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SPECTROPHOTOMETRY
UV/VISIBLE SPECTROPHOTOMETRY

PRASHANT PANDEY
M.PHARM | PHARMACEUTICS

1

Spectrophotometry-
UV/ Visible Spectroscopy

INTRODUCTION

Spectrophotometric techniques are mainly based on the measurement of interaction of
electromagnetic radiation with the quantized matter at specific energy levels. In general terms,
spectrophotometry is the measurement and interpretation of electromagnetic radiation absorbed
when the molecules of a sample move from one energy state to another energy state (i.e., from
ground state to excited state (or) exited state to ground state).

Electromagnetic waves are usually described in terms of (a) wavelength (λ), the distance
between two successive peaks; (b) wave number (v), the number of waves per centimeter;
frequency (v), the number of waves per second.

Waves Phenomenon

The arithmetic relationship of these three quantities is expressed by the following:

c = λv

The laws of quantum mechanics may be applied to photons to show that

E = hv

where E is the energy of the radiation; n is the frequency; and h is Planck’s constant.
Combining these two equations

E = hc/λ

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In the visible region, it is convenient to define wavelength in nanometers (nm) that is in units

of 10−9m, although other units may be encountered such as mill micron (μm) or Angstrom (Å).

1 nanometer = l nm = l mμ = 10 Å.

The visible spectrum is usually considered to be 380–770 nm and the ultraviolet region is
normally defined as 200–380 nm.

Absorption of light in both ultraviolet and visible regions of the electromagnetic spectrum
occurs when the light matches with the required spectrum to induce an electronic transition in the
molecule and it is assisted with vibrational and rotational transitions.

The diagram below shows a simple UV/visible absorption spectrum for buta-1, 3-diene—a
molecule which will be detailed later. Absorbance (on the vertical axis) is just a measure of the
amount of light absorbed. The higher the value, the more particular the wavelength is being
absorbed.

Absorption maxima diagram

It was invented in 1940 and commercialized in 1961.

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THE ELECTROMAGNETIC SPECTRUM

Table for the electromagnetic spectrums

THEORY

Electrons in the atom can be considered as occupying groups of roughly similar energy levels. In
the more complicated molecular model, electrons associated with more than one nucleus, the so-
called bonding electrons, are particularly susceptible to energy level transitions under the stimulus

Absorption of Different Electromagnetic Radiations by Organic
Molecules

In absorption spectroscopy, although the mechanism of absorption of energy is different in the
ultraviolet, infrared and nuclear magnetic resonance regions, the fundamental process is the
absorption of a discrete amount of energy. The energy required for the transition from a state of
lower energy (E1) to state of higher energy (E2) is exactly equivalent to the energy of
electromagnetic radiation that causes transition.

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Energy difference

Therefore E1 – E2 = E = hv = hc/λ, where E is the energy of electromagnetic radiation being
absorbed; h is universal Planck’s constant, 6.624 × 10−27 erg s and v is the frequency of incident
light in cycles per second (cps or Hertz, Hz), c is the velocity of light 2.998 × 1010 cm s−1 and λ is
the wavelength (cm).

Therefore, higher is the frequency, higher would be the energy and longer is the wavelength,
lower would be the energy. As we move from cosmic radiations to ultraviolet region to infrared
region and then radio frequencies, we are gradually moving to regions of lower energies.

A molecule can only absorb a particular frequency, if there exists within the molecule an energy
transition of magnitude E = hv.

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Electronic excited states diagram

Electronic Transitions

The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There
are three types of electronic transition which can be considered:

1. Transitions involving p (π), s (σ), and n electrons.

2. Transitions involving charge-transfer electrons.

3. Transitions involving d and f electrons (not covered in this unit)

When an atom or molecule absorbs energy, electrons are promoted from their ground state to
an excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These
vibrations and rotations also have discrete energy levels, which can be considered as being packed
on top of each electronic level.

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Electronic levels diagram

Absorbing Species Containing π, σ, and n Electrons

Absorption of ultraviolet and visible radiations in organic molecules is restricted to certain
functional groups (chromophores) that contain valence electrons of low excitation energy. The
spectrum of a molecule containing these chromophores is complex. This is because the
superposition of rotational and vibrational transitions on the electronic transitions gives a
combination of overlapping lines. This appears as a continuous absorption band.

Possible electronic transitions of π, σ, and n electrons are the following:

Types of transitions diagram

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σ σ* Transitions: An electron in a bonding orbital s is excited to the corresponding antibonding

orbital. The energy required is large. For example, methane (which has only C–H bonds, and can
only undergo σ σ* transitions) shows an absorbance maximum at 125 nm. Absorption maxima due
to σ σ* transitions are not seen in typical UV-visible spectra (200–700 nm).

n σ* Transitions: Saturated compounds containing atoms with lone pairs (non-bonding
electrons) are capable of n σ* transitions. These transitions usually need less energy than σ σ*
transitions. They can be initiated by light whose wavelength is in the range 150–250 nm. The
number of organic functional groups with n σ* peaks in the UV region is small.

n π* and π π* Transitions: Most absorption spectroscopy of organic compounds is based on
transitions of n or p electrons to the p* excited state. This is because the absorption peaks for these
transitions fall in an experimentally convenient region of the spectrum (200–700 nm). These
transitions need an unsaturated group in the molecule to provide the p electrons.

