandwidth of light detected by a diode is
related to the size of the polychromator entrance
slit and to the size of the diode.
 Each diode in effect performs the same function
as the exit slit of a monochromator.
 The polychromator disperses the light onto a
diode array, on which each diode measures a
narrow band of the spectrum.
 The polychromator and the diode array are
contained in a unit known as a spectrograph.
 This configuration often is referred to as
reversed optics.
90

Instrum…
 To minimize possible photochemical reactions, a
shutter is used.
 When the measurement is initiated, the shutter is
automatically opened, and light passes through the
sample to the array of diodes.
 The difference in the intensities of the light reaching
the detector with and without the sample is
measured.
 A diode array spectrophotometer :
 inherently very fast owing to its parallel data acquisition
and electronic scanning capabilities
 has excellent wavelength reproducibility, and is highly
reliable.
91

Instrum…
v) Signal Processors/Readout
 Signal Processing
 Amplifying the signal coming from the detector
 Converting the signal coming from detector into a
form that is easily displayed.
e.g. from electron current to (DC) voltage
 Many forms of readout can be used:
 Computer display
 Digital or analog readout
 Strip chart recorders
 Integrators
9
2

Instrum…
Configuration
 Various configurations of spectrophotometers are
available.
i) Single-beam design
 Both conventional and diode array spectrophotometers
are single beam.
 The reference spectrophotometers used by national
standards institutions such as the NIST in the US and
NPL in the UK are single beam.
 Diode array spectrophotometers in particular are well-
suited to single-beam configuration.
93

Instrum…
 Figure below shows the optical system of a modern
diode array spectrophotometer.
94

Instrum…
Dual-beam design
 In a conventional single-beam spectrophotometer,
Lamp drift can result in significant errors over long
time intervals.
 The dual-beam spectrophotometer was developed to
compensate for these changes in lamp intensity
between measurements on blank and sample
cuvettes.
 In this configuration, a chopper is placed in the
optical path, near the light source.
 The chopper switches the light path between a
reference optical path and a sample optical path to
the detector.
 It rotates at a speed such that the alternate
95

Instrum…
 Figure below shows a schematic of a dual-beam
spectrophotometer.
 Compared with single-beam designs, dual-beam
instruments contain more optical components, which
reduces throughput and sensitivity.
96

Instrum…
 In addition, the more complex mechanical design of
the dual-beam spectrophotometer may result in poorer
reliability.
 Single-beam instruments offer higher sensitivity and
greater ease of use, with drift typically only a factor of
two worse than that of dual-beam instruments.
 The first commercially available diode array
Spectrophotometer was a multibeam design (see
Figure below).
97

Instrum…
 The beam director is used to shift the beam alternately
through the reference position and as many as four
sample positions (for clarity only one is shown in the
figure).
98

Instrum…
Split-beam design
 This configuration enables the blank and the sample to
be measured at the same time.
99

Instrum…
 Although the split-beam design is mechanically
simpler than the true dual-beam instrument and
requires fewer optical elements, the use of two
independent detectors introduces another
potential source of drift.
 This design provides high stability, although not
as high as a dual-beam instrument since two
detectors can drift independently, and good
noise, although not as good as a single-beam
instrument since the light is split so that less
than 100 % passes through the sample.
100

Applications for Spectrophotometry
 It is one of the most useful tools available to the experts for
analysis.
 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 106 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.
101

Qualitative…
 UV-visible spectra generally show only a few broad
absorbance bands.
 UV-visible spectroscopy provides a limited amount of
qualitative information.
 Most absorption by organic compounds results from
the presence of π bonds.
 The presence of an absorbance band at a particular
wavelength often is a good indicator of the presence
of a chromophore.
103

Qualitative…
 However, the position of the absorbance maximum
is not fixed but depends partially on the molecular
environment of the chromophore and on the
solvent in which the sample may be dissolved.
 Other parameters, such as pH and temperature,
also may cause changes in both the intensity and
the wavelength of the absorbance maxima.
 Some of the qualitative application of UV-Vis
spectrophotometry are:
104

