Visibleand

Visible andUltraviolet
Ultraviolet
Spectrophotometry
Spectrophotometry

Badr Al-Hadiya, Ph.D.

June 21/2022
Abs = 0.51
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 Ultra‑violet and visible spectrophotometry
(Total number of theoretical Lectures = 5) including:

- Interaction of light with atoms and molecules: (Nature of light, various regions
of electromagnetic spectrum, concept of quantization of energy, different types of
energy of molecules).

- U.V. ‑ visible spectra of compounds: (Chromophores, auxochromes, factors

affecting spectra).

- Lambert ‑ Beer's Law: (Application to single component and two component

mixtures).

- Instrumentation: (U.V. ‑ visible spectrophotometers ‑ components and function

of each).

- Colorimetric reactions: (Classes of reactions, conditions and chromogens).

The electromagnetic spectrum
 Definition of UV-vis Absorption

UV-Visible absorption is a process where

a molecule absorbs ultraviolet or visible light that
excites electrons (makes them high energy). This
energy causes an electronic transition from a ground
state (non excited) to an excited state.

Absorbance
value
Different molecules will absorb different
wavelengths of light. The wavelengths of light
for UV-visible absorption are from about 200
nanometers to 800 nanometers. This is a section
of the electromagnetic spectrum pictured.
Absorption of light starts with energy of a
certain wavelength in this UV-visible region
being exposed to a molecule.
The light/energy then excites the ground state
(non excited) outer or valence electrons to an
excited state (high energy). The outcome of this
can be measured by a UV-visible
spectrophotometer. The data is shown as a
spectrum with absorption versus wavelength.
This pattern can be used to learn properties of
the molecule. Only certain molecules can absorb
light in this region.
 Electronic Transitions:
If light with suitable energy ν hits a molecule in the ground
state ψ1, the energy can be absorbed and the molecule is
raised to an electronically excited state ψ2. The molecule can
return to its ground state via spontaneous emission of a
photon (light).

−− ψ 2 −− ψ 2

↑
Absorption ∆E = E(ψ1) - E(ψ2 ) Emission
= h. ν ↓

−− ψ 1 −− ψ 1
Chromophores: They are molecules that absorb light at these
wavelengths. They are functional groups of a molecule that absorb
light in this UV-Visible region. They are most of the time
characterized by delocalized pi (π)electrons. Pi electrons refer to a
type of bond that occurs between electron orbitals called pi orbitals.
When many of these pi bonds exist in a molecule this allows
electrons to be delocalized or spread out across a molecule. An
example of this type of molecule is pictured below. Many dyes
(colored molecules) are characterized by these delocalized pi
electrons and their color. These molecules can be used for pH
indicators to determine if a solution is acidic or basic. The addition
of acid or base disrupts the delocalized pi electrons. This disruption
causes a color change.
 Laws of light absorption
All spectrophotometric methods that measure absorption,
reside upon two basic rules, which combined are known as
the Beer-Lambert law, as follows:
Beer’s Law:
It claims that the amount of light absorbed is proportional to
the number of molecules of the chromophore through which
the light passes.
The absorption of a molecule can be used to determine
the concentration of the molecule in solution. To find
concentration, Beer’s law is used. This is a mathematical
relationship shown by the equation below:

A = abc
A = Absorption, a = absorptivity coefficient, b = pathlength,
c = concentration
The sample container (named a cuvette) is made of a
material that does not absorb light in this region. The
molar absorptivity coefficient can be calculated by
measuring the absorption of a sample of a known
concentration and known pathlength. The absorptivity
coefficient units are dependent on the pathlength and
concentration units. Molar absorptivity units are liters
per mole centimeter (L mol-1 cm-1) when the
concentration units are molarity. The absorptivity is a
measure of how strongly a molecule absorbs light at a
particular wavelength. Once this constant is
determined, unknown concentrations can be
determined from this relationship.
Lambert's law: states that the fraction of light
absorbed by a transparent medium is independent
of the incident light intensity, and each successive
layer of the medium absorbs an equal fraction of
the light passing through it. This leads to an
exponential decay of the light intensity along the
light path in the sample, which can be expressed
mathematically, as follows:
log 10 (I0/I) = kl
where I0 is the intensity of the incident light, I is
the intensity of transmitted light, l is the length of
the light-path in the spectrophotometer cuvette,
and k is a constant for the medium, which is
deciphered by Beer's law.
law
 Lambert-Beer Law:
A = log10 (I0/I) = ε × c × l
absorbance ε : molar extinction coefficient,
A: absorbance, coefficient l: path length of sample
cuvette The term Iog10 (I0/I) is called the absorbance (A).
cuvette.
The above equation may be rewritten:
∆A = ∆ ε.c.l
where ∆A is the difference in absorption for the chromophore in the
two environments.
Sometimes the passage of light through the cuvette is described in terms
of transmittance, or transmission (T): T = I/I0 and is generally expressed as a
percentage. It is important to note, however, that only absorbance, not
transmittance, is linearly proportional to the chromophore concentration. In
quantitative analysis, where it is required to obtain the concentration of
substance, therefore, absorbance is more commonly used. The relation between
these two parameters is given by the following:
A = log10(1/T)
Thus, when A = 2 only 1% of the incident light is transmitted, while at A = 3,
only 0.1% is transmitted.
SPECTROPHOTOMETERS (Instruments):
Single-beam Spectrophotometers: are often sufficient for
making quantitative absorption measurements in the UV-Vis
spectral region. The concentration of an analyte in solution
can be determined by measuring the absorbance at a single
wavelength and applying the Beer-Lambert Law.
Single-beam Spectrophotometers are widely used for routine laboratory
measurements at a single wavelength. As shown in Figure 4, only a single light beam
from the monochromator passes through the sample compartment. The absorbance
zero or 100 per cent transmittance is adjusted with a cuvette containing buffer or
solvent. Then the absorbance (or transmittance) value of a sample cuvette containing
sample solution is measured. It is necessary to put the sample and the reference in
the optical beam alternately, by manual operation, typically with an interval of a few
seconds required to change the cuvettes.

