1.
Pharmaceutical Analysis-II
Part I
SPECtrOSCOPIC MEtHODS
Uv-Visible spectrophotometer 1

2.
Introduction to Spectroscopic Methods
• This chapter presents a brief review of electromagnetic
radiation and discusses how molecules and elements
absorb and emit electromagnetic radiation.
• Absorption and emission of electromagnetic radiation
are the basis for identification and quantitative
determinations in spectroscopic methods such as
– UV spectrophotometry,
– IR spectrophotometry,
– NIR spectrophotometry,
– atomic absorption spectrometry and
– atomic emission spectrometry.
• These methods are presented in subsequent chapters.
Uv-Visible spectrophotometer 2

3.
INTRODUCTION…
INTERACTION B/N RADIATION AND MATTER
• To understand how light interacts with matter, let’s see the
following example
• If beam of white light is passed through a beaker of water, it
remains white.
• If KMnO4 is added to the water---purple
• KMnO4 allows blue and red color to pass and absorbs the
other colors.
• This is an example of the interaction b/n radiant energy and
matter.
• In this case, the radiant energy is visible and we can see
the effect of absorption with our eyes.
Uv-Visible spectrophotometer 3

4.
INTRODUCTION…
• However, absorption of radiation can take place over
a wide range of radiant energy, most of which can
not be seen.
• Such absorption effects can be measured using
suitable instruments
E.g. UV-Visible and IR instruments
Uv-Visible spectrophotometer 4

5.
INTRODUCTION…
vDefinition
– Spectroscopy is the study of interaction between
electromagnetic radiation (EMR) and matter.
– Because these (uv-vis and IR) techniques use
optical materials to disperse and focus the
radiation, they often are identified as optical
spectroscopies.
– Despite the difference in instrumentation, all
spectroscopic techniques share several common
features.
– Before we consider individual examples in greater
detail, let’s take a moment to consider some of
these similarities.
Uv-Visible spectrophotometer 5

6.
INTRODUCTION…
vAll techniques use electromagnetic radiation as light
source
What is Electromagnetic Radiation (EMR)?
• Electromagnetic radiation is a form of energy
• It has properties of both waves and particles.
– Its refraction when it passes from one medium to
another shows its wave property
Whereas
– Absorption and emission properties are associated
with its particulate property
Uv-Visible spectrophotometer 6

7.
INTRODUCTION…
Now let’s discuss these important properties of EMR
one by one
a. 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
Figure: Plane-polarized electromagnetic radiation showing the
oscillating electric field in red and the oscillating magnetic field
in blue.
Uv-Visible spectrophotometer 7

8.
INTRODUCTION…
vIn a vacuum, electromagnetic radiation travels at
the speed of light, c, which is 3x108m/s
vEMR moves through a medium other than a
vacuum with a velocity, v, less than that of the
speed of light in a vacuum.
vThis is a proof that EMR has wave property.
Uv-Visible spectrophotometer 8

9.
INTRODUCTION…
• The interaction of electromagnetic radiation with
matter can be explained using either the electric
field or the magnetic field.
Uv-Visible spectrophotometer 9

10.
INTRODUCTION…
v An electromagnetic wave, therefore, is characterized by
several fundamental properties. The most important ones
are discussed as follows
A. Wavelength (l, lambda)
v Is defined as the distance between successive maxima, or
successive minima.
v Different units of length are used to express wavelengths
§ E.g. Angstrom, centimeter, micro and nanometer
§ 1 m = 102 cm = 103 mm = 106 m = 109 nm = 1010 o.
§ 10 = 10-1 nm = 10-4  = 10-7 mm = 10-8 cm = 10‑ 10 m.
v For UV-visible EMR the wavelength is usually expressed in
nanometers , for IR radiation is given in microns (µm, 10–6
m).
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11.
INTRODUCTION…
B. Amplitude (A)
v Is the vertical distance from midline of a wave to the peak or
trough.
v It is Measured by units of distance
C. Frequency (v,nu)
Frequency is the number of waves that pass through a
particular point in one second (Hz = 1 cycle/s)
v Relationship b/n wavelength and frequency
D.
Ø This expresses the Number of waves per Centimeter (has a
unit cm-1)
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12.
INTRODUCTION…
Exercise
• The wavelength of the sodium D line is 589 nm.
What are the frequency and the wave number for
this line?
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13.
INTRODUCTION…
b. Particle properties of EMR
vWhen matter absorbs electromagnetic radiation it
undergoes a change in energy.
vEMR consists of a beam of energetic particles
called photons.
vWhen a photon is absorbed by a sample it is
“destroyed,” and its energy is acquired by the
sample.
Uv-Visible spectrophotometer 13

14.
INTRODUCTION…
• The energy of a photon, in joules, is related to its
frequency, wavelength, or wavenumber by the
following equations.
• Where h is Planck’s constant, with a value of 6.626 X 10–34 J · s.
• Example-- What is the energy per photon of the sodium D
line ( λ= 589 nm)?
Uv-Visible spectrophotometer 14

15.
INTRODUCTION…
2. The Electromagnetic Spectrum
vThe frequency and wavelength of EMR vary
over many orders of magnitude.
vFor convenience, EMR is divided into different
regions based on the type of atomic or
molecular transition that gives rise to the
absorption or emission of photons (see the
Figure below).
– T h e s e re g i o n s co l l e c t i ve l y fo r m T h e
Electromagnetic Spectrum
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16.
 The boundaries describing the electromagnetic spectrum
are not rigid, and an overlap between spectral regions is
possible.
Figure :The electromagnetic spectrum showing the boundaries between
different regions and the type of atomic or molecular transition
responsible for the change in energy.
Uv-Visible spectrophotometer 16

17.
INTRODUCTION…
3. How does EMR interact with Matter?
vMatter is in a continuous motion
vMotion could be
• rotational,
• vibrational
• electronic or
• translational motion or combination of
these.
vEach motion is associated with different level of
energy.
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18.
INTRODUCTION…
3. How does EMR interact with Matter?...
vEach motion can be made to occur at a faster rate
(at higher energy level) by applying an external
energy.
vThis can be achieved by applying one of the regions
of the EMR , since each consists energetic particles
called photons.
vAfter absorbing energy, each type of motion are
promoted from the lower energy level (Ground
state) to higher energy level (Excited Level).
Uv-Visible spectrophotometer 18

19.
3. How does EMR interact with Matter?...
v The source of the energetic state depends on the photon’s
energy.
Example
v Absorption of UV/Visible affects all types of motions, the
major effect being transition of valence electrons
v Absorption of IR radiation results in vibrational and
rotational energy transitions
Uv-Visible spectrophotometer 19

20.
UV-Visible spectrophotometry
Principles
• Radiation in the wavelength range 200–800 nm is
passed through a solution of a compound.
• The electrons in the bonds within the molecule
become excited so that they occupy a higher
quantum state and in the process absorb some of
the energy passing through the solution.
• The more loosely held the electrons are within
the bonds of the molecule, the longer the
wavelength (lower the energy) of the radiation
absorbed.
Uv-Visible spectrophotometer 20