1. Color Tests:
 The color of matter is related to its absorptivity or
reflectivity
a) Substance may have a characteristic color of its own.
Eg. Vitamin B12, indicators, Cr salts and KMnO4 etc…
b) Substance may be treated with a certain selected
reagent to give
characteristic colored product (e.g.):
Cu2+ + 4 NH3 [Cu(NH3)4]2+
Azur blue color
Fe3+ + SCN- [Fe(SCN)]2+
Bloody red color
105
Qualitative…

c) Substance may be converted to some derivatives that
can react with a special reagent to give a colored
product;
Red color
10
6
Qualitative…

2. Identification with absorption spectrum :
 UV-visible spectra do not enable absolute identification
of an unknown,
 They frequently are used to confirm the identity of a
substance
 Where spectra are highly similar, derivative spectra may
be used.
 The number of bands increases with higher orders of
derivatives.
 This increase in complexity of the derivative spectra can
be useful in qualitative analysis
 For example, the absorbance spectrum of the steroid
testosterone shows a single, broad, featureless band
centered at around 330 nm, whereas the second derivative
shows six distinct peaks.
10
7
Qualitative…

108
Chromophore Example λmax (nm) єmax
Alkene R.CH=CH2 177 13,000
Conjugated alkene CH2=CH.CH=CH2 214 21,000
Carbonyl CH3.CO.CH3 186 1000
Carboxyl CH3.COOH 204 41
Azo R.CH2.N=N.CH3 339 5
Aromatic Benzene 204
255
7,900
200
 UV-Visible spectrum give useful information about substance
via
examination of its max and emax, which could be correlated
with the
structural features (See the following table).
Qualitative…
Absorption characteristics of some common organic
chromophores:

NH2 NH3
In alkaline medium in acid medium
Aniline, max= 280 nm Anilinium ion max= 254 nm
+
+ H+
- H+
-
+
H
in acid medium in alkaline medium
O
O
OH
OH
(Phenol)max = 270 nm (phenate anion) max= 290 nm
3. Detection of some functional groups:
10
9
Qualitative…

Laws of light absorption
 When a monochromatic light passes through a cell
containing an
absorbing substance, then effects occurring will include:
 Io = Ia + Ir + It + If + Is
Where I0 is the total light entering.
 For clear solutions, Is = 0, If and Ir can be cancelled by
using blank
solution. So, under experimental conditions the equation
become;
Io = Ia + It
111
Quantitative …

Quantitative …
112
 A beam of parallel radiation before and after it has
passed through a layer of solution with a thickness of b
cm and a conc. of c of an absorbing species.
 As a consequence of interactions b/n the photons and
absorbing particles, the power of the beam is attenuated
from Po to P.
 The T of the solution is the fraction of incident radiation
transmitted by the solution.
 That is,
 The absorbance of a solution is defined by the equation

Quantitative …
Lambert’s law
 According to this law, each layer of equal thickness
absorbs an equal
fraction of light passing through it (i.e.) the rate of
decrease in I0 with
the thickness of medium is proportional to I0 itself.
or -δI0/ δ t α I0
 After mathematical treatment the equation become
ln I0/It = µ t
 Where ln is the natural logarithm log I0/It = µ /2.303 .
t = Kt
Where K is the extinction coefficient, t is
thickness
113

Quantitative …
Beer’s Law:
 It stated that absorption is proportional to the number of
absorbant
molecules in the light path
log Io/It =K C , Where K is proportionality constant
corresponding to m
and C is the concentration in g/L.
115

Beer’s-Lambert’s Law
This is a combination of both laws ; Log I0/It = A =
abC
where a is a constant called absorptivity, b is the path-length in
cm and C is the
concentration in grams/Liter.
 The value of a will clearly depend upon the method of expression
of the conc.
 If C is expressed in moles/liter, and b in cm then a is
given the
symbol  (Epsilon, l.mol-1cm-1) and is called the molar
absorption coefficient or molar absorptivity.
A =  b C. When  is measured at max it is
116
Quantitative …

Quantitative …
118
 The extinction coefficient (ε) is characteristic of a given
substance under a precisely defined set of conditions,
such as wavelength, solvent, and temperature.
 In practice, the measured extinction coefficient also
depends partially on the characteristics of the instrument
used.
 For these reasons, predetermined values for the
extinction coefficient usually are not used for quantitative
analysis.
 Instead, a calibration or working curve for the substance
to be analyzed is constructed using one or more
standard solutions with known concentrations of the
analyte.

