UV-Visible Spectroscopy

Dr. Asim Kumar Bepari

Department of Pharmaceutical Sciences
North South University
 References
●
Pharmaceutical Analysis: A Textbook for Pharmacy Students and
Pharmaceutical Chemists. David G. Watson
●
Spectroscopy: International Edition. Donald L. Pavia and Gary M. Lampman
●
https://www.ssi.shimadzu.com/products/uv-vis-spectrophotometers/faqs/index.
html
●
JoVE Science Education Database. Analytical Chemistry. Ultraviolet-Visible
(UV-Vis) Spectroscopy. JoVE, Cambridge, MA, (2020).
– https://www.jove.com/science-education/10204/ultraviolet-visible-uv-vis-sp
ectroscopy
UV-Vis Spectra: Example
UV-Vis Spectra: Example
 What is spectroscopy?
●
Spectroscopy is the technique of measuring the interaction of light with
matter using an instrument called a spectrophotometer.
●
The two primary concerns are
●
the wavelength of light incident on the sample and
●
the type of physical interaction with the sample.
●
These physical interactions can be as varied as absorption, reflection,
refraction, and scattering.
●
Spectroscopy can measure all common phases of matter (solid, liquid, gas).
Physical Interactions of Light
 Spectrophotometer
●
An instrument is any device employed to measure something
●
A spectrophotometer is a type of instrument.
●
The word spectrum is singular, the word spectra is plural.
Spectrophotometer
 Spectrophotometer

Agilent Cary 60 UV-Vis Spectrophotometer

PerkinElmer LAMBDA 1050+ UV/Vis/NIR spectrophotometers
Electromagnetic Spectrum
Molecular Orbitals
Electronic Energy Levels and Transitions
Electronic Energy Levels and Transitions
Principles of Absorption Spectroscopy
●
The greater the number of molecules
capable of absorbing light of a given
wavelength, the greater the extent of light
absorption.
●
The more effectively a molecule absorbs
light of a given wavelength, the greater
the extent of light absorption.
●
From these guiding ideas, the empirical
expression, known as the
Beer–Lambert Law, may be formulated.
Principles of Absorption Spectroscopy
 Principles of Absorption Spectroscopy
●
The Beer–Lambert Law is rigorously obeyed
when a single species gives rise to the observed
absorption.
●
The law may not be obeyed, however,
– when different forms of the absorbing molecule are in
equilibrium,
– when solute and solvent form complexes through
some sort of association,
– when thermal equilibrium exists between the ground
electronic state and a low-lying excited state, or
– when fluorescent compounds or compounds changed
by irradiation are present.
 Choice of Solvents
●
Cut-off
●
Fine structure
●
Influence on
wavelength
via
stabilization
 Choice of Solvents
●
Cut-off
●
Fine structure
●
Influence on
wavelength
via
stabilization
 The Configuration of a Spectrophotometer

●
There are four basic components to a simple
single beam UV/Vis spectrophotometer;
1)a light source,
2)a monochromator,
3)a sample, and
4)a detector.
 The Light Source
The requirements for a spectrophotometer light source include:
●
Bright across a wide wavelength range
●
Stable over time
●
Long service life
●
Low cost
Many light sources meet some of the requirements on this slide, but no light source can
meet them all.
Many spectrophotometers switch between a halogen lamp for the visible range and a
deuterium lamp for the ultraviolet range according to the wavelength setting.
 The Light Source

●
The tungsten lamp emits light from about 340 nm in the UV region up to over 3500
nm in the near infrared. These lamps are similar to the high intensity tungsten/halogen
lamps for household uses.
●
The deuterium lamp is a gas discharge light source that uses deuterium gas (an isotope
of hydrogen that contains an additional neutron in its nucleus). A deuterium lamp emits
light in the near ultraviolet and ultraviolet regions from 150 nm to 400 nm.
 Monochromator