21.
Applications in pharmaceutical analysis
• A robust, workhorse method for the
quantification of drugs in formulations where
there is no interference from excipients.
• Determination of the pKa values of some drugs.
• Determination of partition coefficients and
solubilities of drugs.
• Used to determine the release of drugs from
formulations with time, e.g. in dissolution testing.
• Can be used to monitor the reaction kinetics of
drug degradation.
• The UV spectrum of a drug is often used as one
of a number of pharmacopoeial identity checks.
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22.
Strengths
• An easy-to-use, cheap and robust method offering good
precision for making quantitative measurements of drugs in
formulations.
• Routine method for determining some of the physico-
chemical properties of drugs, which need to be known for
the purposes of formulation.
• Some of the problems of the basic method can be solved
by the use of derivative spectra.
Limitations
• Only moderately selective. The selectivity of the method
depends on the chromophore of the individual drugs, e.g a
coloured drug with an extended chromophore is more
distinctive than a drug with a simple benzene ring
chromophore.
• Not readily applicable to the analysis of mixtures
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23.
Introduction
§ A spectroscopic technique which utilizes the
UV/Visible region of the EMR is known as UV/visible
spectroscopy /spectrophtometery/.
§ Near UV region 200 nm-400 nm
§ Visible region 400-800 nm
§ Absorption of light in these region mainly causes
electronic transition.
§ The outer electrons in an organic molecule may
occupy one of three different energy levels (- , - or
n- energy level).
Uv-Visible spectrophotometer 23

24.
Accordingly there are three types of electrons.
a) σ-electrons;
• They are bonding electrons
• They represent valence bonds and possess the
lowest energy level ( the most stable)
b) π-electrons;
• They are bonding electrons, forming the pi-bonds
(double bounds), and
• possess higher energy than sigma-electrons.
Uv-Visible spectrophotometer 24

25.
c) n-electrons;
• They are nonbonding electrons,
• Present in atomic orbitals of hetero atoms (N, O, S or
halogens).
• They usually occupy the highest energy level of the
ground state.
Uv-Visible spectrophotometer 25

26.
Electronic transitions of organic compounds
§ Electrons reside in orbitals. A molecule also posseses
normally unoccupied orbitals called antibonding
orbitals; these corresponds to excited state energy
levels and are either * or *.
Uv-Visible spectrophotometer 26

27.
• In excited state the  -electrons occupy an anti-
bonding energy level ( *) and the transition is
termed  -  * transition.
•  -electrons occupy an anti-bonding energy level (
*) and the transition is termed  -  * transition,
• While the n-electrons may occupy  * or  * levels to
give n-  * or n-  * transition.
Uv-Visible spectrophotometer 27

28.
Organic compounds containing -Electrons:
vCompounds contain -electrons only are the
saturated hydrocarbons, which absorb below 170
nm.
vThey are transparent in the near UV region (200 -
400 nm)
– And this make them ideal solvents for other
compounds studied in this range.
vThey are characterized by --* transition only.
Uv-Visible spectrophotometer 28

29.
Organic compounds containing n-Electrons :
vSaturated organic compounds containing hetero
atoms, possess n-electrons in addition to sigma-
electrons.
vThey are characterized by the  -* and n – *
transitions.
vn-electrons can also be transited to * when they
exist in unsaturated compounds
Uv-Visible spectrophotometer 29

30.
Organic compounds containing -Electrons :
vUnsaturated compounds containing no hetero
atoms are characterized by the -* and -*
transitions, such as ethylene (CH2=CH2).
vWhen these compounds contain hetero atoms,
they can undergo -*, -*, n-* and n-*
transitions
example: acetone (CH3-COCH3).
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31.
• Increasing order in absorption wavelength
-* <n-* < -*< n-*
Table: Electronic transitions involving n,  and  molecular
orbitals
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32.
Of these transitions, the most important are the
n-π* and π - π *, because they involve functional
groups that are characteristic of the analyte and
wavelengths that are easily accessible.
The bonds and functional groups that give rise to
the absorption of ultraviolet and visible radiation
are called chromophores.
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33.
Factors governing absorption of radiation in the
UV-Vis region
vFactors leading to spectral changes are the following
vConjugation
vAttachment of auxochromes and chromophres
vSolvent polarity
vPH of the medium
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34.
vπ to π * transitions, when occurring in isolated
groups in a molecule, give rise to absorptions of
fairly low intensity.
vHowever, conjugation of unsaturated groups in
a molecule produces a remarkable effect upon
the absorption spectrum.
• The wavelength of maximum absorption moves
to a longer wavelength and the absorption
intensity may often increase.
Uv-Visible spectrophotometer 34

35.
Uv-Visible spectrophotometer 35

36.
vThe same effect occurs when groups containing n
electrons are conjugated with a π electron group;
e.g.,
vThus, the characteristic energy of a transition and
hence the wavelength of absorption is a property of a
group of atoms rather than the electrons themselves.
Uv-Visible spectrophotometer 36

37.
• When such absorption occurs, two types of groups
can influence the resulting absorption spectrum of
the molecule:
• chromophores and
• auxochromes
Chromophores
v are functional groups, not conjugated with another
group, which exhibit a characteristic absorption
spectrum in the ultraviolet or visible region.
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38.
vSome of the more important chromophoric groups are:
vIf any of the simple chromophores is conjugated with
another (of the same type or different type) a multiple
chromophore is formed having a new absorption band.
Uv-Visible spectrophotometer 38

39.
Auxochromes
vThey Intensify the absorption of a molecule
vAuxochromes do not absorb significantly in the 200-
800nm region,
– but will affect the spectrum of the chromophore when
attached to it.
vThese include OH, NH2, CH3
, alkoxy and Halogens
Uv-Visible spectrophotometer 39

40.
Auxochromes cause two types of shifts
• Bathochromic (Red) shift: shift of absorption to longer
wavelength due to substitution and solvent effects.
 Hypsochromic (Blue) shift: it is shift of absorption to
shorter wavelength.
 Hyperchromic & hypochromic effects: it is the increase and
decrease in absorption intensity respectively.
Uv-Visible spectrophotometer 40

41.
Absorption characteristics of chromophores
1- Ethylenic chromophores:
vTheir bands are difficult to observe in near UV region, so
they are not Useful analytically.
v 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.
Attachement to auxochromes: cause red shift and increased
absorption intensity due to extension of conjugation.
Uv-Visible spectrophotometer 41

42.
2- Carbon-hetero atom chromophores:
Such as -C=O, -C=N, -C=S, -N=O, ….etc.
vThey exhibit some common characteristics; n-pi* band in the
range of 275-300 nm., which is the most apparent band, has
low intensity and long wavelength.
v This band undergoes a blue shift on increasing the solvent
polarity
– due to increasing the energy of transition as a result of
hydrogen bonding
vAlkyl substitution; Cause red shift due to hyper-conjugation.
Uv-Visible spectrophotometer 42

43.
A] 1,3-Butadienes and conjugated enones
Uv-Visible spectrophotometer
43
Separated chromophores (by two or
more single bonds)
CH2 = CH – CH2 – CH = CH2
have additive effect only because
there is little or no electronic
interaction between separated
chromophores.
CH2 = CH2
CH2 = CH – CH2 – CH = CH2
170-180 nm

44.
• If the two chromophoric
groups are present in a
molecule and they are
separated by only one single
bond (a conjugated system), a
large effect on the spectrum
results, more than found by
mere addition.
CH2 = CH – CH = CH2 or CH2 = CH
– CH = O
Uv-Visible spectrophotometer 44
CH2 = CH – CH = CH2
170-180 205-215 nm