Molar absorbtivities from n π* transitions are relatively low, and range from 10 to 100 L mol−1
cm−1. π π*transitions normally give molar absorbtivities between 1,000 and 10,000 L mol−1 cm−1.

The solvent in which the absorbing species is dissolved also has an effect on the spectrum of
the species. Peaks resulting from n π* transitions are shifted to shorter wavelengths (blue shift)
with increasing solvent polarity. This arises from increased solvation of the lone pair, which lowers
the energy of the n orbital. Often (but not always), the reverse (i.e. red shift) is seen for π π*
transitions. This is caused by attractive polarization forces between the solvent and the absorber,
which lower the energy levels of both the excited and unexcited states. This effect is greater for
the excited state, and so the energy difference between the excited and unexcited states is slightly
reduced—resulting in a small red shift. This effect also influences n π* transitions but is
overshadowed by the blue shift resulting from the solvation of lone pairs.

Charge-transfer Absorption

Many inorganic species show charge-transfer absorption and are called charge-transfer complexes.
For a complex to demonstrate charge-transfer behavior, one of its components must have electron
donating properties and another component must have electrons absorbing properties. Absorption
of radiation then involves the transfer of an electron from the donor to an orbital associated with
the acceptor.

Molar absorptivities from charge-transfer absorption are large (greater than 10,000 L mol−1
cm−1).

Vibration and Rotation

The internal structure of a molecule may respond to radiant energy by more than just electronic
transitions. In some molecules, the bonding electrons also have natural resonant frequencies that
give rise to molecular vibration while others exhibit a phenomenon known as rotation. Because
the differences in energy levels associated with vibration and rotation are much smaller than those
involved in electronic transitions, excitation will occur at correspondingly longer wavelengths.

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Different electronic transitions

Vibrational absorption is typically associated with the infrared region while the differences
between the energy levels related to molecular rotation are so small that far infrared or even
microwave wavelengths are effective. Because vibrational and rotational absorptions are primarily
associated with spectral regions other than UV/visible, it is necessary here to note only the effect
on electronic absorption spectra. The principal effect is of “peak broadening”, i.e. the deviation of
an observed absorption peak from the predicted shape.

For most absorbing species, especially in solution, absorption peaks do not appear as sharp lines
at highly differentiated wavelengths, but rather as bands of absorption over a range of wavelengths.
A principal reason is that an electronic transition is frequently accompanied by vibrational
transitions between electronic levels (vibrational fine structure). In the same way, each vibrational
level may have associated rotational levels so that an absorption spectrum due to an electronic
transition may well be a complex structure, with contributing components from vibrational and
rotational absorptions.

Generally, when light falls upon a homogenous medium, reduction of the intensity of the light
may occur due to the following:

 A portion of the incident light is reflected.

 A portion is absorbed within the medium.

 Remaining is transmitted.

I0 = Ia + It + Ir

I0 is the intensity of incident light; Ia, t and r are the intensity of absorbed, transmitted and reflected
light.

Generally reflection is not observed in the case of clear medium.

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Flow of the radiation through the solution

The change of absorption of light with the thickness of the medium is given by Lambert and
extended concepts are developed by Bouguer. Beer later applied that to different concentrations.
The two separate laws governing absorption are known as Lambert’s law and Beer’s law and in
combined, known as Beer–Lambert’s law.

Beer’s law: The intensity of a beam of monochromes in light decreases exponentially with
increase in the concentration of absorbing species arithmetically. In quantitative analysis mainly
concerned with solutions which Beer studied, the effect of concentration of the coloured
constituent in solution upon the light transmission (or) absorption.

−dI/dc ∞ I

where I is the intensity of the incident light; dI is the decrease in the intensity of the incident
light; dcis the decrease in the concentration.

dI/dc = KI (−K is proportionality constant)

dI/I = Kdc

On integration

– lnI – Kc + b(b = constant of integration) (1)

When the concentration is “0”, there is no absorbance, hence I = I0

−lnI0 = Kx0 +b

−lnI0 = b

By substituting the “b” value in Eq. (1)

−lnI = Kc −ln I0

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lnI0 – lnI = Kc

lnI0/I = Kc (since log x − log y = log x/y)

I0/I =eKc

On inversing both sides

I/I0 = e−Kc

I = I0e−Kc Beer’s law

Lambert’s law: The rate of decrease in the intensity of the incident light with the thickness of
the medium is directly proportional to the intensity of the incident light this is equivalent to stating
that the intensity of emitted light decreased exponentially as the thickness of the absorbing medium
increases arithmetically.

−dI/dt ∞ I

Where dI is the decrease in the intensity of the incident light; dt is the decrease in the thickens
of the medium.

Same as Beer’s law we will get

I = I0e−Kt Lambert’s law

By combining both Beer’s and Lambert’s equations, we get

I = I0e
−kct

I = I010−kct (converting natural logarithm to base 10)

I/I0 = 10−kct

On inversing both sides

I0/I = 10−kct

log I0/I = Kct (taking log on both sides)

The quantity log I0/I is called as absorbance (A) and it is equal to the reciprocal of the common
logarithm of transmittance (T)

A = log 1/I/I0

Therefore, A = log I0/I

T = log I/I0 = KCT

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A = KCT

BEER–LAMBER’S LAW

When “C” is in moles/l, the constant is called molar absorptivity (or) molar extinction coefficient
(ε):

A = εCT
ε can also be written as

where is the absorbance of 1% W/V solution using a path length of 1 cm.

