INTRODUCTION

TO
INSTRUMENTAL METHODS
OF ANALYSIS

Habtamu Abuye (B Pharm., MSc.), Department of

Pharmacy, College of Medicine and Health
Sciences, Wachemo University
OUTLINE
 Introduction to instrumental methods of analysis
 Classification of analytical methods
 LEARNING OBJECTIVES

 To introduce to the different methods used for drug analysis.

 To differentiate instrumental methods of analysis from classical methods.

 To understand the principles behind different instrumental methods of analysis.

 INTRODUCTION

 The need of the sophisticated analytical instruments and

determinations using them is almost a routine process for the modern
chemical laboratories.
 It has been a vast expanding area of knowledge as—
 the instrument and computer manufacturers are producing
analytical machines,
 which are in ever-increase of power and scope.
 All the manual techniques in the line of the analytical studies had
steadily been transferred to the instrumental techniques.
 INTRODUCTION….
 Basically, chemical analysis can be divided into three broad categories as are almost
invariably applied to major areas such as
o Fundamental Research,
o Product Development,
o Product Quality Control,
o Monitoring & Control of Pollutants,
o Medical & Clinical Studies, etc:
1. Qualitative analysis—
 Chemical analysis which just identifies one or more species present in a sample.
2. Quantitative analysis—
 Chemical analysis which finds out the total amount of the particular species
present in a sample.
3. Structural analysis—
 Chemical analysis which helps in finding the spatial arrangement of atoms in a
molecule and the presence or position of certain organic functional groups in a
given compound
 INTRODUCTION….
 Chemical analysis has some basic steps like—
o choice of method,
o sampling,
o preliminary sample treatment,
o separations,
o final measurement and
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 CLASSIFICATION
Analytical Techniques

Instrumental Analysis Classical Analysis

Qualitative Analysis Quantitative Analysis Separation Analysis

Chemical Reaction Titration Distillation

Physical Character Gravimetric Precipitation

7
 CLASSIFICATION…
Analytical Techniques

Classical Analysis Instrumental Analysis

Spectroscopy Analysis Quantitative Analysis Separation Analysis

UV/VIS Spec
Electrochemistry Chromatography
IR Spec
Mass Spec o Qualitative—Amount present in sample/mixture

NMR Spec o Qualitative— Identity species present in the sample

o Structural—Determination of structure of drug
AA/AE Spec molecule
o Separation and/ or Purification, Identification,
 UV- VISIBLE
SPECTROPHOTOMETRY
 OUTLINE
Introduction
oThe concept of Chromophore and Auxochrome
o Absorption intensity shifts
o Effect of pH on absorption
o Conjugated dienes and Wood Ward and Fiesher’s
rule
Instrumentation
o Radiation sources
o Monochromators
o Sampling cells and compartments
o Detectors
o Recording systems
o Double and single beam instruments
Qualitative spectrophotometry
Quantitative spectrophotometry
o Beer’s law and its limitations
Applications
Colorimetry
 INTRODUCTION
Spectroscopy—
 the study of physical systems by the electromagnetic radiation with which they
interact or that they produce.
 is the study of (measures) the interaction between matter (molecules) and
radiated energy (electro magnetic waves).
 science of studying the interactions of matter and radiation
 To understand spectroscopy, one must first understand the electromagnetic radiation and spectrum
that stretch from Radio waves to Cosmic rays . The energy of these waves is dependent on the
wavelength or the frequency of the wave
 INTRODUCTION…
Electromagnetic Radiation/spectrum
 is a form of energy that propagates as both electrical and magnetic waves traveling in
packets of energy called photons.
 There is a spectrum of EMR with variable wavelengths and frequency, which in turn
imparts different characteristics.
 INTRODUCTION…
EMR Spectrum…

Spin Orientation Molecular Vibration Nuclear Disruption

Molecular Rotation
Transition of outer most
Valence Electrons

NMR IR Spectroscopy UV/VIS AA Spectroscopy

Spectroscopy Spectroscopy
 INTRODUCTION…
EMR Spectrum…

 Spectra originate when atoms, ions, and molecules emit or absorb energy.
 Atoms, ions, and molecules emit or absorb characteristically; only certain energies of
these species are possible;
 the energy of the photon (quantum of radiant energy) emitted or absorbed
corresponds to the difference between two permitted values of the energy of the
species, or energy levels.
 Thus the energy levels may be studied by observing the differences between them.
 The absorption of radiant energy is accompanied by the promotion of the species
from a lower to a higher energy level;
 The emission of radiant energy is accompanied by falling from a higher to a lower
state; and
 If both processes occur together, the condition is called resonance.
 INTRODUCTION…
Spectrum—
 is, a plot of the intensity of emitted or transmitted radiant energy (or some
function of the intensity) versus the energy of that light
 Interpretation of spectra provides
o fundamental information on atomic and molecular energy levels,
o the distribution of species within those levels,
o the nature of processes involving change from one level to another,
o molecular geometries,
o chemical bonding, and
o interaction of molecules in solution.
 At the practical level, comparisons of spectra provide
o a basis for the determination of qualitative chemical composition
and chemical structure, and
o for quantitative chemical analysis.
 INTRODUCTION…
 When light is absorbed by a sample, the irradiance of the beam of light is decreased.
Irradiance, I, is the energy per second per unit area of the light beam.
 Light is passed through a monochromator (a prism, a grating, or even a filter) to select one
wavelength.
 Light with a very narrow range of wavelength is said to be monochromatic (one color)
 The monochromatic light, with irradiance Io, strikes a sample of length b.
 The irradiance of the beam emerging from the other side of the sample is I. Some of the
light may be absorbed by the sample, so I ≤ Io.
b, length of path through sample
Light Wavelength Sample Light
Source Selector Io I Detector
(Monochromator)