Problems
1. A 5 X 10–4M 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?
2.
3.
4.
119

Quantitative …
Deviations from Beer-Lambert’s Law
 As per the Beer’s law discussed above, there is a
direct proportionality between the absorbance and
concentration.
 A plot of absorbance versus concentration is expected
to be a straight line passing through origin.
 However, this is not always true; there are certain
limitations.
 The law does not hold for all species under every
condition.
 Many a times instead of a straight line, a curvature in
the plot may be observed as shown in fig below.
120

Quantitative …
 Some of the factors
responsible for the deviation
from Beer’s law are as
follows.
Presence of Electrolytes
 The presence of small amounts
of colourless electrolytes which
do not react chemically with the
coloured components does not
affect the light absorption as a
rule.
 However, large amounts of
electrolytes may affect the
absorption spectrum
qualitatively as well as
121

Quantitative …
 This is due to the physical interaction between the ions
of the electrolyte and the coloured ions or molecules.
 This interaction results in a deformation of the coloured
ions or molecules
 Thereby causing a change in its light absorption
property.
H+ Concentration
 There are a number of substances whose ionic
state in solution is greatly influenced by the
presence of H+.
122
Yellow orange

Quantitative …
 In some cases, two absorbing species are in
equilibrium and have a common value of absorptivity
at a certain wavelength.
 For example, in case of bromothymol blue the
absorption spectra at different pH values are
different.
 However, at wavelength of 501 nm, we see that all
species have same molar absorptivity (see Fig).
123

Quantitative …
 Such a wavelength is known
as isosbestic point.
 At this wavelength the Beer’s
law holds, though the
measurements have low
sensitivity.
 However, such wavelengths
should be avoided for the
quantitative work.
124

Quantitative …
Complexation, Association or Dissociation
 Some salts have a tendency to form complexes whose
colours are different from those of the simple compounds.
 For example, the colour of cobalt chloride changes from
pink to blue due to the following complex formation.
 The degree of complex formation increases with increase in
conc., therefore, Beer’s law does not hold at high
concentrations.
 Similar discrepancies are found when the absorbing solute
dissociates or associates in solution because the nature of
the species in solution depends on the concentration.
125

Quantitative …
Non-monochromatic Nature of the Radiation
 In order for the Beer’s law to hold, it is necessary that
monochromatic light is used.
 The wider the bandwidth of radiation passed by the filter or
other dispersing devices, the greater will be the apparent
deviation of a system from adherence to Beer’s law.
Concentration of the Analyte
 As per Beer and Lambert’s law, the plot of A versus the
conc. should be a straight line when µ and b are constant.
 Therefore, at higher conc. (>10-3 mol dm-3) there may be
deviation from the law.
126

Quantitative …
 This is because interactions b/n absorbing species at
higher conc affect ability to absorb radiation due to:
 crowding,
 molecular interaction and association
 charge distribution
Temperature
 The T is not considered as an important factor since ordinarily
the measurements are made at a constant T.
 However, changes in T sometimes may shift ionic equilibrium
and the absorptivity.
 For example, the colour of acidic FeCl3 solution changes from
yellow to reddish brown on heating due to change in λmax and
absorptivity.
127

(1) Real deviation:
 Valid only for dilute (< 10-3 M) solutions. Why?
 Because interactions between absorbing species at higher
concentrations affect ability to absorb radiation due to crowding,
molecular interaction and association as well as charge
distribution
 Because also e depends on refractive index which depends
on concentration of the solution ( mostly at high concentration
levels)
(2) Instrumental deviation(errors):
a) Irregular deviation due to: Unmatched cells, unclean handling and
unclean optics.
b) Regular deviation: due to:
1
2
8
Quantitative …

.
2- Stray light
Stray light is light which falls on detecor within a UV instruments
without passing
through the sample
 It can arise either from light scattering within the instrument or by entry
of light into
the instrumrnte 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.
This is most serious where the sample has a high absorbance e.g. at
an absorbance of 2
1
3
0
Quantitative …