Application of Beer’s Law

Consider the case of two solutions of a colored substance with concentrations C1 and C2 placed in
an instrument in which the thickness of the layer can be altered and measured easily. When two
layers have the same color intensity

It1 = I0 × 10– ∊L = It2 = I0 × 10– ∊L2C2

where L1 and L2 are the lengths of columns of solutions

L1C1= L2C2

Hence it can be possible to investigate the validity of Beer’s law by varying C1 and C2 and also
for the determination of an unknown concentration.

Hence by plotting “A” as ordinate against concentration as abscissa, a straight line will be
obtained and which will pass through the origin. This calibration line is used to determine the
unknown concentration of solutions by measuring the absorbances.

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Beer–Lambert’s law plot

Deviation from Beer–Lambert’s Law

Generally, positive deviation (upward carve) (or) negative deviation (downward curve) is observed
in graphs of absorbance versus concentration (Beer–Lambert’s Law plot) (or) of absorbance versus
path length.

Beer–Lambert’s Law plot

Positive deviation results when a small change in concentration produces a greater change in
absorbance

Negative deviation results when a large change in concentration produces a smaller change in
absorbance.

Several reasons for the observed deviation form Beer’s law. They are as follows:

1. Instrumental deviations such as stray light, improper slit width, fluctuation in single beam.

2. Effect of stray light on Beer’s law plots

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3. Chemical effects such as association, dissociation polymerization, complex formation, etc.

as a result of the variation in the concentration.

Examples:

 A solution of benzoic acid of high concentration in a sample solution has a lower pH and
contains a higher proportion of unionized form than a solution of low concentration. The
ionized and unionized forms of benzoic acid have different absorption characteristics.

 Hence increasing the concentration of benzoic acid gives max of 273 nm with positive
deviation from Beer’s law and lower absorption of 268 nm with negative deviation from
Beer’s Law.

 In un-buffered solution of potassium dichromate, the dissociation of the dichromate ions
are observed by lowing the pH:

 Methylene blue at concentration of 105 M exists as a monomer and has max of 660 nm.
But methylene blue at concentration above 10−4 M exists as dimer which has λmax of 600
nm.

 The Beer–Lambert’s law does not hold when the solute forms complexes, the composition
of which depends on the concentration.

 In complete reactions such as insufficient time for the completion of reaction also produces
deviations from Beer’s law.

 Example: Determination of iron using thioglycollic acid before completion of reaction.

INSTRUMENTATION

The different components are the following:

2. Monochromators

3. Sample cells

4. Detector

5. Recorder (or) display

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Flow chart of instrument of UV spectroscopy

1. Tungsten–Halogen lamp: This lamp covers the wavelength ranging from the red
end of the visible spectrum (750–800 nm) to the near ultraviolet (300–320 nm).
The lamp is provided with a quartz outer sheath to permit the use of the ultraviolet
part of the emission.

2. Hydrogen (or) deuterium lamp: This is mainly used for the measurement of far
ultraviolet (down to 200 nm). It consists of two electrodes dipped in a deuterium
filled silica envelope.

Lamps Diagrams

Monochromator

The function of a monochromator is to produce a beam of monochromatic (single wavelength)
radiation that can be selected from a wide range of wavelengths. The essential components are (1)
entrance slit, (2) collimating device (to produce parallel light), (3) a wavelength selection or
dispersing system, (4) a focusing lens or mirror and (5) an exit slit.

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Two basic methods of wavelength selection may be noted: filters and a dispersing system (e.g.,

a prism or diffraction grating).

Filters: Filters of colored glass or gelatin are the simplest form of selection, but they are severely
limited in usefulness because they are restricted to the visible region and they have wide spectral
band-widths. Typical bandwidths are rarely better than 30–40 nm.

Interference filters, essentially a substrate (glass normally, but may be silica) on which materials
of different refractive indices have been deposited can be constructed with bandwidths of the order
of 10 nm or less. However, the comparatively wide bandwidth – and therefore limited resolution
– of filters, together with their inability to provide a continuous spectrum (except in special form
such as wedge filters) make them inappropriate for use in routine laboratory spectrophotometry,
in spite of low cost and technical simplicity.

Prisms: A prism of suitable material and geometry will provide a continuous spectrum in which
the component wavelengths are separated in space. It is usual to improve the definition of the light
between the source and the prism by using an entrance slit (to define the incident beam) and
acollimator (to produce a parallel beam at the prism). After dispersion the spectrum is focused at
the exit slit which may be scanned across the beam to isolate the required wavelength. In practice
the prism is normally rotated to cause the spectrum to move across the exit slit. A typical prism
monochromator is shown in below figure. Reflecting components, i.e. mirrors instead of lenses,
are desirable in UV systems for both efficiency and cost considerations.