Transmittance, T, is defined as the fraction of the original light that passes through the sample.
irradiance of beam emerging from sample;
irradiance of beam entering sample;
 INTRODUCTION…
 Therefore, T has the range 0 to 1.
 The percent transmittance is simply 100T and ranges between 0 and 100%.
 Absorbance is defined as:

Relation between transmittance, T and absorbance, A:

I/Io T% A
1 100 0
0.1 10 1
0.01 0 2
 Absorbance is so important because it is directly proportional to the concentration, c,
of the light-absorbing species in the sample
 INTRODUCTION…
UV/VIS Spectrophotometry - is technique that uses UV/VIS light to measure analyte
concentrations since Ultraviolet (UV) and visible radiation comprise only a small part of the
electromagnetic spectrum.
 The basic principle is that each compound absorbs or transmits light over a
certain range of wavelength.
 A procedure based on absorption of visible light is called colorimetry
 INTRODUCTION…
 The absorption of EMR of wavelengths between 200 and 800 nm by molecules can
be employed for both qualitative and quantitative analysis;.
 As a wide variety of pharmaceutical substances absorb radiation in the
 near-ultraviolet (200–380 nm) and
 visible (380–800 nm) regions of the electromagnetic spectrum,
 the technique is widely employed in pharmaceutical analysis.
1. Quantitative:
 The relationship between :
 the concentration of analyte and
 the intensity of light absorbed
 is the basis of quantitative applications of spectrophotometry.

2. Qualitative:
 Features of absorption spectra such as the
 molar absorptivity,
 spectral position, and
 shape and breadth of the absorption band are
 related to molecular structure and environment
 can be used for qualitative analysis.
 INTRODUCTION…
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 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;

20
 INTRODUCTION…
 The basic principle is that each compound absorbs or transmits light over a certain
range of wavelength.
 Electronic absorption spectra originate with the excitation of the electrons which
form the bonds holding the molecule together.
 The electronically excited states of organic molecules which absorb in the near-
ultraviolet and visible regions are created by the promotion of—
1. π-electrons to π* and
2. n-electrons to π*- or σ*-orbitals.

21
 CHROMOPHORE-color bearing
 The energy of radiation being absorbed during excitation of electrons from ground state to excited
state primarily depends on the nuclei that hold the electrons together in a bond.
Chromophore—
 is the group of atoms containing electrons responsible for the absorption and
exhibits a characteristic absorption spectrum in the ultraviolet or visible region.
 Most of the simple un-conjugated chromophores give rise to high energy transitions of little use

Ketones, Imine

Carbonyl Acetamide Ester

 AUXOCHROMES
Auxochromes—
 are the substituents that themselves do not absorb UV radiations but their presence
shifts the absorption maximum to longer wavelength.
 eg. methyl, hydroxyl, alkoxy, halogen, amino group etc.
 If any of the simple chromophores is conjugated with another (of the same type or different type)
 a multiple chromophore is formed having a new absorption band which is—
1. more intense and
2. at a longer wavelength that the strong bands of the simple chromophores.

Shifting of 𝜆max
EFFECTS of Auxochromes and Conjugated Chromophors
1. Bathochromic Shift or Red shift:
 A shift of an absorption maximum towards longer wavelength or lower energy.
2. Hypsochromic Shift or Blue Shift:
 A shift of an absorption maximum towards shorter wavelength or higher energy.
3. Hypochromic Effect:
 An effect that results in decreased absorption intensity.
4. Hyperchromic Effect:
 An effect that results in increased absorption intensity.

Hypsochromic Shift Bathochromic Shift

 EFFECTS of Auxochromes and Conjugated
Chromophors…
• The actual effect of an auxochrome on a chromophore depends on—
 the polarity of the auxochrome,
1. e.g. groups like CH3-, CH3CH2 - and Cl- have very little effect, usually a small red

shift of 5-10 nm.

2. groups such as -NH2 and -NO2 are very popular and completely alter the spectra

of chromophores such as: benzene.