Schematic of a wavelength-selectable, single-beam UV-Vis spectrophotometer

 Split beam or Double-beam Spectrophotometers:
The nomenclature 'split beam‘ emphasizes that a single beam of monochromatic light
is split spatially. The monochromatic light from the monochromator is split (chopped)
into reference and sample beams by a chopper mirror. The two transmitted light
beams of identical wavelength are separated in time and space, one beam passes
through the sample and the other through a standard or reference. The beams are
sequentially detected as the reference signal I0 and the sample signal I, and the sample
absorbance is obtained as the logarithm of the ratio of the two signals, which,
according to the Beer-Lambert law, is independent of the incident light intensity.
'Double beam' is better reserved for two beams of different wavelengths. The double-
beam (or split beam) design greatly simplifies this process by measuring the
transmittance of the sample and solvent simultaneously. The detection electronics can
then manipulate the measurements to give the absorbance.
All modern spectrophotometers are based on the double-beam design.
It consists of source of light, wavelength selector, a V-shaped mirror
called a beam splitter forms two matched light beams, one of them
passes through the sample solution and the second through the
reference solution (blank) in two matched cells, two photodetectors,
an amplifier for the signals and recorder as shown in Figure below.
Schematic of single- and double-beam UV-Vis spectrophotometers

Blank

Light source mono- Detector Meter

chromator
Sample
Figure 17: Schematic diagram of a single beam spectrophotometer

Blank Detector

Light source mono- Amplifier Meter

chromator
Sample Detector

Figure 18: Schematic diagram of a double-beam spectrophotometer

 (Dual-wavelength spectrophotometers):
The dual-beam design greatly simplifies this process by simultaneously measuring P
and Po of the sample and reference cells, respectively. Most spectrometers use a
mirrored rotating chopper wheel to alternately direct the light beam through the
sample and reference cells. The detection electronics or software program can then
manipulate the P and Po values as the wavelength scans to produce the spectrum of
absorbance or transmittance as a function of wavelength.

Schematic of a dual-beam UV-vis spectrophotometer

 Diode-Array Detector Spectrophotometers:
Are particularly suited to the single-beam mode since spectra
are acquired very quickly. Here a photomultiplier or
photodiode is used as the detector which is sensitive to a wide
range of wavelengths. It allows rapid recording
of absorption spectra. Dispersing the source light after it passes
through a sample allows the use of an array detector to
simultaneously record the transmitted light power at multiple
wavelengths. There are a large number of applications where
absorbance spectra must be recorded very quickly. Some
examples include HPLC detection, process monitoring, and
measurement of reaction kinetics. These spectrometers use
photodiode arrays (PDAs) or charge-coupled devices (CCDs) as
the detector. The spectral range of these array detectors is
typically 200 to 1000 nm. The light source is a continuum source
such as a tungsten lamp. All wavelengths pass through the
sample. The light is dispersed by a diffraction grating after the
sample and the separated wavelengths fall on different pixels of
the array detector. The resolution depends on the grating,
spectrometer design, and pixel size, and is usually fixed for a
given instrument. Besides allowing rapid spectral recording,
these instruments are relatively small and robust.
Portable spectrometers have been developed that use optical
fibers to deliver light to and from a sample. These instruments
use only a single light beam, so a reference spectrum is
recorded and stored in memory to produce transmittance or
absorbance spectra after recording the sample spectrum (see
figure below):