Simple condenser prism monochromator

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Prism monochromators with bandwidths in the UV/visible of 1 nm or better are achieved

without great difficulty and so performance is greatly improved compared with filter-based
designs. However, there are drawbacks associated when using prisms: (1) their non-linear
dispersion, (2) the temperature-related characteristics of the commonly used prism materials and
(3) the complicated prism drive mechanism necessary to provide a convenient wavelength control

Diffraction gratings: Gratings provide an alternative means of producing monochromatic light.
A diffraction grating consists of a series of parallel grooves (lines) on a reflecting surface that is
produced by taking a replica from a master carefully prepared using a machine or, increasingly,
from one which is holographically generated. The grooves can be considered as separate mirrors
from which the reflected light interacts with light reflected from neighboring grooves to produce
interference, and so to select preferentially the wavelength that is reflected when the angle of the
grating to the incident beam is changed. Among the advantages that gratings offer (compared to
prisms) are better resolution, linear dispersion and therefore constant bandwidth and simpler
mechanical design for wavelength selection.

Gratings monochromator diagram

When parallel radiation illuminates a reflecting diffraction grating, the multiple reflections from
the mirror grooves will overlap and interfere with each other. If the reflected waves are in phase,
interference is said to be constructive and the reflected light is not affected. If the reflected waves
are out of phase, there is destructive interference and light of the wavelength at which such
interference occurs will not be propagated.

The relationship that determines the wavelength of the reflected light is expressed by the
following:

nλ = 2d sin θ

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where, n is the order (see below); d is the separation of the reflecting surfaces (or lines) and θ is
the angle of incidence of the radiation. Rotating the grating in the light beam changes θ and so
selects the wavelength to be reflected.

Two additional characteristics of gratings may be noted:

1. If wavelength λ is reflected for a given angle q, then λ/2, λ/3 and so on are also reflected at
that angle. These overlapping spectra, known as second and third orders, etc., can be
removed with filters or with a pre-monochromator. Careful selection of the blaze angle (the
angle at which the groove is cut) will peak the energy at the wavelength of the blaze,
typically 250 nm for instruments of the kind under discussion.

2. Both the energy and the resolution of a grating are directly proportional to the number of
lines. For maximum efficiency, the line separation should be as close as possible to one
wavelength, and for UV/visible gratings, the line density is typically 1200 per mm.

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Efficiency of the gratings diagram

Gratings have the following advantages over prisms:

1. Better resolution and energy transfer.

2. Linear dispersion and therefore constant bandwidth.

3. Less complicated wavelength drive mechanism is required.

4. Stray light is limited to imperfections at the grating surface.

Optical Geometry

As all absorption measurements are ratio dependent (I/I0), it is necessary to record a reference
solution before bringing the sample under test into the light path. These measurements are done
using a cuvette (matched, if possible, to that containing the test sample) in the light path filled with
the appropriate solvent. The reference intensity (I0) varies with wavelength in a complicated multi-
function way (due mainly to source energy, monochromator transmission, slit width and detector
response), so it is essential, when measuring absorption, to re-measure the reference for each
discrete wavelength at which measurement is to be made. All modern instruments are
microprocessor based and have the facility to store a baseline, that is, 100% T or 0 A set at each
wavelength in the range, overcoming this requirement. This has allowed single beam
spectrophotometers to compete on performance with the more expensive double beam instruments.

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Additional advantages of microprocessor handling of the detector output are the ability to

introduce component factors (e.g., concentration or molar absorption data) and to present results
in alternative formats without additional manual calculation. An important consideration in some
laboratories is the ability to interface with personal computers, so that results can be incorporated
into a laboratory information management system or transferred to disk for archiving or data
manipulation purposes.

Single-beam optics: The development of microprocessor has made it possible to achieve
excellent results using a single-beam configuration when compared to a double-beam
configuration; these results in greater optical and mechanical simplicity. The process of
comparison between reference and sample cells can be achieved with single-beam instrumentation
by feeding the post-detector signal to a microprocessor which stores the reference data for
subtraction from the sample signal prior to printing or displaying the reference corrected result
(the baseline). Signal levels can be compared between different samples at one wavelength, at a
series of predetermined wavelengths or, if wavelength drive is provided, a complete absorption
spectrum can be obtained.

Split (reference) beam optics: With the introduction of xenon flash lamps into
spectrophotometers, the split beam configuration has become necessary; this is because, the high-
intensity flashes from the xenon pulse lamp are not always of equal magnitude. Thus
approximately 70% of the energy from the monochromator is passed through the sample, with the
rest going to a separate feedback detector, enabling a means of taking into account drops/gains in
energy via a feedback gain loop in the detector electronics. This stabilises the system, and there
are no large extra cost elements involved.

Double-beam optics: Traditionally, the preferred technique was a double-beam geometry in the
sample handling area. Double-beam operation is achieved by a time-sharing system in which the
light path is directed (by rotating sectional mirror or similar device) alternately through the sample
and the reference cell. The wavelength-dependent functions of the instrument are significantly
reduced to give much improved operating characteristics by a feedback system in the reference
channel that adjusts the detector gain to compensate for source and detector variations. To make
full use of the potential of double-beam operation, it is usual to add wavelength scanning and some
form of output recording. UV/visible spectrophotometers of this type will, after initiation, produce
automatically an absorption spectrum of the kind shown in the below figure.