In general it should be possible to predict the effect of non-polar or weakly polar auxochromes, but the effect
of strongly polar auxochromes is difficult to predict. In addition, the availability of non-bonding electrons
which may enter into transitions also contributes greatly to the effect of an auxochrome
 ABSORPTION INTENSITY

 Molar absoptivities may be very large for strongly absorbing chromophores (>10,000) and
very small if absorption is weak (10 to 100).
 The magnitude of ε reflects both
 the size of the chromophore and
 the probability that light of a given wavelength will be absorbed when it
strikes the chromophore.
 A general equation stating this relationship may be written as follows:

ε = 0.87 x 1020 I x a

 Where I is the transition probability (0 to 1) & a is the chromophore area in cm2 )

 The factors that influence transition probabilities are complex, and are treated by what
spectroscopists.
 EFFECTS of pH on ABSORPTION
 The pH of the sample solution can have a significant impact on absorption spectra.
 The mechanism for this is primarily a shift in the equilibrium between the different
chemical forms of an analyte.
 If pH is known to be a factor,
 a remedy is to prepare the sample in a suitable buffer solution so as to maintain the
pH at a steady value
pH 13 Example,
4000
pH 6  Absorption spectrum of tyrosine
4000
at pH 6 and 13

A Λmax. At pH 6= 274nm
Λmax. At pH 13= 295nm

2000
2000

250
250 270
270 290
290 310
310
Wavelength(nm).
 EFFECTS of pH on ABSORPTION…

 The buffer though needs to be transparent over the

wavelength range of the measurements – if the buffer
absorbs radiation—
 absorbance readings attributed to the analyte may be
higher than they should because—
 the buffer and analyte absorptions will add
together at each wavelength.
 High pH solutions absorb at higher wavelengths.
 Whereas low pH solutions absorb at lower
wavelengths
 The pH of the solvent determines the ionization state of
ionizable chromophores.
 SOLVENTS EFFECTS

 Highly pure, non-polar solvents such as 20% ethylene glycol

saturated hydrocarbons—
 do not interact with solute molecules
either in the ground or excited state and
 the absorption spectrum of a compound
in these solvents is similar to the one in a
A
pure gaseous state.
 Polar solvents such as water, alcohols etc. may
stabilize or destabilize the molecular orbitals Solvents water

of a molecule either in the ground state or in

excited state and
 the spectrum of a compound in these
250
250 260
260 270
270 280
280 290
290
solvents may significantly vary from the Wavelength(nm).
Wavelength(nm).
one recorded in a hydrocarbon solvent. 2
9
2
 SOLVENTS EFFECTS…
π  π* Transitions

 In case of π  π* transitions—
 the excited states are more polar than the ground state and
 the dipole-dipole interactions with solvent molecules lower the energy of the
excited state more than that of the ground state.
 Therefore a polar solvent decreases the energy of π  π* transition and
 absorption maximum appears ~10-20 nm red shifted in going from hexane to
ethanol solvent.
 SOLVENTS EFFECTS…
n  π* Transitions
 In case of n π* transitions,
 the polar solvents form hydrogen bonds with the ground state of polar molecules
more readily than with their excited states.
 Therefore, in polar solvents the energies of electronic transitions are increased.
Example,
 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
 ABSORPTION LAW
Beer’s Law
 the intensity of a beam of parallel monochromatic radiation decreases exponentially
with the number of absorbing molecules.

 More simply, it stated that absorbance is proportional to the concentration (c) of the
solution.
 Beer demonstrated the following relationship between absorbance and concentration
of a solution:

 Absorbance is defined as the amount of light which is absorbed by the substance and
is calculated as the negative logarithm of transmittance:
 ABSORPTION LAW…
Lambert Law
 When a beam of light is allowed to pass through a transparent medium, the rate
of decrease of intensity with the thickness (b) of medium is directly proportional
to the intensity of light.
Mathematically:

 where k’ is the proportionality constant, therefore when we take the reciprocal of

the entire equation:

 integrating the above equation we get

 where I is the intensity transmitted at thickness b and C is the constant of

integration.
 ABSORPTION LAW…
Beer-Lambert Law

 Consider a beam of parallel monochromatic light with intensity I0

striking the sample at the surface as shown in the figure above, such
that it is perpendicular to the surface.
 After passing through the path length b of the sample, which
contains N molecules/cm3, the intensity of the light reduces to IT.

A = εbc
 Specific absorbance is—
 the absorbance of a substance in solution of specific concentration in a cell of
specific path length. The most commonly used term for specific absorbance is
A1%1cm, which is the absorbance of a 1 g/100ml (1%) solution in a 1cm cell at a
particular wavelength of light. The beer-lambert equation in such case becomes:
A = A1%1cmbc
 ABSORPTION LAW…
Limitations and Deviations of Beer-Lambert Law
 Beer-Lambert’s law proves a direct correlation between the absorbance (A) of a
molecule to the concentration (c) and the path length (b) of the sample.
This relationship is a linear for the most part.
 However, under certain circumstances the Beer Lambert relationship breaks down
and gives a non-linear relationship.
 These deviations from the Beer Lambert law can be classified into three categories:
1. Real Deviations –
 are fundamental deviations due to the limitations of the law itself.
2. Chemical Deviations–
 are deviations observed due to specific chemical species of the sample which is being
analyzed.
3. Instrument Deviations –
 are deviations which occur due to how the absorbance measurements are made.
 ABSORPTION LAW…

