Chapter 1

Pharamacuetical Analaysis II
(Phar 3122)

1
 UV- Visible
spectrophotometry

2
 Application of instrumental 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.

 (e.g.spectrophotometry, mass spectrometry).

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
 Mass-to-charge ratio Mass spectrometry

4
 Introduction

5
 Intro…
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.

6
 Electromagnetic radiation
 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

7
 EMR…

8
 EMR..
 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.

9
 EMR…
 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

10
 EMR……
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

1
1
 EMR……
 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……

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…
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.

or 6.625 X 10-34 J.sec

15
 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
17 molecules
 How Light Interacts….
 The nature of the interaction depends upon the properties of the
matter.
 Each interaction can disclose certain properties of the matter.

18
 How Light Interacts….

 There are two types of absorption

 Atomic
 Molecular

19
 How Light Interacts….

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.

20
 How Light Interacts….
 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.

21
 How Light Interacts….

ΔE transition = E1 - E0
330 nm
= hv = hc/

590 nm
ΔE transition =
E2 - E0 = hv = hc/

Absorption and emission for the sodium atom in the gas phase, Illustrates discrete energy transfer

22
 How Light Interacts….

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
 How Light Interacts….
Molecular spectrum

24
 How Light Interacts….
 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

25
 Spectrum
 It is the display of the energy level of
EMR as a function of wave number
or
wavelength of EMR energy. A max

The energy level of EMR is usually

cules)
expressed in terms of absorbance,

Line spec.(atoms)
(mole
transmission, Intensity.

spec.
 It may be:

Band
a) line spectrum: occur with atomic
spectra such as sodium metal which 

has a sharp line of  at 589 nm.
26
 Spectrum…

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.

27
 Spectrum
 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.

28
 UV-Visible Spectrophotometry
 What occur to a molecule when absorbing UV-visible photon?
 A UV-visible photon (200-800nm) 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.

29
 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.

30
 Types of electronic…
 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.

31
 Types of electronic…

electronic transitions of formaldhyde

32
 Types of electronic…

*- Antibonding
*- Antibonding
* Transtion

n-* Transtion

* Transtion

n-* Transtion
Energy

n- Nonbonding

 Bonding
 Bonding

150 200 250 300 350

Wavelength, nm
33
34
 Types of electronic…

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.

35
 Types of electronic…

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 - 250 nm region.

36
 Types of electronic…
Cut-off wavelengths of some common solvents:
Solvent , nm Solvent  , nm

Water 190 Chloroform 247

Ether 205 Carbon tetrachloride 257

Ethanol 207 Benzene 280

Methanol 210 Acetone 331

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
37
* and n-* transitions, example acetone (CH -COCH ).
 Some important terms
Chromophores: (Chrome = color, phore = carrier).
 They are functional groups, which confer color on substances capable
of absorbing UV and/or visible light (200 - 780 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.

38
 Some important…
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.

39
 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.

40
 Absorption Characteristics…

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
41  Alkyl substitution; Cause red shift due to hyper-conjugation.
 Absorption Characteristics…
3. Conjugated Chromophores
CH2 = CH – CH2 – CH = CH2

have additive effect only

because there is little or no
electronic interaction b/n
CH2 = CH2 separated Chromophores.

170-180 nm
CH2 = CH – CH = CH2

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 170-180 205-215 nm
42
 Absorption Characteristics…

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

43
 Absorption Characteristics…
 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 254 nm
204 nm

44
 Absorption Characteristics…

(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
D
group.
h
D
 In addition the B band loses most of its fine structure.
h
W X W X

45
 Absorption Characteristics…

 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
6
 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 
electrons.

47
 Effect of pH on….

OH O O
-
OH
H+

in acid medium in alkaline medium

(Phenol) max = 270 nm (phenate anion)  max= 290 nm

48
 Effect Of pH On….
 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
NH2 NH3
this case is
+ H+
similar to that of benzene (thus
- H+ called benzenoid spectrum).

In alkaline medium in acid medium

Aniline,  max= 280 nm Anilinium ion  max= 254 nm
49
 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.

50
 Effect of Solvents…

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, .
S1

S2

G1
G G2
 - * bands of enones n - * bands of enones
51
 Calculation of max of an organic compound
I- According to Woodward Feiser Rules:
Rules For conjugated dienes:

 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.

CH3
R CH2

R CH3 endocyclic exocyclic

Counte d Ring re s idue s a nd a lkyl s ubs titutions
52
 Calculation of .max….
 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.
Auxochrome attachements

 Attachment of AUXOCHROMES to the conjugated system.