Diode array optics: A fourth optical configuration is the diode array; here, light is
monochromated after passing through the sample, which means that no sample compartment lid
is necessary. The other major difference is that the dispersive element (grating) is fixed and does
not move, as in more conventional systems.

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Arrangement of the instrumental components diagram

Sample Cells

Sample presented for spectrophotometric analysis may be in the solid, liquid (or) in the gaseous
state, the material that contains the sample should be ideally transparent at the wavelength of
measurement.

Sample cells diagram

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For the analysis of liquids and gases in UV/visible region above 320 nm, cells constructed with

optically flat fused glass may be used for measurements and below 320 nm requires the use of
more expensive fused silica cells which are transparent to below 180 nm. The standard path lengths
of cells are in the range of 10 mn and also 1–50 nm cells available for special applications.

Detectors

Most commonly used detector in the UV/visible spectrophotometers are photomultiplier tubes. In
order to obtain greater sensitivity to very weak light intensities, multiplication of the initial
photoelectrons by secondary emission is employed. Several anodes at a gradually increasing
potential are used in one bulb.

PMT detector diagram

Electrons from the photocathode are attracted to anode 1 and liberate more electrons which
travel to anode 2 and continues to the last anode and results in final current of 106–108 times greater
than that of primary current.

Silicon diode: Silicon diode detectors have good performance characteristics when (when the
device is integrated with an operational amplifier) compared with those of a photomultiplier, but
having a wider wavelength range but less sensitivity. They are mechanically robust (being solid-
state devices), and electronic benefits include reduced power supply and control circuit
requirements.

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Recorders

The primary function of a spectrophotometer ends with the provision of a signal (normally an
electrical voltage) that is proportional to the absorption by a sample at a given wavelength. The
signal handling and measuring systems can be as simple as an amplifier and a meter or as elaborate
as a personal computer and printer, depending on the application. In the simplest form, a meter
will serve either to indicate the absolute value of the output signal, or in some instances, the null
point in a back-off circuit. Digital readouts (LED or LCD) are favored for clarity and lack of
ambiguity and it may be linked to a microprocessor such that readout is in any preferred terms, for
example, directly in concentration units. Chart (or other) recorders can be used with instruments
equipped with wavelength scanning systems to provide directly an absorption spectrum. They are
also useful in the study of reaction rates where the requirement may be to plot absorption against
time at a fixed wavelength.

A block diagram of the post-detector electronic handling and of the integrated output and drive
systems of a modern sophisticated single-beam spectrophotometer, all controlled via a single
microprocessor, is shown the below figure. Once the operator has defined the parameters (e.g.,
wavelength, output mode and relevant computing factors) the system will ensure the correct and
optimum combination of all the variables available. Selection of source and detector are
automatically determined, any filters (e.g. order suppressing filters) or other components will be
introduced into the optical train at appropriate points and sample and reference cells are correctly
managed in the sample area. Output in the required terms (transmittance, absorbance,
concentration, etc.) will be presented and the relevant sample will be identified. Secondary routines
such as wavelength calibration and other self-tests may be available on demand and interfacing
with external computers or other instrumentation – e.g., automatic sampling devices – is easy.

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Block diagram of a microprocessor controlled spectrophotometer

SPECTROPHOTOMETERS

Single-beam spectrophotometers: An image of light source “A” is focused by the condition mirror
“B” and the diagonal mirror “C” on the entrance slit at “D”. Light falling on the collimation mirror
“E” is rendered parallel and reflected to quartz prism “F”. The back surface of the first surface is
reflected back through the prism via the absorption cell “G” to the photo cells (H): the photocell
response is amplified and recorded on “M”.

Double-beam spectrophotometers: Most UV/visible double-beam spectrophotometers over the
range between about 200 and 800 nm by a continuous automatic scanning.

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In this type, the monochromatic light is split by a rapidly rotating beam chopper into two beams

which are directed to sample and reference.

The Origins of Absorption Spectra

The absorption of radiation is due to fact that molecules contain electrons which can be raised to
higher energy levels by absorbing the energy. Electrons in a molecule can be classified as follows:

1. σ-Electrons: These electrons are present in lightly bound single covalent bond and radiation
of high energy is required to excite them.

2. Π-Electrons: These are in double (or) triple bonds which can be excited relatively easily.

3. n-Electrons: These are electrons attached to chlorine, oxygen (or) nitrogen as lone pairs.
These non-bonding electrons can be excited at a lower energy than “a” electrons

Electronic transition in organic compounds

*Excited orbital.

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SOLVENT EFFECTS

Highly pure, non-polar solvents such as 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 pure gaseous state. However, polar solvents such as water,
alcohols, may stabilize or destabilize the molecular orbitals of a molecule either in the ground state
or in the excited state and the spectrum of a compound in these solvents may significantly vary
from the one recorded in a hydrocarbon solvent.

1. π π* Transitions: In the 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 when
going from hexane to ethanol solvent.

2. n π* Transitions: In the 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.

SELECTION OF SOLVENTS

Solvents have important effects on the determination by UV/visible spectrophotomerty. They
should possess the following:

 It must be a good solvent.