45.
[B] Aromatic Systems
I) Benzene ring :
vBenzene has three maxima at 184 nm ( the most intense),
204 nm and at 254 nm.
vThe first two bands have their origin in the ethylenic -*
transition, while the longest B-band is a specific feature of
benzenoid compounds.
vThis band abbreviated B-band, is characterized by
vibrational fine structures.
vIn structure elucidation both the B-band and the 204-nm
ethylenic band, termed E-band are useful while the far UV
band (184 nm) is unsuitable for analytical purposes.
Uv-Visible spectrophotometer 45

46.
[B] Aromatic Systems…
I) Benzene ring :
Uv-Visible spectrophotometer 46
184 nm
204 nm 254 nm

47.
[B] Aromatic Systems…
II) Monosubstituted benzenes :
vWhen 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.
vThis occurs whether the substituent is an electron
donating or electron withdrawing group.
vIn addition the B band loses most of its fine structure.
Uv-Visible spectrophotometer 47

48.
Effect of pH on absorption spectra
vThe spectra of compounds containing acidic or basic
groups are dependent on the pH of the medium
e.g. phenols and amines.
vUV-spectrum of phenol in acid medium (where the
molecular form predominates) is completely different
from its spectrum in alkaline medium (where the
phenolate anion predominates).
vSpectrum in alkaline medium exhibits bathochromic
shift with hyperchromic effect.
Uv-Visible spectrophotometer 48

49.
Effect of pH on absorption spectra…
vThe red shift is due to the participation of the pair
of electrons in resonance with the  electrons of
the benzene ring,
vthus increasing the delocalization of the 
electrons.
Uv-Visible spectrophotometer 49
-
+
H
in acid medium in alkaline medium
O
O
OH
OH
(Phenol)lmax = 270 nm (phenate anion) lmax= 290 nm

50.
v On the other hand, UV spectrum of aniline in acid medium
show hypsochromic (blue) shift with hypochromic effect
vThis blue shift is due to the protonation of the amino group,
hence the pair of electrons is no longer available
vand the spectrum in this case is similar to that of benzene
(thus called benzenoid spectrum).
Uv-Visible spectrophotometer 50
NH2 NH3
In alkaline medium in acid medium
Aniline, lmax= 280 nm Anilinium ion lmax= 254 nm
+
+ H+
- H+

51.
Effect of Solvent on absorption spectra
vThe solvent in which the absorbing species is
dissolved also has an effect on the spectrum of the
species.
v Peaks resulting from n → π* transitions are shifted to
shorter wavelengths (blue shift) with increasing solvent
polarity.
vThe ground state is more polar than the excited state
vHydrogen bonding solvents with unshared electron
pairs in the ground state molecule lowers the energy of
the n-orbital
Uv-Visible spectrophotometer 51

52.
Effect of Solvent on absorption spectra…
vOften the reverse (i.e. red shift) is seen for π → π*
transitions.
vThe ground state of the molecule is relatively non-
polar, and the excited state is often more polar than
the ground state.
vAs a result, when a polar solvent is used, it interacts
more strongly with the excited state than with the
ground state, and the transition is shifted to longer
wavelength.
Uv-Visible spectrophotometer 52

53.
Effect of Solvent on absorption spectra…
vFor example, the figure below shows that the
absorption maximum of acetone in hexane appears
at 279 nm which in water is shifted to 264 nm, with a
blue shift of 15 nm.
Uv-Visible spectrophotometer 53

54.
Calculation of λmax of an organic compound
I. Woodward's rules:
v Named after Robert Burns Woodward
vThese rules are several sets of empirically derived
rules
vThey attempt to predict the wavelength of the
absorption maximum ( λmax ) in an ultraviolet-
visible spectrum of a given compound.
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55.
I. Woodward's rules…
A. Rules for conjugated dienes
v These rules specify a base value (214 nm) for the
parent diene which is 1,3-butadiene.
v The value is red shifted upon
v alkyl substitution or
v attachment of ring carbons or
v ring residues or olefin
Uv-Visible spectrophotometer 55
R2C=CR-CR=CR2

56.
A. Rules for conjugated dienes…
vIt is also affected by the presence of double bonds out
side a ring (exocyclic), extra double bonds in
conjugation, and auxochromes.
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57.
A. Rules for conjugated dienes…
Examples
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58.
Examples…
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59.
B. Rules for enones
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60.
B. Rules for enones…
• Examples
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61.
B. Rules for enones…
• Examples
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62.
vα, β -unsaturated aldehydes, acids and esters follow
the same general trends as enones, but have different
base values.
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63.
C. Rules for Benzoyl Derivatives
Uv-Visible spectrophotometer 63

64.
C. Rules for Benzoyl Derivatives…
• Example
v The Woodward’s rules work well only for conjugated
polyenes having four double bonds or less.
v For conjugated polyenes with more than four double
bonds the Kuhn rules are used.
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65.
II. Simplified Kuhn and Hausser rule
v According to this rule
λmax = 134(n)1/2 +31
• Where n is the number of conjugated double bonds
Example
• λmax =476 nm
• λmax =476 nm
•
Uv-Visible spectrophotometer 65

66.
II. Simplified Kuhn and Hausser rules…
vThis rule is also useful for calculating number of
double bonds from the observed λmax.
Example, a compound with λmax of 433 nm will
have 9 conjugated double bonds.
Uv-Visible spectrophotometer 66

67.
Information from UV VIS Data
Qualitative or Quantitative
ü UV-VIS data alone gives little structural information
1. Single band of low intensity with (ε 100 to 10,000) and λmax <
220 nm:
Usually n → σ* transition possible and the groups present may
be alcohols, ethers, amines, thiols, etc.
2. A single band of low intensity (ε= 10 to 100) λmax250-
360nm ,with no absorption in the shorter range 200-250nm
indicates n--> π* transition of a simple or unconjugated
chromophore.
Egs.C=O, C=N, N=N, NO2,COOH, CONH2.
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68.
3. Two bands of medium intensity with (ε 1,000 to
10,000) and both λmax > 200 nm: Usually is π → π*
transition of aromatic system: Look for fine structure
in longer wavelength band.
4 . Bands of high intensity with (ε 10,000 to 20,000) and
λmax > 220 nm:
Usually conjugated π system: Check dienes and α,β-
unsaturated carbonyls
5. Band of low intensity with λmax > 300 nm (n → π*)
and band of high intensity with λmax < 250 nm (π →
π*):
Show unconjugated ketones, esters, acids, etc.
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69.
Quantitative UV-Visible Spectrophotometer
v The attenuation of EMR as it passes through a sample is
described quantitatively by two separate, but related
terms: transmittance and absorbance.
v Transmittance is defined as the ratio of the EMR’s power
exiting the sample, PT , to that incident on the sample
from the source, P0,
v Multiplying the transmittance by 100 gives the percent
transmittance (%T), which varies between 100% (no
absorption) and 0% (complete absorption).
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70.
Quantitative UV-Visible Spectrophotometer…
vAn alternative method for expressing the attenuation
of electromagnetic radiation is absorbance, A, which
is defined as
vAbsorbance is the more common because it is a
linear function of the analyte’s concentration.
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71.
Quantitative UV-Visible Spectrophotometer…
vBesides absorption by the analyte, several
additional phenomena contribute to the net
attenuation of radiation, including
§ reflection and absorption by the sample
container,
§ absorption by components of the sample matrix
and
§ the scattering of radiation.
vTo compensate for this loss of the electromagnetic
radiation’s power, we use a method called blank.
Uv-Visible spectrophotometer 71