Q? Why absorption measurements are taken at wavelength of maximum

absorbance λmax?
 If the band of wavelength selected on the spectrometer is such that the
molar absorptivities of the analyte is essentially constant, deviations from
Beer-Lambert law are minimal.
 However, if a band is chosen such that the molar absorptivity of the
analyte at these wavelengths changes a lot, the absorbance of the analyte
will not follow Beer-Lambert law.
 It is observed that the deviations in absorbance over wavelengths is
minimal when the wavelength observed is at the λmax.
 Due to this reason absorption measurements are taken at wavelengths.
 ABSORPTION LAW…
Why absorption measurements are taken at wavelength of maximum absorbance λ max?

Figure : Shows the difference in deviations

Figure: shows the deviations in Beer-
in absorbance when values are obtained at
Lambert law due to observations made at
maximum wavelength of absorbance (band
wavelengths other than lambda max.
A) vs other wavelengths of absorbance
(band B).
 CONJUGATED DIENES and WOODWARD &
FIESER’S RULE
Conjugated Dienes—
 are compounds having double bonds joined by one a bond.
 are also called 1,3-dienes
 1,3-Butadiene (CH2=CH-CH=CH2) is the simplest conjugated
diene.
 Three stereoisomers are possible for1,3-dienes with alkyl
groups bonded to. each end carbon of the diene.
 CONJUGATED DIENES, TRIENS & POLYENES and WOODWARD & FIESER’S RULE..

Conjugated Dienes….

 The presence of conjugate double bond decreases the energy difference between
HOMO and LUMO of resulting diene.
 As a result, the radiations of longer wavelength are absorbed.
  increases the intensity of absorption.
 Therefore, the increase in size of the conjugated system gradually
 shifts the absorption maximum (λmax) to longer wavelength and
 also increases the absorption.
Example,
 Ethylene absorbs at 175 nm (ε = 1000) and the conjugation in butadiene gives a strong
absorption at longer wavelength at 230 nm and with higher intensity (ε = >1000).
 The presence of alkyl substituents on double bond also produces
 bathochromic shift and hyperchromic effect.
 These effects are additive in dienes and up to some extent in trienes.
 CONJUGATED DIENES, TRIENS & POLYENES and WOODWARD & FIESER’S RULE..

Conjugated Dienes…

 Woodward suggested empirical rules for predicting the absorption of—

1. open chain and
2. six-membered ring dienes
3. which have been later on extended to large number of dienes and trienes.
 Several sets of empirically derived rules which attempt to predict –
 the wavelength of the absorption maximum (λmax) in an UV-VIS
spectrum of a given compound.
 Inputs used in the calculation are—
 the type of chromophores present,
 the substituents on the chromophores, and
 shifts due to the solvent
CONJUGATED DIENES, TRIENS & POLYENES and WOODWARD & FIESER’S RULE..

Conjugated Dienes, ….
 CONJUGATED DIENES, TRIENS & POLYENES and WOODWARD & FIESER’S RULE..

Conjugated Dienes, …

Example,
 Here the absorption maxima for dienes 1 and 2 have been calculated according to Woodward rules.
The comparison of calculated λmax values with observed λmax values highlights the importance of
these rules.

 Parent value = 214 nm  Parent value = 253 nm

 Three ring residues (3 x 5) = 15
 Three substituents = 15 nm
 Exocyclic double bond = 5 nm
 Ring residue = 5
 Total = 234 nm  Total = 273
 Observed value = 235 nm
 Observed value = 275 nm
 CONJUGATED DIENES, TRIENS & POLYENES and WOODWARD & FIESER’S RULE..
Conjugated Dienes,...

 As the number of double bonds in conjugation increases, the

bathochromic (towards longer wavelength) shift in lowest energy
absorption maxima is observed.
 The increase in conjugation gradually shifts the maxima to visible
region (> 400 nm) and imparts colour to the sample.
EXAMPLE
 the λmax shift in Me(CH=CH)nMe with increasing number of
conjugated double bonds.