 It should not interfere with the solute.

 It should not show significant absorption.

Examples of Solvents

Solvent λ max (nm)

Water 190

Hexane 199

Ethanol 207

Methanol 210

Cyclohexane 212

Chloroform 247

Carbon tetra chloride 257

Benzene 280

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Some Important Terms and Definitions

Chromophore: 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.
The group of atoms containing electrons responsible for the absorption is called chromophore.
Most of the simple un-conjugated chromophore gives rise to high-energy transitions of little use.

Example: σ-bonded electrons (C-C, C-H, etc.) σ σ* transition shows λ max of ~150 nm.

Lone pair of electrons (–O –, -N-, -S-) n σ* transition shows λ max of ~ 190 nm.

Auxochrome: The substituents that do not absorb ultraviolet radiations but their presence shifts
the absorption maximum to longer wavelength are called auxochromes. The substituents such as
methyl, hydroxyl, alkoxy, halogen, amino group, etc., are some examples of auxochromes.

Bathochromic shift or red shift: A shift of an absorption maximum towards longer wavelength
or lower energy.

Hypsochromic shift or blue shift: A shift of an absorption maximum towards shorter wavelength
or higher energy.

Hypochromic effect: An effect that results in decreased absorption intensity.

Hyperchromic effect: An effect that results in increased absorption intensity.

Woodward-Fieser’s Rules

Woodward’s and Fieser’s performed extensive studies on terpene and steroidal alkenes and noted
similar substituents and structural features would predictably lead to an empirical prediction of the
wavelength for the lowest energy π π* electronic transition.

This work was distilled by Scott in 1964 into an extensive treatise on the Woodward–Fieser
rules for the determination of structure.

Dienes

Example:

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Cyclic dienes: There are two major types of cyclic dienes, with two different base values.

Three common errors:

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This compound has three exocyclic double bonds; the indicated bond is exocyclic to two rings.

This is not a heteroannular diene; we should use the base value for an acyclic diene.

Likewise, this is not a homoannular diene; we should use the base value for an acyclic diene.

Aromatic Compounds

Substitution, auxochromic, conjugation and solvent effects can cause shifts in wavelength and
intensity of aromatic systems similar to dienes and enones.

However, these shifts are difficult to predict—the formulation of empirical rules is not efficient
for most of the part (there are more exceptions than rules).

There are some general qualitative observations that can be made by classifying substituent
groups:

1. Substituents with unshared electrons:

o If the group attached to the ring bears n electrons, they can induce a shift in the
primary and secondary absorption bands.

o Non-bonding electrons extend the p-system through resonance—lowering the
energy of transition π π*. More available n-pairs of electrons give greater shifts.

o pH can change the nature of the substituent group. Deprotonation of oxygen gives
more available n-pairs, lowering transition energy protonation of nitrogen
eliminates the n-pair, raising transition energy.

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2. Substituents capable of p-conjugation:

o When the substituent is a p-chromophore, it can interact with the benzene p-system.

o With benzoic acids, this causes an appreciable shift in the primary and secondary
bands.

o For the benzoate ion, the effect of extra n-electrons from the anion reduces the
effect slightly.

3. Electron-donating and electron-withdrawing effects:

o No matter what electronic influence a group exerts, the presence shifts the primary
absorption band to longer l.

o Electron-withdrawing groups exert no influence on the position of the secondary
absorption band.

o Electron-donating groups increase the l and e of the secondary absorption band.

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4. Di-substituted and multiple group effects:

o With di-substituted aromatics, it is necessary to consider both groups.

o If both groups are electron donating or withdrawing, the effect is similar to the
effect of the stronger of the two groups as if it were a mono-substituted ring.

o If one group is electron withdrawing and one group electron donating and they are
para-to one another, then the magnitude of the shift is greater than the sum of both
the group effects.

o Consider p-nitroaniline:

– If the two electronically dissimilar groups are ortho or meta- to one another, then the effect is
usually the sum of the two individual effects (meta-resonance ortho-steric bind).

– For the case of substituted benzoyl derivatives, an empirical correlation of structure with
observed λmax has been developed. This is slightly less accurate than the Woodward–Fisher rules,
but can usually predict within an error of 5 nm.

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Methods Available for Assays of Samples

1. Standard absorptivity value method: The use of standard “A” values (or) “E” values avoids
the preparation of standard solution of reference substance in order to determine its
absorptivity.

2. Example: Calculation of the concentration of methylstearate unknown absorbance is 0.890
at 241 nm. The STD value is 540 at 241 nm:

A = A 1%
1 cm bc

0.890 = 540 × 1 × C += 0.00165 g/100 ml

3. Calibration graph method: In this, the absorbances of a number of substance are measured
and a calibration graph is plotted from the graph to obtain the regression line.

y = α + βx can be estimated by the method of least squares.

where y = absorbance value; x = concentration; N = number of pairs of values.

4. Single (or) double point method: The single point precedence involves the measurement of
the absorbance of a sample salutation of a slandered solution of the reference substance.

where Ctest and Cstd are concentrations of sample and standard solutions, respectively. Atest
andAstd are absorbance’s of the sample and standard solutions.