72.
Absorbance and Concentration: Beer’s Law
vBeer’s law states that, using a monochromatic
wavelength, Absorbance is directly proportional to
concentration.
v Where
vA is absorbance
va is absorptivity where the concentration is
expressed in gm/L
v ɛ is molar absorptivity where the
concnetration is expressed in mol/L
vC is concentration
v b is the path length of sample cell
Uv-Visible spectrophotometer 72

73.
Absorbance and Concentration: Beer’s Law…
Examples:
• A 5.00x10–4 M solution of an analyte is placed in a sample
cell that has a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the absorbance of the solution is
found to be 0.338. What is the analyte’s molar absorptivity
at this wavelength? Ans(ɛ = 676 cm-1 M-1
)
v A sample has a percent transmittance of 50.0%. What is its
absorbance? Ans (A= 0.301)
v The molar absorptivity of a substance is 2.0 × 104 cm-1 mol-
1 L. Calculate the transmittance through a cuvette of path
length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of the
substance. Ans (T= 0.63)
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74.
Limitations to Beer’s Law
vDeviations from the direct proportionality between the
measured absorbance and concentration when path
length is constant may be encountered.
vAssumptions of the absorption law:
vThe incident beam is monochromatic
vThe absorbers absorb independently of each
other.
vIncident radiation consists of parallel rays
perpendicular to the surface of the absorbing
medium.
vPath length traversed is uniform over the cross
section of the beam.
vAbsorbing medium is homogenous and does not
scatter the radiation.
Uv-Visible spectrophotometer 74

75.
Limitations to Beer’s Law…
vDeviations from linearity are divided into three
categories:
vFundamental
vChemical and
vInstrumental
Uv-Visible spectrophotometer 75

76.
I. Fundamental Limitations:
– Beer’s law is valid only for low
concentrations/diluted solutions/ of analyte.
– At higher concentrations the individual
particles of analyte no longer behave
independently of one another.
– There will be reflection, Refraction and
scattering
– Positive deviations
Uv-Visible spectrophotometer 76

77.
II. Chemical Limitations
vDeviations from Beer’s law also arise when an analyte
associates, dissociates or reacts with a solvent to
produce a product having a different absorption
spectrum from the analyte.
vDepending on the resulting products, it may result in
positive or negative deviations.
Uv-Visible spectrophotometer 77

78.
III. Instrumental Limitations
• Using polychromatic radiation always gives a
negative deviation from Beer’s
• Stray light causes negative deviations
Uv-Visible spectrophotometer 78

79.
UV-Visible spectrophotometer…
INStrUMENtatION FOr UV-VIS
SPECtrOMEtrY
Uv-Visible spectrophotometer 79

80.
INSTRUMENTATION
v Today a wide range of instruments are available for making
molecular absorption measurements in the UV-visible range.
v These vary from simple and inexpensive machines for
routine work to highly sophisticated devices.
v However, the basic components of these instruments
remain the same.
v The five essential components of UV-VIS instruments are
– A stable radiation source
– Wavelength selector
– Sample holder
– Radiation detector or transducer , and
– Signal processing and output device
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81.
Uv-Visible spectrophotometer 81
INSTRUMENTATION…
vThe general layout of the essential components in a
simple absorption instrument is

82.
INSTRUMENTATION…
1. Radiation Sources
– A deuterium discharge lamp for UV region (160-375
nm)
– A tungsten filament lamp or tungsten-halogen lamp
for Visible and NIR regions (350 - 2500 nm)
– The instruments automatically swap lamps when
scanning between the UV and VIS-NIR regions
Uv-Visible spectrophotometer 82

83.
INSTRUMENTATION…
2. Wavelength Selectors
vIn spectrophotometric measurements we need to use
a narrow band of wavelengths of light.
vThis enhances the selectivity and sensitivity of the
instrument and give a more linear relationship
vThere are different types of wavelength
selectors.
vThese include Filters and moncochromators
Uv-Visible spectrophotometer 83

84.
INSTRUMENTATION…
A. Filters
v Either absorption or interference filters are
used for wavelength selection:
Absorption filters
v Usually function via selective absorption of unwanted
wavelengths and transmitting the complementary color.
v The most common type consists of colored glass or a dye
suspended in gelatin and sandwiched between two glass
plates.
v They are Inexpensive and widely used for band selection
in the visible region.
Uv-Visible spectrophotometer 84

85.
INSTRUMENTATION…
Absorption filters…
• If a solution appears orange, this implies that orange light is
not being absorbed.
Uv-Visible spectrophotometer 85

86.
INSTRUMENTATION…
Interference filters
vAs the name implies, an interference filter relies on
optical interference to provide a relatively narrow
band of radiation.
vIt consists of a transparent material (calcium or
magnesium fluoride) sandwiched between two
semitransparent metallic films coated on the inside
surface of two glass plates.
vWhen it is subjected to a perpendicular beam of
light, a fraction passes through the first metallic layer
and the other is reflected.
Uv-Visible spectrophotometer 86

87.
INSTRUMENTATION…
Interference filters…
vFraction that is passed undergoes a similar
partitioning upon passing through the second
metallic film, thus narrower bandwidths are obtained.
Uv-Visible spectrophotometer 87

88.
INSTRUMENTATION…
B. Monochromators
vOne limitation of an absorption or interference filter is
that they do not allow for a continuous selection of
wavelength.
vIf measurements need to be made at two wavelengths,
then the filter must be changed in between
measurements.
vAnother limitation is that they do not give narrow
band of wavelength.
Uv-Visible spectrophotometer 88

89.
INSTRUMENTATION…
B. Monochromators…
vAn alternative approach to wavelength selection,
which provides for a continuous variation of
wavelength, is the monochromator.
vThese are of two types;
vthe prism and
vgrating monochromators.
Uv-Visible spectrophotometer 89

90.
INSTRUMENTATION…
Prisms
v The radiations of different colors having different
wavelengths are refracted to different extent due to the
difference in the refractive index of glass for different
wavelengths.
Uv-Visible spectrophotometer 90

91.
INSTRUMENTATION…
Prisms…
v In a prism monochromator, shown below fine beam of the
light from the source is obtained by passing through an
entrance slit. This is then collimated on the prism with the
help of a lens.
v The refracted beams are then focused on an exit slit. The
prism is then rotated in a predetermined way to provide the
desired wavelength from the exit slit.
Uv-Visible spectrophotometer 91

92.
INSTRUMENTATION…
Gratings
v A grating is made by cutting or etching a series of closely
spaced parallel grooves on the smooth reflective surface of a
solid material as shown below
v The surface is made reflective by making a thin film of
aluminum on it and the etching is done with the help of a
suitably shaped diamond tool.
Uv-Visible spectrophotometer 92

93.
INSTRUMENTATION…
Gratings…
vIn grating monochromator (Fig. above), a fine
beam of the light from the source falls on a
concave mirror through an entrance slit.
vThis is then reflected on the grating which
disperses it.
vThe dispersed radiation is then directed to an exit
slit.
Uv-Visible spectrophotometer 93