Diode-Array spectrophotometer is a single-beam, microprocessor-

controlled, with visible/UV-range of 190 to 820 nm with 2 nm
resolution. It is used when speed of measurement is essential. It is
faster, more sensitive and has more precision than a conventional
spectrometer due to the photo diode-array detection system. This
spectrometer is ideal for kinetics.
 Multi-Wavelength spectrophotometer
(or Multi Wavelength Measurement):
This is an extension of single wavelength measurements where
sample absorbance is recorded at multiple wavelengths.
Mathematical manipulations of this data can often reveal details
about the sample's composition or purity. Nucleic acid measurement
at 260nm and 280 nm is an example of a multi wavelength
application to check sample purity.
 Spectrum:
Absorption spectroscopy refers to spectroscopic techniques that

measure the absorption of radiation, as a function

of frequency or wavelength, due to its interaction with a sample.

The sample absorbs energy, i.e., photons, from the radiating field.
The intensity of the absorption varies as a function of frequency,
and this variation is the absorption spectrum.

The Spectrum is a plot of absorption intensity versus  or , it may

be:

a) Line spectrum: Occur with atomic spectra such as sodium metal,

which have a sharp line of  at 589 nm. (Figure 4.a):
b) Band spectrum: occurs with molecules due to the presence of different
vibrational and rotational sub-levels, which the molecules may occupy on
transition to excited state. That is, rotational and vibrational modes will
be found combined with electronic transition result in band rather than
line spectra (Figure 4.a & b).
 Revision: Types of Electronic Transitions
in the UV-VIS Region
The absorption of radiation in the UV-VIS. region depends upon the
number and arrangement of electrons in absorbing molecules.

The outer electrons in an organic molecule may occupy one of three

different energy levels (- , - or n- energy level), Accordingly, there
are three types of electrons:

a) sigma -electrons: they are bonding electrons, represent valence

bonds and possess the lowest energy level (the most stable).
Compounds containing only -electrons are the saturated
hydrocarbons, which absorb below 170 nm (in the far UV region). They
are transparent in the near UV region (200 - 400 nm) and this make
them ideal solvents for other compounds studied in this range. They
characterized by  – * transition only.
 b) n-electrons: they are nonbonding electrons, present in atomic orbitals of
heteroatoms (N, O, S or halogens). They usually occupy the highest energy
level of the ground state. Saturated organic compounds containing
heteroatoms, possess n-electrons in addition to  -electrons. So they
characterized by the – * and n – * transitions. The majority of these
compounds show no absorption in the near UV region. They are useful also
as common solvents in the near UV region. However, their intense
absorption usually extends to the edge of the near UV producing what is
called end absorption (cut off wavelength) mostly in the 200 - 250 nm
region, Table 1.

Table 1: Cut-off wavelengths of some common solvents

Solvent , nm Solvent , nm
Water 190 Chloroform 247
Ether 205 Carbon tetrachloride 257
Ethanol 207 Benzene 280
Methanol 210 Acetone 331
 c) pi  -electrons: they are bonding electrons,
forming the pi-bonds (double bounds), and

possess higher energy than -electrons.

Unsaturated compounds containing no
heteroatoms are characterized by the -* and
-* transitions, such as ethylene (CH2=CH2).
When these compounds containing
heteroatoms, they can undergo -*, -*, n-*
and n-* transitions, example acetone (CH3-CO-

CH3).
In excited state the s-electrons occupy an anti-bonding
energy level (s*) and the transition is termed s-s*
transition. p-electrons occupy an anti-bonding energy level
(p*) and the transition is termed p-p* transition. While the
n-electrons may occupy s* or p* levels to give n-s* or n-p*
transition. A summary of the different kinds of electronic
levels and transitions is found in Figure 5.
 Absorption characteristics of chromophores
 1- Ethylenic chromophores
 Their bands are difficult to observe in near UV region,
so they are not useful analytically. However,
substitution and certain structural features may cause red
shift rendering the band observable in the near UV region.
Examples;
a) Alkyl substitution; Cause red shift due to hyper-
conjugation and stabilization of excited state.
b) Exocyclic nature, Cause red shift due to relaxation of
strain upon excitation.
c) Attachment to auxochromes, Cause red shift and
increased absorption intensity due to extension of
conjugation.
2- Carbon-heteroatom chromophores
 Such as -C=O, -C=N, -C=S, -N=O ….etc. They exhibit some
common characteristics;
 n-p* band in the range of 275-300 nm., which is the most
apparent band, has low intensity and long wavelength. This
band undergoes a blue shift on increasing the solvent polarity
due to increasing the energy of transition as a result of
hydrogen bonding
 Alkyl substitution; Cause red shift due to hyper-conjugation.
 In cyclic ketones an equatorial -halogen results in a small blue
shift (5-10 nm) due to the inductive effect. While axial a-
halogen causes red shift (10-30 nm) due to direct resonance
participation in the excited state, thus lowering its energy.
a) Attachment of heteroatom or group to -C=O e.g. Cl, -NH 2 or -OH
causes blue shift due to resonance elevation of the p * level
hence increase energy of n-p* transition.
3- Conjugated chromophores
 Separated chromophores (by two or more single bonds)
have additive effect only because there is little or no
electronic interaction between separated
chromophores, Table 2.
Table 2: Absorption characteristics of selected chromophores
Position of the band, Intensity, 
Compound  (nm) (liters, cm-1.mol-1)