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5. Two-point Backing standardization is required to determine that the concentration of

sample is greater than that of the sample while the other standard. Solution has a lower
concentration than the sample. The concentration of the sample solution is given as

Std1 and Std2 refer to more concentrated standard and concentrated standard respectively.

Simultaneous equations method: If a sample contains two absorbing drugs (X and Y) each
absorbs at the X max X2, then it is possible to determine both drugs by Vierodt’s method.

The information required as follows:

The absorptivities of X at λ1 are ax1 and ax2 respectively.

The absorptivities of Y at λ2 are ay1 and ay2 respectively.

Cx and Cy are the concentrations x and y

At λ1 A1 = ax1 bcx + ay1 bcy

At λ2 A2 = ax2 bcx + ay2 bcy

in cm cells b = 1

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substituting the Cy value

and

And this equation is concised and

The absorbances of the liquid sample A1 and A2, respectively.

6. Absorbance ratio method: The absorbance ratio method is a modification of the
simultaneous equation procedure. It depends on the property that for a substance which
obeys Beer’s law at all wavelengths, the ratio of absorbances at any two wavelengths is a
constant value.

For example, two different dilutions of the same substance give the same absorbance ratio
A1/A2is 2. This ratio is referred a Q value.

7. Geometric correction method: A number of mathematical correction procedures have been
developed which reduce (or) eliminate the background irrelevant absorption that may be
present in the sample of biological origin. The simplest of these procedures is the three-
point geometric procedure which may be applied on it and the irrelevant absorption is linear
at the three wavelengths selected.

8. Difference spectrophotometry: The essential feature of a difference spectrophotometry is

that the measured value is the difference absorbance (A) between two equivocal solutions
of the analyte in different chemical forms which exhibit different spectral characteristics.

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9. The most commonly employed technique is the adjustment of P by means of acid, alkali
(or) H buffer. The wavelength at equal absorptive of the two species is called isopiestic
points.

A = Aalkali −Aacid

Also obtained by the derived equation:

A = abc

where a is the difference absorptivity; b is the path length; C is the concentration.

10. Derivative spectrophotometric method: Derivative spectrophotometry involves the
conversion of a normal spectrum to its first, second (or) higher derivative spectrum.

The normal absorption spectrum is referred as zeroth order (or) D0 spectrum.

The first derivative spectrum (D1) is a plot between the rate of change of absorbance with
wavelength and wavelength.

The second derivative (D2) spectrum is a plot between the curvature of the D0 spectrum
and wavelength.

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A derivative spectrum therefore shows better resolution of overlapping bands of the
fundamental spectrum and permits the accurate determination of the X max of the
individual bands.

1. Chemical derivatisation methods: These methods are based on the conversion of the
analyte by a chemical reagent to a derivative that has different spectral properties. The
following are the mainly used methods for chemical derivatisation.

1. Diazotization: The amine is first diazotized with an aqueous solution of HN02 (by

the reaction of HCL and NANO2) at 0–5 °C.

Ar – NH2 + HNO2 Ar – N+ = N + 2H2O

The colorless diazonium salt is very reactive when treated with a suitable coupling
agent.

Example: Phenol (or) aromatic amine undergoes an electrophilic substitution and
produces an azo derivative.

Ar − N+ ≡ N + Ar′ − H Ar − N = N − Ar + H+

The azo derivatives are colored and consequently have an absorption maximum in
the visible region. Examples of coupling reagents are Borltan and Marshall’s
reagent which absorbs at 545 nm.

2. Condensation reactions: These reactions involve the nuclophilhic attack by the
amine on carbonyl carbon with elimination of H2O.

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3. Acid dye method: The addition of an amine in its ionized form to form an ionized
audic dye.

Example: Methyl orange (or) bromocresol purple yields a salt that will be extracted
into organic solvents such as CHCL3 or dicholoromethane. The absorbance is
measured against reagent blank.

GOOD OPERATING PRACTICE

The good operating procedure is mainly required for the spectrophotometric assays. The materials,
equipment and the sample cuvette should be properly cleaned. The reference and sample cuvette
should be identical in all parameters that is, in volume, shape and optical parameters. The cells
should have the transmission characteristics matched to 1% or better over a defined wavelength
range.

Preferred Absorption Range

The spectrophotometer should be able to measure the wide absorption range by avoiding the noise
in the detector. The error of the detectors should be in the range of 0.8–1.5 A. Spectrophotometers
which are equipped with silicon diode detectors do not suffer from this limitation. In such
instruments, performance limits are usually dependent on the stray light and a quality system will
measure absorbance up to 3 A with accuracy and reliability.

Absorbance Measurement

The unwanted effects like spectral bandwidth on peak absorbance are eliminated by constructing
the calibration curve at a known concentration. It should obey the Beer’s law so that a plot of
concentration can be determined. The importance of measuring absorbance precisely at the
wavelength of an absorption peak, i.e. at λmax is demonstrated in the below figure. Any wavelength
setting within the narrow band indicates that there is no significant effect on the absorbance at the
peak. The band of wavelengths displaced to shorter wavelength would be a major error.
Wavelength setting or instrument calibration errors will be minimum when measurements are
made at the wavelength of maximum absorption.

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Importance of measurement at lmax

Solvent Selection

The solvents available show the decrease in the transmission of the shorter wavelengths. Care
should be taken when working below 250 nm.

