101T MPAT- Modern Pharmaceutical Analytical Techniques

UNIT-I – 10 hrs

UV Visible Spectroscopy
IR Spectroscopy

Spectrofluorimetry
Flame Photometry and AAS

UNIT-II 10 hrs

NMR Spectroscopy

UNIT-II 12 hrs

Mass Spectroscopy

101T MPAT

UNIT-IV – 10 hrs

High Performance Liquid chromatography
High Performance Thin Layer Chromatography

Ion exchange chromatography
Gas chromatography

Ultra High Performance Liquid chromatography
Affinity chromatography

Gel Chromatography

UNIT-V 10 hrs

Electrophoresis and X Ray

UNIT-Vi 08 hrs

Thermal Methods

Molecular Spectroscopy
Ultraviolet and Visible Spectroscopy

－UV/VIS Spectroscopy

－UV/VIS Spectrometer

－Application for Quantitative Analysis

ANALYTICAL DISCIPLINES

• Spectroscopic methods

• Chromatographic methods

• Electrochemical methods

• Thermometric methods

• Radiochemical methods

SPECTROSCOPY
Spectroscopy involves the measurement and

interpretation of electromagnetic radiations
absorbed/emitted when molecules/atoms/ions of a
sample move from one energy state to another.

 It may be ground state to excited state or excited
state to ground state.

 In ground state’ energy of a molecule is the sum of
rotational, vibrational and electronic energies.

 Spectroscopy measures the changes in rotational,
vibrational and/or electronic energies.

INTERNAL ENERGY OF MOLECULES

Etotal=Etrans+Eelec+Evib+Erot+Enucl

Eelec: electronic transitions (UV, X-ray)

Evib: vibrational transitions (Infrared)

Erot: rotational transitions (Microwave)

Enucl: nucleus spin (Nuclear Magnetic

Resonance or NMR)

ELECTROMAGNETIC RADIATIONS

• Form of energy transmitted through space at an
enormous velocity.

• Made up of discrete particles called photons.

• Has dual nature, exhibiting both wave and particle
properties.

• EMR is an alternating electrical and associated
magnetic force field in space.

CHARACTERSTICS OF EMR

• Wave length (  )
• Frequency ( )
• Wave number ( υ )
• Relationship between frequency, velocity and

wave number is
•  = c/λ

• EMR consists of a discrete packets or particles of
pure energy, called photons.

• E = h 

Relationship Between Wavelength and Energy of
EMR

• E = h 

• E = h c/λ = h c υ

• Energy of the radiation is directly
proportional to frequency and inversely
proportional to wave length of
electromagnetic radiation

TYPES OF SPECTROSCOPY
 Atomic spectroscopy :

Atomic absorption spectroscopy, Flame photometry

 Molecular spectroscopy
UV spectroscopy, IR spectroscopy, Fluorimetry

Whether the study is based on absorption or emission of EMR

 Absorption spectroscopy
UV spectroscopy, colorimetry, IR spectroscopy, NMR, AAS

 Emission spectroscopy
Flame photometry, Fluorimetry

Whether the study is at electronic or magnetic levels

 Electronic spectroscopy : UV, Visible, Flourimetry
 Magnetic spectroscopy : NMR, ESR

ELECTROMAGNETIC SPECTRUM

UV-Visible region
• Vacuum UV : 1-180 nm  Violet: 400 – 420 nm
• UV : 180-400 nm
• Visible : 400-750 nm  Indigo: 420 – 440 nm

 Blue: 440 – 490 nm

 Green: 490 – 570 nm

 Yellow: 570 – 585 nm

 Orange: 585 – 620 nm

 Red: 620 – 780 nm

UV- VISIBLE SPECTROSCOPY

 UV-visible spectroscopy is based upon the absorption of energy in the
UV-Visible region (190-800 nm)

 Causes changes in the energy of valence electrons accompanied by
rotational-vibrational changes