Quantitative …
 c) Other errors:
 Non-linear response of photo cells
 Radio and TV interferences
 Unstabilized power supply
131

Multicomponent analysis
13
2
 Multicomponent analyses using UV-visible spectra were
not widely applied.
 Modern instruments yield more precise data, and
modern curve-fitting techniques give more accurate
results.
 For these reasons, multicomponent UV-visible analyses
are becoming more popular.
 According to Beer’s law absorbance is proportional to
the number of molecules that absorb radiation at the
specified wavelength.
 This principle is true if more than one absorbing
species is present.

Multicomponent…
 It is based on the principle that the absorbance at
any wavelength of a mixture is equal to the sum of
the absorbance of each component in the mixture at
that wavelength.
 The simple approach to multicomponent analysis is
based on measurements at a number of
wavelengths equal to the number of components in
the mixture.
 The wavelengths chosen usually are those of the
absorbance maximum of each component.
 Determine the extinction coefficient for each
component at each wavelength selected by
calibration .
133

Multicomponent…
 The absorbance of the mixture at each wavelength is
the sum of the absorbance of each component at that
wavelength
 Thus for two components x and y, the equations are:
where
 A′ is absorbance at wavelength ′
 A′′ is absorbance at wavelength ′′
 e′ and e′′ is molar absorptivity at wavelength ′ and ′′
 c is concentration, and b is path length.
 If measurements were always perfect, accurate results
could be obtained even for complex mixtures of
components with very similar spectra.
134

Multicomponent…
135
 In practice, however, measurement errors always occur.
 Such errors can affect significantly the accuracy of results
when spectra
overlap significantly.
 Figure below shows a simulated two-component mixture
with no
overlap of the spectra at the absorbance maxima.

Multicomponent…
13
6
 In contrast, Figure below shows a simulated two-
component mixture with significant overlap of the spectra
at the absorbance maxima.
 For a mixture of x and y where cx = cy = 1, the measured
absorbances should be:

Multicomponent…
 A 10 % error occurs in the measurement of A′(x + y)
and A′′(x + y), the quantitative calculation yields the
results shown in table
137

Instrum…
Measuring a spectrum
 The degree of interaction of the sample with radiation
(T or A) is determined by measuring both the intensity
of the incident radiation with and without the sample
 Because most samples measured are in solution, the
blank should be measured on a cuvette containing the
pure solvent.
 This process eliminates from the sample
measurement any absorbance due to the solvent.
 With a single-beam instrument, the cuvette containing
the solvent is placed in the spectrophotometer, and
the blank is measured.
 The sample solution is then measured in the same
cuvette.
138

Instrum…
 All modern instruments automatically store the
reference Io values, which are used to calculate
absorbance values for the sample.
 With a dual- or split-beam instrument, two cuvettes
are required.
 Both cuvettes are initially filled with pure solvent, and
a so-called balance measurement is performed.
 This measurement reflects the difference in
absorbance between the two optical paths in use.
 The sample cuvette is then filled with the sample
solution for measurement, and Io and I are
measured virtually simultaneously.
 The resulting spectrum is corrected by subtracting
the balance spectrum.
139

Spectrophotometric titration
 In photometric method of equivalent point detection of titrations, the
appearance of an absorbing species will give a linear or conc
dependent change in absorbance, w/c will yield two straight lines that
intersect at the equivalent point.
 There are at least three components w/c may absorb light: the original
sub, the titrant & the resulting product (s). The usual procedure is to
select some wave length at w/c only one component absorb.
 In spectrophotometric titration, the absorbance of the so/n at a
specified λ is measured after each addition of the titrant.
 The results are plotted ( A vs ml of titrant ), and the end point is
determined graphically.
 The point at w/c the two straight lines intersects the end point.
140