94.
INSTRUMENTATION…
Gratings…
vThe range of wavelengths isolated by the
monochromator is determined by the extent of
dispersion by the grating and the width of the
exit slit.
vRotation of the grating in a predetermined way
can be used to obtain the desired wavelength
from the exit slit.
Uv-Visible spectrophotometer 94

95.
INSTRUMENTATION…
3. Sample cells
vThe UV-VIS absorption spectra are usually determined
either in vapor phase or in solution.
vSample containing the analyte is taken in a cell called a
cuvette
– which is transparent to the wavelength of light
passing through it.
vA variety of quartz cuvettes are available
vThese are of varying path lengths and are equipped
with inlet and outlets.
Uv-Visible spectrophotometer 95

96.
INSTRUMENTATION…
3. Sample cells…
vFor measurements in the visible region the
cuvettes made of glass can also be used.
vHowever, since glass absorbs the ultraviolet
radiations, these cannot be used in the UV
region.
vTherefore, most of the spectrophotometers employ
quartz cuvettes (Fig below), as these can be used
for both visible and UV region.
Uv-Visible spectrophotometer 96

97.
INSTRUMENTATION…
3. Sample cells…
v Usually square cuvettes having internal path length 1.0 cm
are used.
v Though cuvettes of much smaller path lengths say of 0.1
mm or 0.05 mm are also available.
Uv-Visible spectrophotometer 97
qThe faces of these cells
t h r o u g h w h i c h t h e
radiation passes are
highly polished to keep
reflection and scatter
losses to a minimum.

98.
INSTRUMENTATION…
3. Sample cells…
vNow a days ‘spectral grade’ solvents are available
which have
– high purity and
– negligible absorption in the region of absorption
by the chromophore.
vIn a typical measurement of absorption spectrum, the
solution of the sample is taken in a suitable cuvette
and the spectrum is run in the desired range of the
wavelengths.
Uv-Visible spectrophotometer 98

99.
INSTRUMENTATION…
3. Sample cells…
v The absorption by the solvent, if any, is compensated
by
– running the spectrum for the solvent alone in the same or
identical cuvette and subtracting it from the spectrum of
the solution.
vThis gives the spectrum only due to the absorption
of the species under investigation.
vIn double beam spectrometers, the sample and
the solvent are scanned simultaneously
Uv-Visible spectrophotometer 99

100.
INSTRUMENTATION…
4. Detectors
vconvert a light signal to an electrical signal
vThis is suitably measured and transformed into an output.
vThey generate a signal, which is linear in transmittance
i.e. they respond linearly to radiant power falling on them.
vThe transmittance values can be changed logarithmically
into absorbance units by an electrical or mechanical
arrangement in the signal read out device.
vThere are three types of detectors which are used in
modern spectrophotometers.
Uv-Visible spectrophotometer 100

101.
INSTRUMENTATION…
4. Detectors…
i. Phototube
• A phototube consists of a photoemissive cathode and an
anode in an evacuated tube with a quartz window.
• These two electrodes are subjected to high voltage (about
100 V) difference.
• When a photon enters the tube and strikes the cathode, an
electron is ejected and is attracted to the anode resulting in
a flow of current which can be amplified and measured.
Uv-Visible spectrophotometer 101

102.
INSTRUMENTATION…
ii. Photomultiplier (PM) Tube
vA photomultiplier tube consists of a series of
electrodes, called dynodes.
v The voltage of successive electrodes is maintained
50 to 90 volt more positive than the previous one.
vWhen a radiation falls on the cathode an electron is
emitted from it.
vThis is accelerated towards the next photo emissive
electrode by the potential difference between the
two. Here, it releases many more secondary
electrons.
Uv-Visible spectrophotometer 102

103.
INSTRUMENTATION…
ii. Photomultiplier (PM) Tube…
v These, in turn are accelerated to the next electrode where
each secondary electron releases more electrons.
v The process continues up to about 10 stages of
amplification. The final output of the photomultiplier tube
gives a much larger signal than the photocell.
Uv-Visible spectrophotometer 103

104.
INSTRUMENTATION…
iii. Diode Array Detectors
• Employ a large number of silicon diodes arranged
side by side on a single chip.
• When a UV-VIS radiation falls on the diode, its
conductivity increases significantly.
• This increase in conductivity is proportional to
the intensity of the radiation and can be readily
measured.
Uv-Visible spectrophotometer 104

105.
INSTRUMENTATION…
iii. Diode Array Detector…
• Since a large number of diodes can be arranged
together, the intensity at a number of wavelengths
can be measure simultaneously.
Uv-Visible spectrophotometer 105

106.
INSTRUMENTATION…
5. Signal Processing and Output Devices
vThe electrical signal from the transducer is suitably
amplified or processed before it is sent to the recorder
to give an output.
vThe subtraction of the solvent spectrum from that of
the solution is done electronically.
vThe output plot between the wavelength and the
intensity of absorption is the resultant of the
subtraction process and is characteristic of the
absorbing species.
Uv-Visible spectrophotometer 106

107.
TYPES OF UV-VISIBLE SPECTROMETERS
vBroadly speaking there are three types of
spectrometers.
1. Single Beam Spectrometers
vcontain a single beam of light.
üThe same beam is used for reading the absorption
of the sample as well as the reference.
vThe radiation from the source is passed through a
filter or a suitable monochromator to get a band on
monochromatic radiation.
Uv-Visible spectrophotometer 107

108.
I. Single Beam Spectrometers…
v It is then passed through the sample (or the reference)
and the transmitted radiation is detected by the
photodetector.
v The signal so obtained is sent as a read out or is
recorded.
v Typically, two operations have to be performed –
first, the cuvette is filled with the reference solution
and the absorbance reading at a given wavelength or
the spectrum over the desired range is recorded.
Uv-Visible spectrophotometer 108

109.
1. Single Beam Spectrometers…
Second, the cuvette is taken out and rinsed and filled
with sample solution and the process is repeated.
v The spectrum of the sample is obtained by subtracting
the spectrum of the reference from that of the sample
solution.
Uv-Visible spectrophotometer 109

110.
2. Double Beam Spectrometers
v In a double beam spectrometer, the radiation coming from
the monochromator is split into two beams with the help
of a beam splitter.
v These are passed simultaneously through the reference
and the sample cell.
v The transmitted radiations are detected by the detectors
and the difference in the signal at all the wavelengths is
suitably amplified and sent for the output.
Uv-Visible spectrophotometer 110

111.
3. Photodiode Array Spectrometer
v In a photodiode array instrument, also called a multi-
channel instrument, the radiation output from the source
is focused directly on the sample.
v This allows the radiations of all the wavelengths to
simultaneously fall on the sample.
v The radiation coming out of the sample after absorption
(if any) is then made to fall on a reflection grating.
Uv-Visible spectrophotometer 111

112.
Uv-Visible spectrophotometer 112
3. Photodiode Array Spectrometer…
vThe grating disperses all the wavelengths
simultaneously.
vThese then fall on the array of the photodiodes
arranged side by side.
vIn this way the intensities of all the radiations in the
range of the spectrum are measured in one go.
vThe advantage of such instruments is that a scan of
the whole range can be accomplished in a short time.