 Leads to transitions of electrons in the electronic levels of a molecule

 When △E = hν, a molecule can only absorb the particular wavelength
and undergo transition form GS to ES

 Molecular absorption in the UV and visible region of spectrum is
dependent on electronic structure of a molecule

 Limited to conjugated systems

FUNDAMENTAL LAWS OF SPECTROPHOTOMETRY
 When a light is incident upon a homogenous medium, part of incident

light is reflected, absorbed, and a part is transmitted.
 I0 = Ia + It +Ir
 Io = Ia + It as Ir is negligible

 Beer’s Law
The intensity of a beam of monochromatic light decreases exponentially
with the increase in the concentration of absorbing species
arithmetically.
Io / It = ekc

It/ Io = e-kc

It = Io e-kc

 Lambert’s Law
When a beam of light is allowed to pass through a transparent medium,
the rate of decrease of intensity with the thickness of medium is directly
proportional to the intensity of the light.

 It = Io e-kt

Beer Lambert’s law
• It = Io e-kct

• Io / It = e kct

• Log Io/It = kct

• Transmittance (T) = It/Io
• Absorbance (A) = log 1/T
• A = log10 (1/T) = log10 (Io/I).
• Absorbance ( A ) = Kct or εct
• A=abc

A = absorbance /optical density
a = extinction coefficient/ absorptivity
c = concentration of the drug (g/L)
b = path length ( normally 1 cm )

• A= ε bc where ε = molar extinction coefficient
• A=A 1%1cm bc where A1% = absorptivity of a solution of concentration

1g/100ml

Path length
0 0.2 0.4 0.6 0.8 1.0

/ cm

%T 100 50 25 12.5 6.25 3.125

Absorbance 0 0.3 0.6 0.9 1.2 1.5

Type of electrons present in any molecule
• σ electrons

Present in the saturated compounds.
Do not absorb near UV, but absorb vacuum UV radiation.

• π electrons
Present in unsaturated compounds.
E.g. double and triple bonds.

• ‘n’ electrons
Non bonded electrons, not involved in any bonding.
Eg. lone pairs of electrons in S, O, N etc.

• Any molecule has either n, π, σ or the combination of these electrons.

• The bonding and non bonding electrons absorb the characteristic radiation
and undergo transition from ground state to excited state.

• By characteristic absorption peaks, the nature of electron present and hence
molecular structure can be elucidated.

Chromophores
A molecule or part of a molecule that can be excited by absorption or is
capable of absorbing UV Visible radiation is called a chromophore.

E.g. OH and NH2 groups

Chlorophyll a chromophore

It absorbs blue and red light and
therefore appears green from
reflected light.

Electron
Chromoph

Type Example Excitation λ
ore max, nm ε Solvent

C=C Ethene π __> π* 171 15,000 hexane

Sigma (σ) Bond
C≡C 1-Hexyne π __> π* 180 10,000 hexane

n __> π* 290 15 hexane
C=O Ethanal

π __> π* 180 10,000 hexane

Pi (π) Bond

Nitrometh n __> π* 275 17 ethanol
N=O

ane π __> π* 200 5,000 ethanol

σ > π >
Nonbonding(n) electron

n

Vacuum UV or Far UV
Electronic
Transitions (λ<190 nm )

UV/VIS

UV/Vis Spectrum – Acetone (carbonyl chromophore)
Band Band 2
1

max = the wavelength corresponding to
the maximum of the absorption band

max for acetone = 187 nm and 270 nm

Acetone has  and nonbonded electrons. Therefore there are 2 possible
transitions


antibonding
(an*ti)bonding E E

(n*) to *  to *
non-bonding

 bonding

Which band is which? – The lower the energy, the longer the


UV/Vis Spectrum – Acetone (carbonyl
chromophore)

 to * n to *

187 nm is 270 nm is n to π*

π to π*

Why are the two max absorption
peaks of different heights?

Important distinguishing
characteristic of n to π* transitions

The lone-pair (n) electrons are
concentrated in a different region of
space from the π electrons.