CH2=CH-(CH)2-CH3 180 10,000

CH2=CH-CH2- CH2-CH=CH2 180 20,000

CH3-CH=CH- CH=CH- CH3 227 25,000

 However, if two chromophoric groups are present in a molecule and
they are separated by only one single bond (a conjugated system), a
large effect on the spectrum results, more than found by more
addition, Table 3. The reason is that the -electron system is
interacting and spread over at least four atomic centers. When two
chromophoric groups are conjugated such as in dienes, the high
intensity * transition is generally red shifted by about 15 - 45 nm
with respect to the single unconjugated chromophore.
Table 3: Effects of conjugation on electronic absorption spectra

n Position of the band,  Intensity, 

CH3-(CH=CH)n-COOH (nm) (liters, cm-1.mol-1)

Acetic acid 0 197 60

Crotonic acid 1 208 12,500
Sorbic acid 2 261 25,600
2,4,6-Octatrienoic acid 3 303 36,500
2,4,6,8-Decatetraenoic acid 4 332 50,000
The reason for red shift of conjugated system:
1- An isolated double bond (pair of -electrons)
gives rise to an absorption maximum at about 190
nm.
2- When the molecular orbitals of two isolated
double bonds are brought into conjugation, the
energy level of the highest occupied molecular
orbital (HOMO) is raised and that of lowest
unoccupied (empty) anti-bonding orbital (LEMO) is
lowered.
3- The new * transition, is now associated with
a smaller energy value corresponding to longer
wavelength. Thus butadiene now absorbs around
215 nm instead of 190 nm for the isolated ethylenic
groups.
4- Aromatic system
(a) Benzene ring: Benzene has three maxima at 184 nm ( the most
intense), 204 nm and at 254 nm. The first two bands have their
origin in the ethylenic –  transition, while the longest  band is
a specific feature of benzenoid compounds. This band abbreviated
B-band, which is characterized by vibrational fine structures
(Figure 7). In structure elucidation both the B-band and the 204-
nm ethylenic band, termed E-band are useful while the far UV
band (184 nm) is unsuitable for analytical purposes.
Figure 7: Ultraviolet spectrum of benzene showing the vibrational fine structures.
(b) Monosubstituted benzenes
When the benzene ring is substituted with a
single functional group a red shift occurs for
both the E- and B-bands with increase in the
absorption intensity. This occurs whether the
substituent is an electron donating or electron
withdrawing group. In addition the B-band
loses most of its fine structure. Effects of
auxochromes on the electronic spectrum of
monosubstituted benzene is shown in Table 4.
Table 4: Effects of auxochromes on the electronic spectrum of benzene
Position of the band,  Intensity, 
Compound (nm) (liters, cm-1.mol-1)
255 215

270 1,450
OH

280 1,430
NH2

269 700
SH

265 240
Cl

261 300
CH3
ANALYTICAL REPORT
Summary (Title, and Aim of the work)
Equipment (Manufacturer, Calibration date)
Material and Analytical Standards (Source)
Chromatographic condition (Column, Flow rate, pH, wavelength,
temperature, mobile system)
Sample preparation
Selectivity / Stability indicating studies
Forced degradation (for stability indicating assay)
Linearity and Range
Limits of detection / quantitation
Repeatability (system suitability),
Accuracy and Recovery
Ruggedness
Robustness
 TABLE OF CONTENTS
Summary (Title, and Aim of the work)

The electromagnetic spectrum

Definition UV-Vis absorption
Electronic Transitions and Chromophores
Basic Spectrophotometric absorption rules
Equipment (Manufacturer, Calibration date)
Material and Analytical Standards (Source)
Chromatographic condition (Column, Flow rate, pH, wavelength,
temperature, mobile system)
Sample preparation
Selectivity / Stability indicating studies
Forced degradation (for stability indicating assay)