113.
UV-Visible spectrophotometer…
aPPLICatIONS OF UV-VISIBLE
SPECtrOPHOtOMEtEr
Uv-Visible spectrophotometer 113

114.
UV-Visible Spectrophotometer…
Principles: radiation in the wavelength range 200-800nm is
passed through a solution of a compound.
Ø The electrons in the molecule become excited so that they
occupy a higher quantum state and in process absorb some of the
energy passing through the solution.
Ø The wavelength at which the solution (analyte) absorbs and the
Intensity of absorption is determined by the structure and the
concentration of the analyte respectively.
ØCan be used for qualitative and quantitative analysis if
appropriate Instrument is used
114

115.
Important advantages of spectrophotometric methods
1- Wide applicability; large number of organic and inorganic species
absorb light in the UV-Visible ranges.
2- High sensitivity; analysis for concentrations in the range from 10-
4 to 10- 6 M are ordinary in the Spectrophotometric
determinations.
3- Moderate to high selectivity; Due to selective reactions,
selective measurements and different mathematical
treatments.
4- Good accuracy; Relative errors in concentration measurement lie
in the range of 0.1 to 2 %.
5- Ease and convenience; Easily and rapidly performed with modern
instruments.
115

116.
Qualitative Applications
1. Identification of chromophores
2. Confirmation of identity
3. Detection of some functional groups
4. Determination of approximate number of conjugated
double bonds
5. Identification of the position and/or conformation of
certain functional groups
Uv-Visible spectrophotometer 116

117.
1- Identification of chromophores
• Example, the presence of an absorbance band at a
particular wavelength often is a good indicator of the
presence of a chromophore.
• Useful information about substance can be obtained
via examination of its lmax and εmax, which could be
correlated with the structural features (See the
following table).
Uv-Visible spectrophotometer 117

118.
1. Identification of chromophores…
Uv-Visible spectrophotometer 118
Absorption characteristics of some common organic
chromophores:

119.
2-Confirmation of identity
• The spectrum is a physical constant, which along with
melting & boiling points, refractive index and other
properties may be used for characterization of
compounds
• Although UV-visible spectra do not enable absolute
identification of an unknown, they frequently are used
to confirm the identity of a substance:
119

120.
2-Confirmation of identity…
2.1 Through comparison of the measured spectrum
with a reference spectrum.
a) An absorption band at 254 nm with characteristic
vibrational fine structures may be an evidence for
existence of aromatic structure.
b) Three characteristic bands at 278, 361 &550 nm with
absorbance ratio of 2:3:1 is very characteristic for
cyanocobalamin.
120

121.
2.2 Identification by using Absorbance ratio
• Absorbance ratio of a given drug at two different
wavelength is constant, provided that
– beer’s Law is obeyed at the selected wavelengths
– The same concentration of the sample is used for
both wavelengths
121
2-Confirmation of identity…

122.
3- Detection of some functional groups
a) An absorption band at about 280-290 nm, which is displaced
toward shorter wavelength with increasing solvent polarity
strongly, indicates the presence of aromatic carbonyl group.
b) Confirmation of presence of aromatic amine or phenolic
structure may be obtained by testing the pH effect on their
spectra.
122
NH2 NH3
In alkaline medium in acid medium
Aniline, lmax= 280 nm Anilinium ion lmax= 254 nm
+
+ H+
- H+
-
+
H
in acid medium in alkaline medium
O
O
OH
OH
(Phenol)lmax = 270 nm (phenate anion) lmax= 290 nm

123.
4- Approximate determination of the number of double
bonds:
By using Simplified Kuhn and Hausser rule :
lmax (nm) = 134 n + 31
where n is the number of conjugated double bonds.
5-Identification of the position and/or conformation of
certain functional groups:
d g b a
C = C – C = C – C = O enones
• a-Alkyl cause red shift about 10 nm & a-OH about 35 nm
• b-Alkyl cause red shift about 12 nm & b-OH about 30 nm
• g-Alkyl cause red shift about 18 nm & g-OH about 50 nm
123

124.
II. Quantitative Analysis
Scope
- Applications of spectrophotometric methods are so
numerous and touch every field in which quantitative
chemical information are required.
- In general, about 90% of all the quantitative
determinations are performed by spectroscopic
techniques.
- In the field of health alone , 95 % of all quantitative
determinations are performed by UV-Visible
spectrophotometer and similar techniques.
Uv-Visible spectrophotometer 124

125.
II. Quantitative Analysis ...
A. Assay of single components
• The assay of an absorbing substance may be quickly carried
out by preparing a solution in a transparent solvent and
measuring its absorbance at a suitable wavelength.
– Wavelength of maximum absorbance
• The concentration of the absorbing species is then
calculated from the measured absorbance using the
following procedures.
i. Use of standard absorptivity value- used for
substances whose reference standards are expensive
or difficult to obtain
Uv-Visible spectrophotometer 125

126.
Using Beer-Lambert Law
A = abc
a can be ɛ (molar absorptivity) when unit of
concentration is in moles per liter or A (1%, 1cm) (specific
absorbance) when unit of concentration is in g/L.
A (1%, 1cm) is absorbance of a 1g/100ml(1% w/v) solution
in 1cm cell.
A simple easily derived equation allows interconversion
of ɛ and A (1%, 1cm)
Uv-Visible spectrophotometer 126
� =
�1⊂�
1%
10
� ��������� ����ℎ�

127.
A. Assay of single components…
Example 1: Calculate the concentration of methyl testosterone in
an ethanolic solution of which the absorbance in a 1 cm cell at
its λmax, 241 nm, was found to be 0.890. The Specific
Absorptivity in the B.P. is 540 at 241 nm. (0.00165 g/100ml)
Example 2: calculate the concentration in µg/ml of a solution of
tryptophan (M.wt = 204.2) in 0.1 M HCl, giving an
absorbance at its λ max, 277 nm, of 0.613 in 4 cm cell.
( molar absorptivity is 5432). (5.76 µg/ml)
Uv-Visible spectrophotometer 127

128.
A. Assay of single components…
Example-3: A 5.00x10–4 M solution of an analyte is placed
in a sample cell that has a path length of 1.00 cm. When
measured at a wavelength of 490 nm, the absorbance of
the solution is found to be 0.338. What is the analyte’s
molar absorptivity at this wavelength? Ans(Molar A. =
676 cm-1 M-1
)
Example -4: A sample has a percent transmittance of 50.0%.
What is its absorbance? Ans (A= 0.301)
Example-5: The molar absorptivity of a substance is 2.0 × 104
cm-1 mol-1 L. Calculate the transmittance through a cuvette of
path length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of
the substance. Ans (T= 0.63)
Uv-Visible spectrophotometer 128

129.
A. Assay of single components…
ii. Use of calibration graph ,
Y = ax + b
Example: the absorbance values at 250 nm of 5 standard solutions, and
sample solution of a drug are given below:
Conc. (ug/ml) A 250 nm
10 0.168
20 0.329
30 0.508
40 0.660
50 0.846
Sample 0.661
Ø Calculate the concentration of the sample.
(Y= 0.01679X-0.0008, C= 36.5 µg/ml)
Uv-Visible spectrophotometer 129
 
 

-
 2
2
)
(
X
N
Y)
X)(
(
-
XY
N
X
a
 
 