This makes the
n to π* transition less probable than
the π to π* .

Effects of conjugation
1. Conjugation decreases the energy gap between HOMO and

LUMO.

2. Hence less energy is required for electronic transitions.

3. Transitions occur at longer wavelengths.

4. If a compound has enough double bonds it will absorb visible
light

5. Compound will be colored, e.g. -carotene which is orange
and is found in carrots and tomatoes has max = 455nm

-carotene absorbs most strongly
between 400- 500 nm (green/blue
part of the spectrum) So appears
orange, because the red/yellow

Effect of conjugation on wavelength

Acetone Methyl vinyl

 max = 187 nm and ketone

270 nm max = 219 nm and

•The π system of methyl vinyl k3e2to4nnem is more extended
than that of acetone.

•More extensive π-system of conjugated double bonds
in methyl vinyl ketone leads to both the n to π* and
π to π* transitions of methyl vinyl ketone occuring at
longer wavelengths. LUM

*
 O

* 
E E

 HO
MO

Requirement For a Molecule to Absorb in UV Visible
Region

• Chromophore
Chromophore is the species or system responsible for imparting color
to a compound.
Chromophoric group is responsible for characteristic absorption at a
wavelength, whether a ‘color’ is produced or not .

• Chromophores are covalently unsaturated compound with /without
lone pairs of electrons.

Chromophore Excitation max, nm Solvent

C=C →* 171 hexane

n→* 290 hexane
C=O

→* 180 hexane

n→* 275 ethanol
N=O

→* 200 ethanol

C-X n→* 205 hexane
X=Br, I n→* 255 hexane

Auxochrome
 Is a saturated group with

non bonded electrons

which themselves do

not absorb UV radiation.

 However when attached to a

chromophore, they change

both the wavelength and

intensity of absorption.

 Eg. OH,NH2, Cl

Electronic transitions
 The absorption of UV or Visible radiation by an atomic or molecular species

M can be considered as two step process.
M + hν ——— M+.
M+ ———- M + Heat

 Covalent bonding occurs between two atomic centers in such a manner so as
to minimize repulsive columbic forces between them

 The non localized fields between atoms that are occupied by bonding
electrons are called molecular orbitals

 It can be considered as the result from overlap of atomic orbitals
 When two atomic orbitals combine, either

A lower energy bonding molecular orbital ( LBMO)
High energy antibonding molecular orbitals (HABMO)

 The electrons of the molecule occupy the former in the ground state.

• The σ, π, and n electrons can be excited from
ground state by the absorption of UV radiation.

• The different transitions are:

• n -π*

• π -π *

• n – σ*

• σ -σ*

• The energy required for excitation of
different transitions are
n —— π*< π —— π *< n —— σ*<σ ——σ*

• Polar solvents shift n —— π* and n ——
σ* to shorter wave length and π —— π *
to longer wave length.

n – π* transition

• Requires lowest energy (longer wavelength).
• Peaks due to these transition is also called R bands.
• Occur in molecules containing n electrons and a double or

triple bond.
• Eg. Aldehydes, ketones,

nitro compounds

π —— π *

• Promotion of an electron from a bonding π orbital to the π*
orbital

• Gives rise to B, E and K bands
• B (benzenoid) bands due to aromatic and heteroaromatic

systems
• E (ethylenic) bands due to C=C systems
• K bands due to conjugated systems.

n —— σ*

• This transition occurs in saturated compounds, with hetero
atoms like S,O, N, or halogens.

• These compounds also undergo σ -σ* in addition to n – σ*
transitions.

• Normally peaks due to this transition occurs from 180 nm
to 220 nm.

• Since peaks are observed at lower end of spectrum it can
be called as the end spectrum.

σ ——σ*
• Occur in compounds in which all the electrons are involved

as single bonds and there is no lone pair of electrons.
• Examples are saturated hydrocarbons.
• Since energy requirement for transition is very large, the

absorption bands occur in far UV region (126-135nm).
• Saturated hydrocarbons like cyclohexane can be used as

solvents, as it does not give solvent peak.