β – Carotene, responsible for red color in carrots is a typical example of polyene with 11
conjugated double bonds and exhibits λmax at 445 nm
INSTRUMENTATION: Internal Parts
 INSTRUMENTATION
 The instruments used in spectroscopy consist of several common components,
including
1. a source of energy that can be input to the sample,
2. a means for isolating a narrow range of wavelengths,
3. a detector for measuring the signal, and
4. a signal processor to display the signal in a form convenient for the analyst
 INSTRUMENTATION…
I. Sources of Energy:
 All forms of spectroscopy require a source of energy.
a l yt e
 In absorption and scattering spectroscopy this energy is t he an igher
 supplied by photons. m ote ble, h
ro ta
 Emission and luminescence spectroscopy use to p less s te
 oa sta
 thermal, radiant (photon), or t y
rg
 chemical energy ene

1. Sources of Electromagnetic Radiation:

• Must provide an output that is both intense and stable in the desired region of the
electromagnetic spectrum.
• Is classified as either continuum or line sources.
Continuum source - a source that emits • Line sources – a source that emits
radiation over a wide range of radiation at only select wavelength.
wavelengths.
 INSTRUMENTATION…
2. Sources of Thermal Energy
 The most common sources of thermal energy are flames and plasmas.
1. Flame sources use the combustion of a fuel and an oxidant such as
acetylene and air, to achieve temperatures of 2000–3400 K.
2. Plasmas, which are hot, ionized gases, provide temperatures of 6000–
10,000 K.
3. Chemical Sources of Energy
 Exothermic reactions also may serve as a source of energy.
 In chemiluminescence the analyte is raised to a higher-energy state by means of a
chemical reaction, emitting characteristic radiation when it returns to a lower-energy
state.
 When the chemical reaction results from a biological or enzymatic reaction, the
emission of radiation is called bioluminescence.
 E.g. light sticks and the flash of light
 INSTRUMENTATION…
II. Wavelength Selection:
 Wavelength selector passes a narrow band of radiation characterized by:
a. a nominal wavelength, The wavelength which a wavelength selector is set to pass

b. an effective bandwidth, and The width of the radiation at half the maximum throughput
c. a maximum throughput of radiation.
 Effective bandwidth - defined as the width of the band of radiation passing through a wavelength
selector measured at half the band’s height
 The ideal wavelength selector has a
 high throughput of radiation and
 a narrow effective bandwidth.
1. High throughput is desirable because more photons pass through the wavelength selector,
giving a stronger signal with less background noise.
2. Narrow effective bandwidth provides a higher resolution, with spectral features separated by
more than twice the effective bandwidth being
resolved.
 Resolution— the separation between two spectral features, such as absorption or
emission lines.
 INSTRUMENTATION…
 Generally these two features of a wavelength selector are in a
opposition.
 Conditions favoring a higher throughput of radiation usually
provide less resolution.
 Decreasing the effective bandwidth b

 improves resolution, but at the cost of a noisier

signal.
 For a qualitative analysis, c
 resolution is more important than the throughput of
radiation;
thus, smaller effective bandwidths are desirable.
d
 In a quantitative analysis
 a higher throughput of radiation is usually desirable.
Effect of the monochromator’s slit width on noise and resolution for the
UV absorption spectrum of benzene.
The slit width increases from spectrum (a) to spectrum (d) with
effective band passes of 0.25 nm, 1.0 nm, 2.0 nm, and 4.0 nm.
 INSTRUMENTATION…
1.Filters
• A wavelength selector that uses either absorption, or constructive and destructive interference to
control the range of selected wavelengths.
A. Absorption filters- work by selectively absorbing radiation from a narrow region of the
electromagnetic spectrum.
 E.g. a piece of colored glass.
 A purple filter - removes the complementary color green from 500–560 nm.
 Have effective bandwidths from 30–250 nm.
 The maximum throughput of only 10%
B. Interference filters- use constructive and destructive interference to isolate a narrow range of
wavelengths.
 are more expensive than absorption filters, but
 have narrower effective bandwidths, typically 10–20 nm,
 with maximum throughputs of at least 40%.
Filters—
 do not allow for a continuous selection of wavelength.
 are available for only selected nominal ranges of wavelengths
 INSTRUMENTATION…
2. Monochromator—
 wavelength selector that uses a diffraction grating or prism, and
 that allows for a continuous variation of the nominal wavelength

 Radiation from the source enters the monochromator through an entrance slit. The radiation is
collected by a collimating mirror, which reflects a parallel beam of radiation to a diffraction
grating. The diffraction grating is an optically reflecting surface with a large number of parallel
grooves. Diffraction by the grating disperses the radiation in space, where a second mirror
focuses the radiation onto a planar surface containing an exit slit. In some monochromators a
prism is used in place of the diffraction grating.
 INSTRUMENTATION…
 The choice of which wavelength exits the monochromator is determined by rotating the
diffraction grating. 1. smaller effective bandwidth and
 A narrower exit slit provides a 2. better resolution, but
3. allows a smaller throughput of radiation.
 Monochromators are classified as
1. either fixed-wavelength
2. or scanning.
1. In a fixed-wavelength monochromator, the wavelength is selected by manually rotating the
grating.
 Normally, a fixed-wavelength monochromator is only used for quantitative analyses
where measurements are made at one or two wavelengths.
2. A scanning monochromator includes a drive mechanism that continuously rotates the
grating, allowing successive wavelengths to exit from the monochromator.
 are used to acquire spectra and,
 when operated in a fixed wavelength mode, for quantitative analysis.
 INSTRUMENTATION…
3. Interferometers
 A device that allows all wavelengths of light to be measured simultaneously, eliminating the need for a
wavelength selector.