 The graph is constructed on the basis of data obtained well
before and well after the end pt.
 Some typical titration curves for the reaction X + Y → Z where X
the component to be determined, Y titrant & Z the product (s) of
the reaction.
 If X absorbs radiation energy at a specified λ and it is
contaminated with other absorbing sub, a spectrophotometric
titration can be carried out if a titrant can be found w/c react
selectively with X.
 If X & the contaminant are the only species that absorb, the
absorbance of the so/n will decrease as titrant is added.
 If Y is the only species in so/n w/c absorbs, the so/n will not
absorb until the end pt is reached.
 If Z is the only species in so/n w/c absorbs, the absorbance will
increase as product is formed.
141
Spectrophotometric titra…

 The following are some of the spectrophotometric titration curves which
can be
observed in the normal conditions:
Where:
S = Sample , P = titration Product , T = Titrant ,
@ = Absorbing species , X = Non absorbing species.
1
4
2

Examples:
1- Titration of potassium permanganate against ferrous sulfate
MnO4
- + Fe++ + H+ == Mn++ + Fe3+ + H2O
@ at 540 (X) at 540 (X) at 540 (X) at 540 nm
2- Titration of Bi3+ & Cu2+ mixture with EDTA;
 Bi3+ , Cu2+ & Bi-EDTA complex are non absorbing at 745 nm
Cu-EDTA complex is absorbing at 745 nm
1
4
3

Advantages:
1. More accurate results than direct photometric analysis are
obtained.
2. No interference from other absorbing substances because the
end point depends on the change in the absorbance curve
and not on the absorbance value
(affect only the curve shape and sharpness of end point).
3. Can be used for titration of very dilute solutions.
4. Not need favorable equilibrium constants as those required for
titration that depends upon observations near the end point.
5. Can be used for (applied to) all types of reactions (redox, acid-
base, complxometry, pptmetry ….etc.)
144

Derivative spectrophotometry
 It involve the conversion of a normal spectrum to its first, second
or higher derivative spectrum.
 In the context of derivative spectrophotometry, the normal
absorption spectrum is referred to as the fundamental, zero order
or D0.
 The first derivative (D1) spectrum is a plot of the rate of change
of absorbance with wavelength against wavelength, i.e. a plot of
the slope of the fundamental spectrum against wavelength or a
plot of dA / dλ Vs λ.
 It is characterized by a maximum, a minimum, and a cross over point at the
λmax of the band.
145

Derivative…
 The second derivative ( D2 ) spectrum is a plot of the curvature of the
D0 spectrum against wavelength or a plot of d2A / dλ2 Vs λ.
 It is characterized by two satellite maxima and an inverted band of
w/c the minimum corresponding to the λmax of the fundamental
band.
 Advantages of derivative spectrophotometry are enhancing resolution
and bandwidth discrimination w/c increase with increasing derivative
order.
 However, it is also found that
 The concomitant increase in electronic noise inherent in the generation
of the higher order spectra, and
 The consequent reduction of the signal-to-noise ratio, place serious
practical limitation on the higher order spectra.
146

 Determination of conc. of an analyte in presence of interfering
species or other
analytes can sometimes be made more easily and/or more
accurately.
 Ex; A mixture of two substances A and B gave a zero order spectrum as
indicated below which show no well-defined absorption bands, but the second
order spectrum deduced from this curve showed a better peaks resolution.
A

d2A/d2

A
B
1
4
8
Derivative…

Colorimetry
o Colorimetry is the determination of the light absorptive capacity
of a system.
o Quantitative determination carried out by subjecting a colored
so/n to visible region w/c is adsorbed by the so/n.
o Determination can also be carried out by comparative method.
o This approach to Colorimetry is characteristics of the limit tastes
in the pharmacopeia for a numbers of organic & inorganic
contaminants in a certain drugs.
o It is based on the analyst’s ability to distinguish b/n d/t intensities
of the same color.
o It could be direct or indirect method
151

Colorime…
Direct method
 The drug must be self colored. Some pharmaceutical important subs w/c
directly measured colorimetrically are methyl blue, dithiazin iodide,
pamoate etc.
 The substance may be dissolved in a suitable solvent & its absorbance
determined at the wavelength at w/c its absorbs a max of visible energy.
Indirect method
 The drug must react with a reagent to produce a sub, w/c is colored.
 A chromaphore can be introduce in to or prepared from a sub of almost
any chemical class using relatively simple chemical procedure.
152