Vacuum UV or Far UV
(λ<190 nm )

UV/VIS

Auxochromes
Compared to straight-chain conjugated polyenes, aromatic compounds have
relatively complex absorption spectra with several bands in the ultraviolet
region.
Benzene and the alkylbenzenes show two bands in which we shall be
primarily interested, one near 200nm and the other near 260nm.
The 200-nmnm band is of fairly high intensity and corresponds to excitation
of a π electron of the conjugated system to a π∗π∗ orbital (i.e.,
a π→π∗ transition).

The benzene chromophore itself gives rise to a second band at
longer wavelengths.
This band,, is of relatively low intensity and is found under
high resolution to be a composite of several narrow peaks.
It appears to be characteristic of aromatic hydrocarbons
because no analogous band is found in the spectra of
conjugated acyclic polyenes.
For this reason it often is called the benzenoid band.
The position and intensity of this band, like the one at shorter
wavelengths, is affected by the nature of the ring substituents,
particularly by those that extend the conjugated system,

Ultraviolet absorption spectrum of
benzene (in cyclohexane) showing the

“benzenoid” band.

The benzenoid band corresponds
to a low-energy π→π∗ transition
of the benzene molecules.
The absorption intensity is weak
because the π∗π∗ state involved
has the same electronic
symmetry as the ground state of
benzene, and transitions
between symmetrical states
usually are forbidden.
The transitions are observed in
this case only because the
vibrations of the ring cause it to
be slightly distorted at given
instants.

The benzenoid band corresponds to a low-
energy π→π∗π→π∗ transition of the benzene
molecules.
The absorption intensity is weak because
the π∗π∗ state involved has the same electronic
symmetry as the ground state of benzene,
These transitions between symmetrical states
usually are forbidden.

The transitions are observed in this case only
because the vibrations of the ring cause it to be
slightly distorted at given instants.

Selection of solvent
• Drugs should show solubility in the solvent used.

• Drugs should be stable in the selected solvent.

• Drugs should obey Beer-Lambert’s law over an appropriate
range of analytical concentrations.

• The solvent should be to the extent possible economic.

Shifts in UV spectra

• Bathochromic shift
When a molecule dissolves
in a solvent, the energy levels
are decreased. This leads to
shift of absorption max. to
a longer λ. This shift to a
longer λ is called red shift.
Increase in conjugation, addition of alkyl substituents etc. leads to red
shift.

• Blue shift (Hypsochromic shift)

The shift of λmax towards shorter wavelength due to removal of double
or triple bonds by saturation and dealkylation.

• Hyperchromic effect :Increase in intensity of absorption

• Hypochromic effect :Decrease in intensity of absorption.

Effect on conjugation on λmax

Basic components of a UV-Vis spectropotometer

Double Beam UV -Vis spectrophotometer

Photo Diode array instrument

UV-Vis Detectors – Design Principles
UV Lamp Cut-off filter

Variable Wavelength
Holmium oxide

Detector filter

• Single wavelength detection or
multi wavelength detection Slit

possible.
Sample
diode

• Wavelength calibration is done Mirror 1

automatically using a holmium
filter.

Grating

Advantages:
-It is non-destructive, simple Flow cell

,robust
–Wide linear dynamic range

Mirror 2

Reference diode
47

UV-Vis Detector with Spectral
Vis Capability
Lamp

Achromatic
Lens

Diode Array
Detector
Flow Cell

UV
Lamp

Homium
Filter

Grating
Optical
Slit

• Photo Diode Array UV-Vis Detector (PDA) allows online measurement of spectra.
• Wavelength range 190 – 950 nm.
• Wavelength Resolution: Up to 1 nm.
• Wavelength calibration with Holmium oxide filter.