-
 2
2
2
)
(
X
N
XY)
(
X)
(
-
)
X
)(
Y
(
X
b

130.
A. Assay of single components…
iii. Single point standardization
– this method involves the measurement of the absorbance of a
sample solution and of a standard solution of the reference
substance.
– The standard and sample solution are prepared in similar
manner.
– The concentration of the substance in the sample is calculated
from the proportional relationship that exists b/n absorbance
and concentration.
Uv-Visible spectrophotometer 130
std
std
Sample
sample
A
xC
A
C 

131.
iii. Single point standardization…
• It is the procedure adopted in many spectrophotometric
a s s a y s o f t h e B P a n d f o r t h e m a j o r i t y o f
spectrophotometric assays of USP.
• Occasionally, a linear but non proportional r/nship b/n
concentration and absorbance occurs, which is indicated
by a significant negative or positive intercept in a beer’s
law plot….double point standardization
Uv-Visible spectrophotometer 131

132.
iv. Double point standardization
• In the case mentioned before, a two –point bracketing
standardization is therefore required to determine the
concentration of the sample solution.
• The concentration of one of the standard solution is greater
than that of the sample while the other is with a` lower
concentration than the sample.
• The concentration of the test sample is given by:
• Where, Std1is the more concentrated standard and Std2 is less
concentrated standard
Uv-Visible spectrophotometer 132
2
1
2
1
1
2
1
1 )
(
)
)(
(
std
std
std
std
srd
std
std
std
Sample
sample
A
A
A
A
C
C
C
A
A
C
-
-

-
-


133.
B. Assay of Substances in multi component sample
• The spectroscopic analysis of drugs rarely involves
measurement of absorbance of samples containing only
one absorbing component.
• The pharmaceutical analyst frequently encounters the
situation where the concentration of one or more substance
is required in samples known to contain other absorbing
substances which potentially interfere in the assay.
Uv-Visible spectrophotometer 133

134.
B. Assay of Substances in multicomponent sample…
• The basis of all the spectrophotometric techniques for
multicomponent samples is the property that all
wavelengths:
a) The absorbance of a solution is the sum of absorbances
of the individual components or
b) The measured absorbance is the difference b/n the total
absorbance of the solution in the sample cell and that of
the solution in the reference cell.
Uv-Visible spectrophotometer 134

135.
B. Assay of Substances in multicomponent sample…
If the identity, concentration and absorptivity of the absorbing
interferents are known: it is possible to calculate their
contribution to the total absorbance of a mixture.
Example: The max of ephedrine HCl and Chlorocresol are 257 nm
and 279 nm respectively and the specific absorptivity values in
0.1M HCl solution are:
• Ephedrine HCl at 257 nm = 9.0 Chlorocresol at 257 nm = 20
• Ephedrine HCl at 279 nm = 0 Chlorocresol at 279 nm = 105
Ø Calculate the concentration of ephedrine HCl and Chlorocresol
in a batch of Ephedrine HCl injection, diluted 1 to 25 with water,
giving the following absorbance values in 1 cm cells.
(A279 (total) = 0.424, A257 (total) = 0.97)
ü Ans. C. Ephedrine HCl in the injection = 2.475 gm/100ml
C. Chlorocresol = 0. 1010 gm/100ml
Uv-Visible spectrophotometer 135

136.
B. Assay of Substances in multicomponent sample…
Simultaneous equations method
Note: Absorbance is additive
Uv-Visible spectrophotometer 136

137.
B. Assay of Substances in multicomponent sample…
2. Simultaneous equations method…
Example: Palladium (II) and gold (III) can be analyzed
simultaneously through reaction with methiomeprazine
(C19H24N2S2). The absorption maximum for the Pd complex
occurs at 480 nm, while that for the Au complex is at 635 nm.
Molar absorptivity data at these wavelengths are
A 2 5 . 0 - m L s a m p l e w a s t r e a t e d w i t h a n e x c e s s o f
methiomeprazine and subsequently diluted to 50.0 mL. Calculate
the molar concentrations of Pd(II), CPd, and Au(III), CAu, in the
sample if the diluted solution had an absorbance of 0.533 at 480
nm and 0.590 at 635 nm when measured in a 1.00-cm cell.
(CAu = 3. 60X10-5M, Cpd= 2.4X10-4M)
Uv-Visible spectrophotometer 137

138.
B. Assay of Substances in multicomponent…
Difference Spectrophotometer
the selectivity and accuracy of Spectrophotometric analysis of
samples containing absorbing interferents may be markedly
improved by the technique of difference spectrophotometer.
Principle: a component in a mixture is analysed by carrying
out a reaction which is selective for the analyte.
• This could be simply bringing about a shift in wavelength
through adjustment of pH of the solution in which the
analyte is dissolved or a chemical reaction such as
oxidation or reduction.
• The measured value is the difference absorbance (∆A) b/n
two equimolar solutions of the analyte in different
chemical forms which exhibit different spectral
characteristics.
Uv-Visible spectrophotometer 138

139.
B. Assay of Substances in multicomponent…
Difference Spectrophotometer…
ü The criteria for applying difference spectrophotometery
to the assay of a substance in the presence of other
absorbing substances are that:
q Reproducible changes may be induced in the spectrum
of the analyte by the addition of one or more reagents
q The absorbance of the interfering substance is not
altered by the reagents.
ü The simplest and most commonly employed technique
for altering the spectral characteristics of the analyte is
the adjustment of the pH by means of aqueous
solutions of acid, alkali or buffers.
Uv-Visible spectrophotometer 139

140.
B. Assay of Substances in multicomponent…
Difference Spectrophotometer…
∆A = Aalk(total)-Aacid (total)
= Aalk+Aint-(Aacid+Aint)
= Aalk-Aacid
∆A = ∆ε .b. C
• If the substance is not affected by pH, it can be
quantitatively converted by means of a suitable
reagent to a chemical species that has d/t spectral
properties to its unreacted parent species.
Uv-Visible spectrophotometer 140

141.
B. Assay of Substances in multicomponent…
Derivative spectroscopy
• Derivative spectroscopy uses first or higher derivatives of
absorbance with respect to wavelength for qualitative
analysis and for quantification.
• If a spectrum is expressed as
absorbance, A, as a function of
wavelength,, the derivative
spectra are:
Uv-Visible spectrophotometer 141

142.
B. Assay of Substances in multicomponent…
4. Derivative spectroscopy…
• A first-order derivative is the rate of change of absorbance with
respect to wavelength.
• It passes through zero at the same wavelength as λmax of the
absorbance band. This is characteristic of all odd-order
derivatives.
• The most characteristic feature of a second-order derivative is a
negative band with minimum at the same wavelength as the
maximum on the zero-order band.
• A fourth-order derivative shows a positive band.
• A strong negative or positive band with minimum or
maximum at the same wavelength as λ max of the absorbance
band is characteristic of the even-order derivatives.
Uv-Visible spectrophotometer 142

143.
B. Assay of Substances in multicomponent…
4. Derivative spectroscopy…
• Note that the number of bands observed is equal to the derivative
order plus one.
Advantages
ü Derivative spectrum shows better resolution of overlapping bands
the fundamental spectrum and may permit the accurate
determination of the λ max of the individual bands.
ü It permits discrimination against broad band interferences, arising
from turbidity or non-specific matrix absorption.
Ø Thus, the information content of a spectrum is presented in a
potentially more useful form, offering a convenient solution to a
number of analytical problems, such as resolution of multi-
component systems, removal of sample turbidity, matrix
background and enhancement of spectral details.
Uv-Visible spectrophotometer 143