SOURCES OF ERROR

The laboratory requirements should meet the good laboratory practices provided. Validation
should be done for instrument, procedure and apparatus. These instrumental errors which are
caused by the stray light with improper bandwidth are minimized by the validation process.

Instrument-Related Sources of Error

Spectral bandwidth and slit width: The resolution of spectrometers is increased by the
minimization of separation between the absorption bands and which can be controlled by the
following factors:

 Spectral purity.

 Intensity of monochromator light.

 Detector sensitivity over wide range of wavelengths.

 Narrow slit widths.

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Spectral bandwidth

The total energy at the exit slit of a monochromator at wavelength λ assumed as a triangular
function. The half-peak intensity is defined as the spectral bandwidth for a given slit width. As the
ratio of the spectral bandwidth to natural bandwidth increases, the deviation of observed
absorbance from true absorbance will be greater. The natural bandwidth of most commonly
employed compounds in UV/visible is bio-molecules in the Life Sciences, which lies within the
range 5–50 nm. Thus, a spectrophotometer with a fixed bandwidth of 2–6 nm is ideal for bio-
molecule measurement. A narrower bandwidth is mandatory for measurements involving rare
earth and transition metal complexes. A diagrammatic representation of an absorbing species
measured at progressively increasing spectral bandwidths is shown in the below figure. As
bandwidth increases beyond the value, separation of the two bands is less, the apparent absorbance
at the maxima decreases and the observed bandwidth of the peaks increases.

Typical absorption peaks plotted at varying spectral bandwidths

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Effect of slit width on observed absorbance at λmax

Where it is necessary to determine accurately the absorbance at λmax, it may be desirable first
to plot apparent absorbance against slit width; figure above shows that slit widths greater than
about 0.75 mm may introduce significant error into the measurement of the absorbance concerned.

Stray light: Most important error occurred by the instrument is the stray light which is defined
as the radiation emerging from the monochromator of all wavelengths other than the bandwidth at
the selected wavelength. It is minimized by the removal of extraneous light.

The stray light will cause apparent negative deviations from Beer’s law and a level of 0.1% stray
light at any wavelength will prevent accurate absorption measurements of greater than 3A. The
primary effect of the stray light is to reduce the observed peak height (below figure). Where
absorbance is high (e.g., at an absorption peak) or where instrument sensitivity is low (e.g., at the
wavelength limits or near 190 nm where atmospheric oxygen absorbs strongly), the errors
introduced by the stray light will be relatively enhanced.

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Effect of stray light on observed peak height

Absorbance accuracy: The photo detector systems of most modern instruments are linear to less
than 1 % by design. Hence, the only factor which has any significant effect on absorbance accuracy
is the stray light, which is described above.

Wavelength accuracy: The effects of wavelength inaccuracies are noticed by the measurements
taken from absorbance peak, at the absorbance maximum where the rate of change is at a
minimum. The A260/A280 ratio used in assessing the purity of nucleic acid preparations, this is not
possible and care must be taken in the interpretation of results, especially if the solutions are dilute.

Noise: There is noise which is associated with the fluctuations of the beam reaching the detector
where beam energy is low. Noise problems may be reduced by integration with respect to time or
by storage and enhancement, and microprocessors are used for this purpose.

Non-instrumental Sources of Error

Non-instrumental errors are observed from the nature of the solution to be examined. The effects
of temperature or pressure must be maintained. Multi-component mixtures where more than one
constituent absorbs at a wavelength are of great interest. Absorbance in these conditions is additive
and a Beer’s law plot for one component may no longer be possible. The number of absorbances
measured at different wavelengths should be equal to the number of components in the mixture.
Providing the absorption coefficients of the components are known for each of the wavelengths
measured, the equations can be solved algebraically. The several processes that may occur when
a beam of radiation meets a cell containing a solution are shown in the below figure.

Total attenuation (i.e. the ratio of I to I0) may include components from the following:

1. Reflection of cell and cell interfaces.

2. Scattering by suspended particles.

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3. Absorption by the solution.

4. Fluorescent component (result of re emission)

Components of total attenuation process

Non-instrumental errors are minimized by the use of quality sample cells and fluorescence is
reduced by chemical inhibition or by the use of cut-off filters.

APPLICATIONS

 Used in the determination of Absorption curve and concentration of a substance.
 Example: KNO3 determination
 Used in the study of substituents effect on the absorption spectrum.
 Example: Comparing the absorption spectrum of benzoic acid with that of 4-

hydroxybenzoic acid and 4-aminobenzoic acid
 Used in the simultaneous spectrophotometric determinations.
 Example: Simultaneous determination of manganese and chronium in steel and other ferro-

alloys.
 Used for determination of molar absorption coefficients.
 Used in the analysis of Binary mixtures.
 Example: Benzene-toluene mixture Binary analysis.
 Used in the determination of phenols in water.
 Used in the determination of the constituents in a medical preparation by derivative

spectroscopy.
 Example: Determination of pseudoephedrine and tripolidine in actifed medicinal

preparation
 Used in the determination of keto-enol tautomerism.
 Following list of drugs are analyzed by UV-visible spectrophotometric method:

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