48

Online Spectra – UV-Vis Detector

Absorbance
Spectra

Wavelength

Time

49

This figure explains the principle of the DAD:

The tungsten lamp emits light in the visible range and the
deuterium lamp, emits light in the UV range.
The polychromatic beam passes the flow cell.
The grating splits up the polychromatic beam to different
wavelengths, the intensities of which are measured by an array of
photodiodes.
The main difference to the VWD is that all available wavelengths
are measured simultaneously,
i.e. that spectra can be acquired and absorbance recorded at
multiple single wavelengths at the same time by different diodes in
the array. As substances can be identified by their spectra, the
DAD has a high selectivity.

Additional advantages of the DAD are:
Tungsten lamp offers extended visible wavelength range.
The optical unit of the DAD is temperature controlled for
optimum signal quality.
The slit width can be changed automatically.
The DAD does not need a reference diode.

Here a detector balance is used, which can be done
automatically when switching on the detector or when starting a
measurement. During a detector balance, absorption values for
all wavelengths are set to zero,

All intensities measured during an experiment are now relative
to this zero absorption intensity.

APPLICATIONS OF UV- VISIBLE SPECTROPHOTOMETRY

Detection of conjugation

Detection of geometrical isomers

Detection of functional groups (chromophores)

Detection of impurities

Qualitative Analysis

Quantitative Analysis

Spectrophotometric Titration

Kinetic Assay

Derivative Spectrophotometry

QUALITATIVE ANALYSIS

UV absorption spectroscopy characterizes those molecules
which absorbs UV radiation

Identification is done by comparing the absorption spectrum with
the spectra of standards

QUANTITATIVE ANALYSIS
Determination is based on Beer Lambert’s law

A=log I0/It =abc

Use of calibration graph

Use of standard absorptivity value

Single or Double point standardization

Group K band () B band() R band

Benzenoid aromatics Alkyl 208(7800) 260(220) —

-OH 211(6200) 270(1450)

-O- 236(9400) 287(2600)

-OCH3 217(6400) 269(1500)
UV of

NH2 230(8600) 280(1400)
Benzene in -F 204(6200) 254(900)

heptane -Cl 210(7500) 257(170)

-Br 210(7500) 257(170)

-I 207(7000) 258/285(610/18
0)

-NH +
3 203(7500) 254(160)

-C=CH2 248(15000) 282(740)

-CCH 248(17000) 278(6500

-C6H6 250(14000)

-C(=O)H 242(14000) 280(1400) 328(55)

-C(=O)R 238(13000) 276(800) 320(40)

-CO2H 226(9800) 272(850)

-CO2- 224(8700) 268(800)

-CN 224(13000) 271(1000)

From Crewes, Rodriguez, Jaspars, Organic Structure Analysis -NO2 252(10000) 280(1000) 330(140
)

SINGLE COMPONENT ANALYSIS
USE OF CALIBRATION GRAPH

Use of calibration graph

Wavelength of maximum absorption is selected

Absorbance is measured for different concentration of the
standard solution and a calibration graph is plotted.

r2 of the calibration curve should be >0.999 to indicate linearity of
data

A

C

Use Of Standard Absorptivity Value

A1%1cm or E value avoids the need to prepare a standard
solution of the reference substance

A=A 1%1cm bc

c = A / A1%1cm b

Single -Point Standardization

Involves the measurement of the absorbance of a sample
solution and a standard solution

Ctest= Atest x Ctest

Astd

Double-point Standardization

Absorbance of the two standard solutions and sample are
measured

Ctest =[Atest-Astd1][Cstd1-cstd2]+Cstd1[Astd1-Astd2]

Astd1 – -Astd2

Multicomponent analysis

Points to be considered before devising new multicomponent methods are:

 Literature Survey:
Existing analytical methods for multicomponent formulations to be analysed
are scanned to avoid duplication of the method. Further the information about
the solubility; absorption maximas and the molar absorptivities in various
solvents of the individual components of the multicomponent formulation are
obtained.

 Selection of a Solvent:
A solvent or solvent mixture in which all the active components are soluble and
stable is chosen.