144.
B. Assay of Substances in multicomponent…
4. Derivative spectroscopy…
Background elimination Resolution
Discrimination
Uv-Visible spectrophotometer 144

145.
B. Assay of Substances in multicomponent…
4. Derivative spectroscopy…
• It is possible to measure the absolute value of the derivative at
an abscissa value (wavelength) corresponding to a zero-
crossing of one of the components in the mixture.
• This is termed a zero-crossing measurement.
• The zero-crossing derivative spectroscopic mode allows the
resolution of binary mixtures of analytes by recording their
derivative spectra at wavelengths at which one of the
components exhibits no signal.
• Zero-crossing measurements for each component of the
mixture are therefore the sole function of the concentration of
the others.
Uv-Visible spectrophotometer 145

146.
III. Other Applications
• Monitoring drug degradation kinetics
• Detection in Chromatography
• Determination of Equilibrium Constants
• Determination of complex stoichiometry
• Spectrophotometeric titrations
Uv-Visible spectrophotometer 146

147.
III. Other Applications
A. Monitoring drug degradation kinetics
Ø Can be simply done when the product has a different
absorption spectrum than that of un-degraded drug.
Ø The rate of disappearance of the spectrum or
appearance of other spectrum (as a function of time )
may be used to determine rate constant for hydrolysis or
degradation.
Ø Oxidation reactions and any other type of reactions that
yield products whose spectra are different from the
reactants , may be followed and their rate constant
estimated.
Uv-Visible spectrophotometer 147

148.
B. Detection in Chromatography
Ø Mainly used in HPLC and HPTLC.
Ø They are the most widely used detectors, because:
ØMost drugs absorb UV-Visible radiation.
ØMore sensitive and more selective than the
bulk property detectors
Ø Some absorbance detectors have one or two fixed
wavelengths (280 and/or 254 nm).
Ø More modern HPLC instruments have variable
wavelength detectors using the photodiodes
Uv-Visible spectrophotometer 148

149.
C. Determination of Equilibrium Constants
Ø Acid dissociation constants and metal ion-ligand stability
constants can be determined spectrophotometrically if the
species involved have absorptivities which differ from one
another.
Example : Determination of the pKa of Methyl red indicator ;
Acidic (HMR) and basic (MR-) forms of methyl red are
shown below
Uv-Visible spectrophotometer 149
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

150.
C. Determination of Equilibrium Constants…
Ø The pKa of methyl red indicator is given by the equation:
Ø Both HMR and MR- have strong absorption peaks in the visible
portion of the spectrum.
Uv-Visible spectrophotometer 150
A
430 nm 520 nm pH
l 5.0
Measured at
520 nm
Measured at
430 nm

151.
Ø The color change interval from pH 4 to pH 6 can be
obtained with acetate buffer system.
Ø At pH = 4, the acid is completely unionized and at pH =
6, the acid is completely ionized
Ø At intermediate pH values, the two species are present.
Ø Plotting absorbance (A) against pH values at l1 and l2
gives two curves.
Ø The pH at the point of intersection represents the pKa of
the indicator.
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152.
D. Determination of complex stoichiometry
Ø The stoichiometry for a metal–ligand complexation
reaction has the following general form.
Ø Can be determined by one of three methods:
– the method of continuous variations
– the mole-ratio method and
– the slope-ratio method.
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153.
D. Determination of complex stoichiometry…
i. Method of continuous variations (CVM)
Ø Also called Job’s method and is the most popular.
Ø In this method a series of solutions is prepared such
that the total moles of metal and ligand, ntot, in each
solution is the same.
Ø Thus, if (nM)iand (nL)i are, respectively, the moles of
metal and ligand in the i-th solution, then
Uv-Visible spectrophotometer 153

154.
i. Method of continuous variations…
Ø The relative amount of ligand and metal in each solution is
expressed as the mole fraction of ligand, (XL)i, and the mole
fraction of metal, (XM)i,
Ø Absorbance versus the mole fraction of ligand will be plotted.
CVM
Uv-Visible spectrophotometer 154
A
L/M ratio
0.0 1.0
A
[L]/[L]+[M]

155.
i. Method of continuous variations…
Ø The intersection of the two lines drawn from both
sides occurs when stoichiometric mixing of metal and
ligand is reached.
Ø Mole fraction of ligand at this intersection is used to
determine the value of y for the metal–ligand complex,
MLy.
Uv-Visible spectrophotometer 155

156.
ii. Mole-ratio method
Ø In the mole-ratio method the moles of one reactant,
usually the metal, are held constant, while the moles of
the other reactant are varied.
Ø The absorbance is monitored at a wavelength at which the
metal–ligand complex absorbs.
Ø A plot of absorbance as a function of the ligand-to-metal
mole ratio (nL/nM) has two linear branches that intersect at
a mole ratio corresponding to the formula of the complex.
Uv-Visible spectrophotometer 156

157.
iii. slope-ratio method
Ø In the slope-ratio method two sets of solutions are
prepared.
Ø The first set consists of a constant amount of metal and a
variable amount of ligand, chosen such that the total
concentration of metal, CM, is much greater than the total
concentration of ligand, CL.
Ø Under these conditions we may assume that essentially all
the ligand is complexed. The concentration of a metal–ligand
complex of the general form MxLy is
Uv-Visible spectrophotometer 157

158.
iii. slope-ratio method …
Ø If absorbance is monitored at a wavelength where only MxLy
absorbs, then
and a plot of absorbance versus CL will be linear with a slope, sL,
of
Ø A second set of solutions is prepared with a fixed concentration
of ligand that is much greater than the variable concentration of
metal; thus
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159.
iii. slope-ratio method …
Ø The mole ratio of ligand-to-metal is determined from the ratio
of the two slopes.
Uv-Visible spectrophotometer 159

160.
E. Spectrophotometeric titrations
Ø One or more of the reactants or products absorb radiation.
Ø They are carried out in a vessel for which the light path is
constant.
Ø The absorbance is directly proportional to concentration.
Titration Curves
• Plot of absorbance as a function of titrant volume and
consists of two straight-line regions with different slopes
Uv-Visible spectrophotometer 160

161.
E. Spectrophotometric titrations…
Advantages
§ More accurate results than direct titrimetric analysis
are obtained.
§ Can be used for the titration of very dilute solutions
(Sensitive)
§ Do not need favorable equilibrium constants as
those required for titration that depends upon
observations near the end point.
§ Can be used for all types of reactions (Redox, acid-
base, complexometric , pptmetry…etc).
Uv-Visible spectrophotometer 161

162.
Colorimetry
Ø Is a technique which involves measurement of absorbance in
the visible region is known as colorimetry.
Ø Involves measurement of color intensity of compounds.
Requirements for colorimetry
ü the substance should be colored or
ü The substance should be able to be derivatized in to colored
product.
ü While derivatizing
§ The reagent should be specific
§ The color produced should be stable enough until the analysis is
completed
§ Color intensity should be directly proportional to the concentration of
the analyte.
Application- colored drugs and those drugs which can be
derivatized.
Uv-Visible spectrophotometer 162

163.
THE END
Uv-Visible spectrophotometer 163