Colorime…
General requirement for the colored sub
 The so/h should be intensely colored.
 The so/n must be unaffected by pH change.
 The so/n should be stable. If the absorbance value changes with time,
the so/n must be at some specified time or the system must be
chemically stabilized.
 The constituent should react quickly & quantitatively with the reagent
at room temp.
 The so/n should obey beer’s law. To check for compliance with beer’s
law, the analyst plots A vs conc.
153

Colorime…
The chemistry of Colorimetry
 Colored sub are formed by reacting the constituent with either
inorganic or organic reagent.
 Rxn may classified on the basis of the type of reagent used & on the
type of rxn, w/c occurs when the constituent is reacted with the
reagent.
 Colorimetric method w/c is based on the chemical derivatization with
the use of color developing reagent is utilized in the assay of drugs in
the pharmacopoeia. Examples w/c official methods in the
pharmacopoeia are based on the chemical derivatization are given
below
154

Colorime…
A) Diazotization & coupling of primary aromatic amines
 The amine functional group is first diazotized with aqueous so/n of nitrous acid
at 0-5 0C .
 The colorless daizonium salt is very reactive and when treated with a suitable
coupling reagent, phenol or aromatic amine, it undergoes an electrophilic
substitution rxn to produce an azo dye.
 The azo cpds all absorb light in the visible region, but the wavelength and
absorptivity depend on
 the structure of the aromatic ring (Ar & Ar’),
 pH and solvent.

Ar-NH2 + NHO2 Ar-N +
N
H+
+ 2H2O
Ar-N +
N + Ar'
-H Ar-N N -Ar'
+ H+
155

Colorime…
 The most widely used coupling reagents N-(1-naphtayl)-ethane-1-2-
diammoniumdichloride ( Bratton-Marshall reagent) w/c give high
absorptivities.
 Sulphamic acid or ammonium sulphamate is added to the so/n of the
diazotized amine before the coupling stage to destroy the excess
nitrous acid w/c inhibits the coupling rxn.
 The sensitivity & selectivity of the procedure permit the assay of low
conc. of impurities that contain a primary aromatic amine group in the
presence of the parent sub lacking amine function.
NHCH2CH2NH2
156

Colorime…
 The BP (1988) employ this method for determination of free
amine impurities in
o Frusemide,
o Iothalemic acid,
o Meglutamine Iothamic inj.
o Meglumine diatrazoate inj. &
o the BP use this method in the determination of Folic
acid and its tablet.
157

Colorime…
B) Reaction of the α-ketal group with tetrazoluim salt
 In the presence of a steroid α-ketal (21-hydroyl-20-keton) side
chain group, tetrazolium salts are reduced to their colored
formazone derivatives.
 The rxn carried out in an alkaline medium (
tetramethylammonium hydroxide) at 30-35 C for 1-2hrs and
the absorbance of the red product is measured around 488nm
 The oxidation of the α-ketalgroup and the reduction of
triphenyltetrazonium chloride to triphenylformazon are
indicated in rxn below.
158

Colorime…
 Interference by reducing sugars is eliminated by alcohol
extraction ( alcohol free of aldhayed).
 Steroids esterified in the C-21 position, e.g. Hydrocortison
acetate, hydrolyse in the alkalin so/n to yield the free 21-
hydroxysteriods and detrmined by the above process.
C=O
CH2OH
OH +
N
N
C
N N
N
H
N
C
N N
C=O
CH=O
OH +
159

Applications for
Spectrophotometry
 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 spectrophotometry and similar techniques.( Over
3,000,000 daily tests
are carried out in USA only).
1
6
0

[A] Analysis of plain compounds :
a) Absorbing species :
 Direct spectrophotometric determination of any organic compound
containing one or more chromophoric groups is potentially feasible.
 Huge number of applications are found in literature including
organic and also inorganic chromogens. Examples:
Spectrophotometric applications of
pharmaceutical interest
Alimemazine tartrate absorbed at 255 nm P-aminobenzoic acid absorbed at 280 nm
1
6
1