 Selecting the Sampling Wavelengths:
Sampling wavelengths are selected considering the peaks and valleys in the UV
spectra of the individual components and the other wavelengths where the
various components show the difference in absorbance.

 Selecting the number of mixed standards and concentration of each
component in mixed standards:
The concentration of each component in the mixed standard is determined from
molar absorptivity values of the component and the ratio in which the different
components are present in the formulation to be analyzed.
The number of mixed standards that should be used is determined by carrying out
the analysis using varying number of mixed standards. The numbers of mixed
standard is then selected keeping into view the accuracy and reproducibility of the
result.

 Sampling analysis and calculation:
The analysis is repeated and accuracy, reproducibility is confirmed.

 Type of Instrument:
It is the heart of the analytical method because more advanced the instrument,
greater will be the accuracy of results and confidence with which the results are
reported.

 Evaluation of reproducibility:
To ensure that proper conditions have been selected and that no important variables
have been overlooked, the tentative method should be critically evaluated with
respect to Beer’s law.

 SIMULTANEOUS EQUATION METHOD

Absorbances of the sample mixture at λ1 and λ2

may be expressed by the formation of two
Simultaneous equations –

 A1 = aX1bcx + aY1bcY at λ1 ———————— (1)

 A2 = aX2bcx + aY2bcY at λ2 ——————— — (2)

 Rearranging equation (1) & (2) gives,

A2 aY1 – A1 aY2

 cx = ……………………………(3)
aX2 aY1 – aX1 aY2

A1 aX2 – A2 aX1

 cY = ……………………………(4)
aX2 aY1 – aX1 aY2

 Using equations (3) and (4), the concentrations of X and Y in the sample mixture
can be determined.





FIRST ORDER DERIVATIVE METHOD
• The absorbance (A) of a sample is differentiated with respect to wavelength λ to generate

first, second or higher order derivatives spectras.

• [A] = f(λ) : Zero order,
• *dA/dλ + : f’(λ) : First order,
• [d2A / dλ2 ] : f’’(λ) : Second order

Photometric Titrations

 Used where an analyte reacts with a reagent so that the
analyte, the reagent or the product absorbs UV-Vis radiation

 Technique used is photometric titration
 A plot of absorbance versus titrant volume is called a

photometric titration curve.
 The titration curve is supposed to consist of two linear lines

intersecting in a point corresponding to the end point of the
reaction.

 Photometric titrations are more accurate than visual titrations.

 Photometric titrations are faster than visual titrations

64

Analyte + Reagent = Product

65

Woodward Feiser Rules:
 From the study of UV absorption spectra of a larger number
of compounds, Woodward gave certain rules for correlating
λmax with molecular structure.

These rules can be used to calculate the λmax for a given
structure by considering various parent structures and degree
of substitutions

Attached group
increment, nm

Extend conjugation +30

Addn exocyclic DB +5

Alkyl +5

Homoannular diene Heteroannular diene
O-Acyl 0

S-alkyl +30

O-alkyl +6

NR2 +60

Cl, Br +5

Endocyclic bouble bond
Exocyclic duuble bond

λmax for Cholesta-3,5,Diene:

Hetero annular diene = 214nm

3ring residues = 15nm

1 exocyclic double bond = 5nm

Predicted λmax = 234nm

Observed λmax =235nm

λmax for Cholesta-2,4-Diene:
Homoannular diene = 253nm

3 ring residues = 15nm

Predicted λmax = 268nm

Observed λmax =275nm

Distinguish Isomers!

Base value 214
4 x alkyl subst. 20
exo DB 5
total 239
Obs. 238

HO C
2

Base value 253
4 x alkyl subst. 20
total 273
Obs. 273

HO C
2

Conclusions

UV-Vis spectroscopy remains one of the most widely used
techniques for both qualitative and quantitative analysis

Applicable to wide range of compounds

Simplicity of analysis

Routine technique used in research and industry