SH OCH3

homoannular heteroannular

5
Extra double bond
3
 OCH3
OH
SH OAc
Cl
Che ck tha t this che mica l compound conta ining
2-Extra double bonds
5- Auxochrome s a tta che me nts
5- Ring re s idue s
No a lkyl s ubs titutions
3- Exocyclic double bounds
One homoa nnula r na ture

5
4
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
5
 The following examples illustrate the use of these rules:
OH OH
SH Cl

Cl
NH2 OR NH2 Cl OCH3

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
5
6
 Calculate the max of the following compounds :

Cl Cl Cl
OCH3

NH2 OR
OH OH

OH OH Br
Cl CH2
OR

OAc
Cl SH NH2
Cl SH

OH
Br
Cl OR Br

Cl
OR OR Br
NH2
5
7
 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 :

CH2OH

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
5
8
  Using the simplified Kuhn and Hausser rules, Calculate the approximate

λmax for the following compounds

C CH OH
CH

OH

CHOH

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
5
9
 Instrumental design
Components
 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

60
 Instrum…
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)

6 Deuterium Lamp Hollow cathode lamp

1 Tungsten Lamp
 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. deuterium arc lamp

 Such a lamp typically has a half-life of

approximately 1,000 h.

62
 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 tungsten-halogen lamp
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.
 Most spectrophotometers used to measure the
UV-visible range contain both types of lamps.
63
 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
 Xenon lamps are used only for
applications in which high intensity is
the primary concern.

64
 Instrum…
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.
6
5
 Instrum…

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.

 They are inexpensive and widely used for band selection in the visible

region.

66
 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.

67
 Instrum…
Fraction that is passed undergoes a similar partitioning upon
passing
through the second metallic film, thus narrower bandwidths are
obtained.
White
Glass plates
radiation
Dielectric layer
Narrow Mealic films
band
radiation
Figure 16; Interference
Interference Filters filter

68
 Instrum…
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

69
70
 Instrum…
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 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.

71
 Instrum…
 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.

72
 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).

73
 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.

74
 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.

75
 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.
 For the Visible region, compartment composed of simple glass or
plastic cells since they absorb in the UV but not absorb in the visible
region.
 In UV any solvent that does not have any Ñ-bonding can be used including
 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.

77
 Instrum…
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
7  The main three types are illustrated in the following part:
8
 Instrum…

(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 done
on
a photoemissive surface by the
7
9 “photoelectric effect”
 Instrum…
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.

80
 Instrum…
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.

81
 Instrum…

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
travel on to the next dynode in the
82 series
and so on to the last (main) anode.
 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.
83
 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.

84
 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.

85
 Instrum…
a)The conventional spectrophotometer

86
 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 process.

87
 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.

88
 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.

89
 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.

90
 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
1
 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.

92
 Instrum…

 Figure below shows the optical system of a modern diode

array spectrophotometer.

93
 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 measurements of
blank and sample occur several times per second.

94
 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.

95
 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).

96
 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).

97
 Instrum…
Split-beam design
 This configuration enables the blank and the sample to be
measured at the same time.

98
 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.

99
 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.
100
 I- Qualitative Analysis

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.

102
 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:

103
 Qualitative…
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

104
 Qualitative…

c) Substance may be converted to some derivatives that can

react with a special reagent to give a colored product;

Red color

10
5
 Qualitative…

10
6
 Qualitative…
 UV-Visible spectrum give useful information about substance
via
examination of its max and emax, which could be correlated
with the
structuralcharacteristics
Absorption features (See of the
somefollowing table).
common organic
chromophores:
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 7,900
107 255 200
 Qualitative…
3. Detection of some functional groups:
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

10
8
 II- Quantitative analysis

109
 Quantitative …
Laws of light absorption
 When a monochromatic light passes through a cell containing
an
absorbing substance, then effects occurring will include:
 I o = Ia + I t
Where I0 is the total light entering.

110
 Quantitative …
 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

111
 Lambert’s law Quantitative …
 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
112
 Quantitative …

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 and C is the concentration in g/L.