 Radiation from the source is focused on a beam splitter that transmits half of the radiation to a fixed
mirror, while reflecting the other half to a movable mirror. The radiation recombines at the beam
splitter, where constructive and destructive interference determines, for each wavelength, the intensity
of light reaching the detector. As the moving mirror changes position, the wavelengths of light
experiencing maximum constructive interference and maximum destructive interference also changes.
The signal at the detector shows intensity as a function of the moving mirror’s position, expressed in
units of distance or time.
 In comparison with a monochromator, interferometers provide two significant advantages.
 results from the higher throughput of source radiation- improved signal-to-noise ratio
 savings in the time needed to obtain a spectrum- an entire spectrum can be recorded in approximately 1 s,
as compared to 10–15 min with a scanning monochromator
 INSTRUMENTATION…
III. Sampling cells and compartments :

 The key features of a spectrophotometer sample compartment are:

a. the optical paths through the compartment;
b. the cell holders that locate samples in the optical path; and
c. the lid that blocks out ambient light.

Cell Holders (Mounts)

 The cell holders (or mounts) can accommodate different sizes of conventional cells
(or cuvettes) for handling liquid and solution samples.
 The holders are often mounted on interchangeable bases, so that a variety of different
accessories can be used.
 INSTRUMENTATION…
• There are two major groups of sample compartments:
• single beam and
• double beam.
 A single beam spectrophotometer contains only one sample and only one beam of light
passes through that sample.
 A double beam spectrophotometer contains slots for two samples to be placed and one
light beam passes through each sample.
 A two beam spectrophotometer has the beam of light from the monochromator split into two
beams before it enters into the sample compartment.
 This two beam setup is more useful when trying to compare the unknown sample with a
known element or reference sample.
 A two beam setup is often used with one of the sample compartments left as blank.
 The measurements obtained from this empty blank compartment will show any inaccuracies
the machine may have so that the analysis of the sample will be proven to be more accurate.
 This calibrating measurement can also be done in a single beam spectrophotometer by
measuring an empty sample compartment before measuring the sample.
 INSTRUMENTATION…

rectangular
Sampling cells
Cuvettes
 can be round, square or rectangular in section and
 constructed from glass, silica or plastic.
 Square or rectangular cuvettes have a constant light path, the most usual being 1 cm in length.
1. For the visible region, compartments composed of simple pyrex glass is sufficient
 since pyrex will absorb in the UV

square
 but will not absorb in the visible region.

round
 are suitable for use between 320 and 950 nm.
2. For UV region, silica (quartz) cuvettes are used below 320 nm and
 they must be clean and free of scratches.
 This makes the sample compartments very expensive.
 Good practice is to fill all cuvettes with distilled water and measure the absorbance for each against a
reference blank over the wavelengths to be used.
This value should be essentially zero.
 Moulded polystyrene disposable cuvettes are available for use in the visible range and part of the
near UV.
 INSTRUMENTATION…
IV. Detectors

 Commonly used types of detector are

 photodiode arrays

 The diodes discharge energy when they are struck by light.

Each diode is then sequentially scanned and recharged to 5 V.
 The amount of energy required for recharging is proportional to the quantity of light
striking that diode
 INSTRUMENTATION…
 photomultiplier tubes

 Photomultiplier tubes are electron tubes that amplify current.

 Photons strike the cathode, which then emits electrons in an amount proportional to the energy
of the photons.
 The emitted electrons jump to a positively charged dynode, which emits four to six electrons
for every one striking it.
 Emitted electrons pass to the next dynode, which is more positively charged than the first.
 The number of emitted electrons is multiplied by each dynode.
 Finally, the electrons are collected at the anode.
 Photomultipliers are very sensitive to low light intensities, have a fast response time and do
not show as much fatigue as other detectors.
 INSTRUMENTATION…
V. Readout devices/Recorders
 Electrical energy from a detector is most usually displayed on a digital reading system that
provides a visual numerical display of absorbance or converted values of concentration.
 Microprocessors are now incorporated in the modern instrument.
 Signal output from a calibrator can be stored; signals from blanks can be subtracted from both
calibrators and unknowns, and the concentration of unknowns readily calculated.
 Data from multiple calibrators may be used to store a complete calibration curve, display or print
out the curve for visible inspection, and calculate results of unknowns based on the curve or
some mathematical transformation of it.
 QUALITATIVE
SPECTROPHOTOMETRY
1- Identification of chromophores…

 Compared with techniques such as IR spectroscopy, which produces many narrow bands,
 UV-visible spectroscopy provides a limited amount of qualitative information.
 However, 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 max
and emax, which could be correlated with the structural features

 Absorption characteristics of some common organic chromophores:

Chromophore Example ʎmax (nm) emax

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, or 255 7,900 or 200
 QUALITATIVE …
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

through

a. Comparison of the measured spectrum with a reference spectrum

and

b. Absorbance ratio
62
 QUALITATIVE …
2- Confirmation of identity…

a. Comparison of the measured spectrum with a reference spectrum

Example,
 An absorption band at 254 nm with characteristic vibrational fine structures may
be an evidence for existence of aromatic structure.
 Three characteristic bands at 278, 361 & 550 nm with absorbance ratio of 2:3:1 is
very characteristic for cyanocobalamin.