Amoxicillin sodium, absorbed at
275 nm
Aspirin absorbed at 298 nm
Carbamazepine , Absorbed at 285 nm
Chlordiazepoxide , Absorbed at 246 & 308 nm
162

Clofazimine , Absorbed at 283 & 487 nm
Chloroquine phosphate, Absorbed at 256, 329 & 342 nm
Cinchocaine HCl , Absorbed at 246 & 319 nm.
163

b) Non-absorbing species :
 Numerous reagents react selectively with non
absorbing species to yield products that
can absorb strongly in the UV-Visible region.
 Color-forming reagents are used also for
determination of absorbing species if there is
an increase in sensitivity and/or selectivity.
Examples: Erythromycin
NH.NH2
NO2
NO2
+ R C
R'
O
NO2
NO2
NH.N C
R'
R
+ H2O
Colourless &has coloured product
no absorbance Orange( 435 nm)
164

NH2
R
N N . HCl
R
OH
R'
NaNO2
HCl
OH
N N
R'
R
Colourless &has Coloured Product
Absorbance ( 480 nm)
To increase sensitivity and/or selectivity e.g. sulfa drugs and salicylic acid.
H2N SO2.NHR
Colourless &has Coloured Product
Absorbance ( 620 nm )
COOH
OH
Cu++
C
O
Cu
O
O
1
6
5

(A) Testing of identity:
 Done by comparison of some characteristic spectral constants of tested
drug solution
with that of an authentic sample of the same drug, e.g.; Absorption
maxima, Molar
absorptivity (ε) or A1%
1cm,
 Absorbance of drug test solution with known concentration or ratio of
absorbances at
two maxima in a known test solution.
(B) Testing of purity:
Purity of a drug can be tested through:
 Comparison of absorptivity at certain wavelength with that of pure
standard solution (+
certain limits).
 Comparison of the difference or the ratio of absorptivities at two
1
6

(C) Analysis of drugs and their metabolites in biological fluids:
 Due to the high sensitivity and selectivity of spectrophoto-metric
techniques, they could
be used for quantitative analysis of some drugs and their metabolites in
biological
materials.
D) Determination of enzyme activity:
 Enzyme activity can be determined through measurements of the
reactants and/or
products in the reaction that involving certain enzyme.
 Example; determination of lactate dehydrogenase enzyme (LDH) activity
in blood.
Enzyme + Lactate = Reaction product
(Measured spectrophotometrically)
1
6
7
Other Important…

NAD+ Absorbed at 260 NADH Absorbed at 260 &340 nm
Where:
S = Oxidized substrate , SH2 = Reduced substrate
NAD+ = Oxidized nicotinamide adenine dinucleotide
NADH = Reduced NAD , R = Ribose , P = Phosphate , A = Adenine
 Also enzymatic activity can be measured via determination of
certain coenzymes.
 Example : Determination of ethanol content in blood via
measuring activity of
alcohol dehyrogenase enzyme (ADH)
CH3 CH2 OH + NAD+
ADH
CH3 CHO + NADH + H+
N
H
H
CONH2
RPPRA
+ S + H+
Hydrogenase
Dehydrogenase
N
RPPRA
CONH2
+ SH2
1
6
8
Other Important…

260 nm 340 nm
NADH
NAD+
169
Other Important…

(E) Monitoring drug degradation kinetics :
 Simply done when the product has a different absorption spectrum
than that of un-degraded drug.
Example: Hydrolysis of Aspirin to salicylic acid and acetic acid
 Aspirin absorbs at 278 nm in aqueous acidic solutions, while
 Salicylic acid absorbs at 303 in the same media.
 Acetic acid has no peaks at the same range.
 Since the spectra of aspirin and salicylic acid are different,
 Thus, the rate of disappearance of aspirin spectrum or appearance of
salicylic acid spectrum (as a function of time ) may be used to
determine the rate constant for aspirin hydrolysis.
170
Other Important…

(F) 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.
171
Other Important…

(G) 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 as
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
172
Other Important…

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
 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 (HB).
At pH = 6, the acid is completely ionized (B-).
 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
173
Other Important…

Other Important…
A
430 nm 520 nm pH
 5.0
Measured at
520 nm
Measured at
430 nm
174

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