114
 This is a combination of both laws ; Log I0/It = A = abC
a(1%,1cm) bC
Quantitative …
where a is a constant called absorptivity, b is the path-length in cm
and C is the
concentration in grams/Liter.
A(1%,1cm) is the absorbance of 1%W/V(1gm/100ml) sln in a 1cm cell, b is
the path length (1cm) C is the conc. in gm/100ml

 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
115
absorption coefficient or molar absorptivity.
 Quantitative …

116
 Quantitative …
 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.

117
 Q. Determine the concentration of glycogen-iodine
complex if the absorbance of light is 0.4. Also, the
absorption coefficient is 0.20 at 450 nm. The size of
the cuvette is 2 cm.
Answer:
Using Beer-Lambert’s law, we know that
A= 0.4 = (0.20 lit/mol.cm)(c)(2cm)
c = 1mole/lit

118
 Q.2. What are the conc of the ff solns of drugs in
g/100ml mg/100 ml
a. carbimazole A(1%,1cm) value =557 at 291nm,
measured absorbance 0.557 at 291nm
b. hydrocortisone sodium phosphate A(1%,1cm) value
333 at 248nm, measured absorbance 0.666 at 248 nm
Answer: A=a(1%,1cm) bc; A=0.557 a=557 , b=1 C=?
C = 0.557/557*1 =0.001g/100ml=1mg/100ml

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 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 n

atio
e vi
for the deviation from Beer’s law iv e d
it
are as follows. po
s

Presence of Electrolytes ti on
via
 The presence of small amounts of e de
tiv
colourless electrolytes which do not e ga
n
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 quantitatively.

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+.

Yellow orange
122
 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 …

127
 Quantitative …
(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 1- Errors in wavelength scale and Slit width control.
 Quantitative …

129
 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 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.
This is most serious where the sample has a high absorbance e.g. at an
1
3absorbance of 2
 Quantitative …
 c) Other errors:

 Non-linear response of photo cells

 Radio and TV interferences
 Unstabilized power supply

131
 Multicomponent analysis
 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.

13
2
 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…
 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.

135
 Multicomponent…
 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:
13
6
 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
 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
 Spectrophotometric titra…
 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 ,

1
@ = Absorbing species , X = Non absorbing species.
4
 Spectrophotometric titra…
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

1
Cu-EDTA complex is absorbing at 745 nm
4
 Spectrophotometric titra…
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
147
 Derivative…
 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
A d2A/d2 B

1  
4
1
5
 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/n 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 .

H+
Ar-NH2 + NHO2 Ar-N + N + 2H2O
 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.+
Ar-N N + Ar'-H Ar-N N -Ar' + H+

 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.


155
 Colorime…
 The most widely used coupling reagents N-(1-naphtayl)-ethane-1-2-

diammoniumdichloride ( Bratton-Marshall reagent) w/c give high absorptivities.

NHCH2CH2NH2

 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.
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.

CH2OH CH=O
C=O C=O N N
N N
OH OH + C H
+ C
N N N N

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
 Spectrophotometric applications of pharmaceutical
interest

[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:

1 Alimemazine tartrate absorbed at 255 nm P-aminobenzoic acid absorbed at 280 nm

6
 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

R'
NH.NH2
NH.N C R
NO 2 R' NO 2
+ R C O + H2O
NO 2
NO 2
Colourle s s & ha s coloure d product
no a bs orba nce Ora nge ( 435 nm)
164
To increase sensitivity and/or selectivity e.g. sulfa drugs and salicylic acid.

H2N SO2.NHR

OH OH
NH2 N N . HCl
R' R'
NaNO2
R R
HCl N N

R
Colourless & has Coloured Product
Absorbance O ( 480 nm)
COOH O
OH C Cu
Cu ++ O

Colourless & has Coloured Product

1
Absorbance ( 620 nm )
6
(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 specified
1
 Other
(C) Analysis of drugs and Important…
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
1
6 (Measured spectrophotometrically)
 Other Important…
 Also enzymatic activity can be measured via determination of certain

coenzymes.
 Example : Determination of ethanol content in blood via measuring
ADH
activity
CH3 ofCH2 OH + NAD+ CH3 CHO + NADH + H+

alcohol dehyrogenase enzyme (ADH)

CONH2 H H
De hydroge na s e CONH2
+ SH2 + S + H+
N Hydroge na s e N
RPP RA
RP PRA

NAD+ Absorbed at 260 NADH Absorbed at 260 &340 nm Where:

S = Oxidized substrate , SH2 = Reduced substrate

NAD+ = Oxidized nicotinamide adenine dinucleotide

1 NADH = Reduced NAD , R = Ribose , P = Phosphate , A = Adenine
6
 Other Important…

NADH

NAD+

260 nm 340 nm

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
170 for aspirin hydrolysis.
 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- 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

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 indicator.
173
 THANK YOU

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