b. 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

63
 QUALITATIVE …
3- Detection of some functional groups:
Example,
 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.
 Confirmation of presence of aromatic amine or phenolic structure may be
obtained by testing the pH effect on their spectra
NH2 +
NH3

+ H+
- H+

In alkaline medium in acid medium

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

OH O O
-
OH
H+

in acid medium in alkaline medium

(Phenol) max = 270 nm (phenate anion)  max= 290 nm64
 Qualitative …
4- Approximate determination of the number of double bonds:
 By using Simplified Kuhn and Hausser rule :

max (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 and a -OH about 35 nm
• b -Alkyl cause red shift about 12 nm and b -OH about 30 nm
• g -Alkyl cause red shift about 18 nm and g -OH about 50 nm
Enone-
also called an α,β-unsaturated carbonyl, is a type of organic compound consisting of an
alkene conjugated to a ketone.
 Qualitative …
6- Empirical Rules for Absorption Wavelengths of Conjugated Systems

Carbonyl Compounds

 Carbonyl compounds have two principal UV radiations

 the allowed π  π* transitions and
 the forbidden n  π* transitions.
 In amides, acids, esters or acid halides, the substituents viz. NR2, OH, OR, or –X on
carbonyl group show pronounced hypsochromic effect on the n  π* transitions.
 due to inductive effect of nitrogen, oxygen or halogen atoms.
 The heteroatom withdraws electrons from carbonyl carbon and makes carbonyl
oxygen lone pair of electrons more stabilized due to its involvement in increasing
C=O bond order.
 As a result, the n  π* transition of these compounds is shifted to 200-215 nm range
relative to 270 nm in aldehydes and ketones.
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…
Carbonyl Compounds…
 Conjugation of the carbonyl group with double bond shifts both n  π* and π π* transitions to
longer wavelengths.
 The effect on π  π* band is more pronounced.
 Woodward formulated rules to predict the position of an absorption maximum in an unknown enone.
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Aromatic Compounds
 The simplest aromatic compound is benzene.
 It shows
 two primary bands at 184 (ε = 47,000) and 202 (ε = 7400) nm and
 a secondary fine structure band at 255 nm (ε = 230 in cyclohexane).
 Substituents on the benzene ring also cause-
 bathochromic and hypsochromic shifts of various peaks.
 Unlike dienes and unsaturated ketones, the effects of various substituents on the
benzene ring are not predictable.
 However, qualitative understanding of the effects of substituents on the
characteristics of UV-Vis spectrum can be considered by classifying the substituents
into
 electron-donating and electron-withdrawing groups
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Aromatic Compounds…

Effect of Substituents with Unshared Electrons:

 The non-bonding electrons increase the length of π-system through resonance and
shift the primary and secondary absorption bands to longer wavelength.
 More is the availability of these non-bonding electrons, greater the shift will be.
 In addition, the presence of non-bonding electrons introduces the possibility of n
π* transitions.
 If non-bonding electron is excited into the extended π*chromophore, the atom from
which it is removed becomes electron-deficient and the π-system of aromatic ring
becomes electron rich.
 This situation causes a separation of charge in the molecule and such excited state is
called a charge-transfer or an electron-transfer excited state
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Effect of Substituents with Unshared Electrons:

 In going from benzene to t-butylphenol, the primary absorption band at 203.5 nm
shifts to 220 nm and secondary absorption band at 254 nm shifts to 275 nm.
 Further, the increased availability of n electrons in negatively charged t-
butylphenoxide ion shifts the primary band from 203.5 to 236 nm (a 32.5 nm shift)
and secondary band shifts from 254 nm to 290 nm (a 36 nm shift)
 Both bands show hyperchromic effect.
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Effect of Substituents with Unshared Electrons:

 On the other hand, in the case of anilinium cation, there are no n electrons for
interaction and absorption properties are quite close to benzene.
 But in aniline, the primary band is shifted to 232 nm from 204 nm in anilinium cation
and the secondary band is shifted to 285 nm from 254 nm
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Aromatic Compounds…

Effect of π Conjugation

 Conjugation of the benzene ring also shifts the primary band at 203.5 nm more effectively to
longer wavelength and secondary band at 254 nm is shifted to longer wavelength to lesser extent.
 In some cases, the primary band overtakes the secondary band.
 For example, in benzaldehyde, the secondary band appears at 282 nm and primary band at
242 nm but in case of cinnamaldehyde, primary band appears at 281 nm and remains
merged with secondary band (figure 10).
 The hyperchromic effect arising due to extended conjugation is also visible
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Aromatic Compounds…

Effect of Electron-withdrawing and Electron-releasing Groups:

 Electron-withdrawing substituents viz. NH3+, SO2NH2, CN, COOH, COCH3, CHO and NO2 etc.
have no effect on the position of secondary absorption band of benzene ring.
 But their conjugation effects with π electrons of the aromatic ring are observed.
 Electron-donating groups such as -CH3, -Cl, -Br, OH, -OCH3, -NH2 etc increase both λmax and
εmax values of the secondary band.
 For example, aniline shows secondary band at 285 nm which due to presence of electron-withdrawing
p-nitro substituent is shifted to 367 nm with a significant increase in absorptivity
 6. Empirical Rules for Absorption Wavelengths of Conjugated Systems…

Aromatic Compounds…

Polycyclic Aromatic Compounds:

 In case of polycyclic aromatic hydrocarbons, due to extended conjugation, both

primary and secondary bands are shifted to longer wavelength.
 These spectra are usually complicated but are characteristic of parent compound.

naphthalene
 The primary band at 184 nm in benzene shifts to
 220 nm in case of naphthalene and
 260 nm in case of anthracene.
 Similarly, the structured secondary band which appears as broad band around 255 nm
in benzene is shifted to
 270 nm and in case of naphthalene
 340 nm in case of anthracene molecules anthracene
 QUANTITATIVE
SPECTROPHOTOMETRY
 UV/VIS spectrophotometry is perhaps the most widely used spectrophotometric
technique for the quantitative analysis of chemical substances.
 It has found increasing usefulness as a means of assaying pharmaceutical
substances described in the pharmacopeias.
 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
 The use of UV-visible spectrophotometry in quantitative analysis is centered
around the Beer-Lambert law, which relates the absorbance of an analyte to its
concentration.
 QUANTITATIVE …
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.
 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

Example 1:
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)

1. Calculate the concentration in ug/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 ug/ml)
 QUANTITATIVE …
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
 Calculate the concentration of the sample.
 (C= 36.5 ug/ml)
 Quantitative …
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.

 C sample = A sample X C standard/A standard

 It is the procedure adopted in many spectrophotometric assays of the BP and for the
majority of 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.
 Quantitative …
A. Assay of single components…

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:

Ctest = (Atest - Astd1) (Cstd1-Cstd2) + Cstd1(Astd1-Astd2)

Astd1-Astd2

Where, Std1is the more concentrated standard and Std2 is less

concentrated standard
 Quantitative …
B. Assay of Substances in Multi-component
 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.
 The basis of all the spectrophotometric techniques for multicomponent samples is the
property that all wavelengths
 The absorbance of a solution is the sum of absorbances of the individual
components or
 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.
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

1. 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 λmax 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
 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)

 Ans. C. Ephedrine HCl in the injection = 2.475 gm/100ml 81

 QUANTITATIVE …
B. Assay of Substances in Multi-component…
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 25.0-mL sample was treated with an excess of 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. (Cpd = 3. 60X10-5M, CAU= 2.4X10-4M) 82
 QUANTITATIVE …
B. Assay of Substances in Multi-component…
3. 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.
83
 QUANTITATIVE …
B. Assay of Substances in Multi-component…
3. Difference Spectrophotometer-
 The criteria for applying difference spectrophotometery to the assay of a substance in the presence of
other absorbing substances are that:
 Reproducible changes may be induced in the spectrum of the analyte by the addition of one or
more reagents
 The absorbance of the interfering substance is not altered by the reagents.
The simplest and most commonly employed technique for altering the spectral characteristics of the analyte
is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers.

∆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 un-reacted parent species. 84
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

4. 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 ʎ, the

derivative spectra are:

85
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

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.

86
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

4. Derivative spectroscopy
• Note that the number of bands observed is equal to the derivative order plus one.

Advantages
 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.
 It permits discrimination against broad band interferences, arising from turbidity
or non-specific matrix absorption.
 Thus, the information content of a spectrum is presented in a potentially more
useful form, offering a convenient solution to a number of analytical problems, such
as resolution of multi-component systems, removal of sample turbidity, matrix
background and enhancement of spectral details.
87
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

4. Derivative spectroscopy

Background elimination Resolution

Discrimination

88
 QUANTITATIVE …
B. Assay of Substances in Multi-component…

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.

89
 OTHER APPLICATION
E. Spectrophotometeric titrations
 One or more of the reactants or products absorb radiation.
 They are carried out in a vessel for which the light path is constant.
 The absorbance is directly proportional to concentration.

Titration Curves
 Plot of absorbance as a function of titrant volume and consists of two straight-line
regions with different slopes.

90
 OTHER APPLICATION..

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).

91
 COLORIMETRY
 Is a technique which involves measurement of absorbance in the visible region is
known as colorimetry.
 Involves measurement of color intensity of compounds.

Requirements for colorimetry

 the substance should be colored or
 The substance should be able to be derivatized in to colored product.
 While derivatizing

 The reagent should be specific

 The color produced should be stable enough until the analysis is completed
 Color intensity should be directly proportional to the concentration of the
analyte.

Application- colored drugs and those drugs which can be derivatized.

92