●
A monochromator is a mechanism that emits monochromatic light from a light
source.
●
A dispersive element, generally a prism or diffraction grating, is used to create
the monochromatic light.
– A prism splits light into a spectrum by exploiting the fact that the refractive index differs
according to the wavelength when light passes through glass.
– A diffraction grating has parallel grating lines ruled on the surface.
 Sample
●
The majority of samples employed in UV/Vis
spectroscopy are solutions placed into a cuvette
for measurement.
●
Cuvettes are small rectangular glass or quartz containers.
●
They are often designed so that the light beam travels a
distance of 1 cm through the contents, but the path length
can vary from 1 or 2 mm all the way up to 10 cm.
●
The sample cell contains a solution of the substance you
are testing, usually very dilute.
 Sample
●
The material of the cell must have no absorption at the measurement wavelength.
●
Two materials often used for cells are glass and quartz.
●
Polystyrene (PS) and polymethylmethacrylate (PMMA) are mainly used for
disposable cells.
 How should cuvettes be cleaned?
●
Due to the wide variety of analytical samples, a wide variety
of corresponding cleaning methods are required.
1) Using Water as a Solvent - After cleaning with purified
water, clean with ethanol and store dry.
2) Using Organic Solvents - After cleaning with the organic
solvent being used, clean with ethanol or acetone and then
clean using the same method as described for the aqueous
solution above.
3) For stubborn contamination - the cell may be scrubbed
lightly with a cotton swab. Avoid using alkaline cleaning
solutions that can dissolve glass or ultrasonic cleaning
devices that can damage the cell.
 Detectors
●
A photomultiplier tube is used for detection in older types of UV/visible
instrument but, increasingly, photodiodes are used as detectors in
spectrophotometers.
●
A photomultiplier is a detector that uses the fact that photoelectrons are
discharged from a photoelectric surface when it is subjected to light (i.e., the
external photoelectric effect).
●
A silicon photodiode is a detector that uses the fact that the electrical properties
of a detector change when it is exposed to light (i.e., the internal photoelectric
effect).
●
This type of detector has no moving parts and can record spectra very quickly.
●
Furthermore, its output can be passed to a computer, which can process the information and provide
a variety of useful output formats.
●
Since the number of photodiodes is limited, the speed and convenience described here are obtained at
some small cost in resolution.
Single beam and double beam instruments
The Configuration of a Spectrophotometer
 Practical aspects of UV/visible spectrophotometry
●
Care should be taken to avoid touching the optical surfaces of sample cells with the fingers
since fingerprints can cause significant absorbance. The optical surfaces of the cell can be wiped
carefully with tissue.

●
The precision of the pathlength of cells is important. Tolerances for cells of good quality are
0.01 mm for pathlength. For maximum quantitative accuracy, the same cell should be used for
measurement of both the standard and the sample.

●
The cell should always face in the same direction in a cell holder to ensure that any cell optical
effects are identical for both blank and sample measurements.

●
Distilled water is the ideal solvent but is not suitable for many organic compounds. Methanol
and ethanol are next best but they cannot be used below a wavelength of 210 nm.
 Practical aspects of UV/visible spectrophotometry
●
The solvent used to dissolve the sample, concentration, pH, and temperature can
affect the position and intensity of absorption bands of molecules. These factors
should be controlled as far as possible.

●
Ideally absorbances measured should be in the range 0.4–1.0 to avoid being outside
the linear range of the instrument.

●
Scattering gives an apparent increase in absorbance and is caused by particles
suspended in solution. It is important that the sample solutions are free from
particles.
 Chromophore
●
Chromophore: Part of the molecule that makes color.
●
The characteristic energy of a transition and the wavelength of radiation
absorbed are properties of a group of atoms rather than of electrons
themselves.
●
The group of atoms producing such an absorption is called a chromophore.
 Chromophore
●
Alkanes:
σ → σ*, Very high energy, shorter wavelength.
●
Alcohols, Ethers, Amines, and Sulfur Compounds.
In saturated molecules that contain atoms bearing nonbonding pairs of electrons,
transitions of the n → σ* type become important.
They are also rather high-energy transitions, but they do absorb radiation that lies
within an experimentally accessible range.
●
Alkenes and Alkynes.
With unsaturated molecules, π → π* transitions become possible. stitution, as will
be clear later.
Alkenes absorb around 175 nm, and alkynes absorb around 170 nm.
 Chromophore
●
Carbonyl Compounds.
●
Unsaturated molecules that contain atoms such as oxygen or nitrogen may also
undergo n → π* transitions.
●
These are perhaps the most interesting and most studied transitions,
particularly among carbonyl compounds.
●
These transitions are also rather sensitive to substitution on the chromophoric
structure.
●
The typical carbonyl compound undergoes an n → π* transition around 280 to
290 nm (ε = 15).
●
Carbonyl compounds also have a π → π* transition at about 188 nm (ε = 900).
 Auxochrome, Shifts and Changes
●
Substituents that increase the
intensity of the absorption, and
possibly the wavelength, are called
auxochromes.
●
Typical auxochromes include
methyl, hydroxyl, alkoxy, halogen,
and amino groups.
●
Other substituents may have any of
four kinds of effects on the
absorption
 Effects of Conjugation

●
One of the best ways to bring about a bathochromic shift is to increase
the extent of conjugation in a double-bonded system.
●
In the presence of conjugated double bonds, the electronic energy levels of
a chromophore move closer together.
●
As a result, the energy required to produce a transition from an occupied
electronic energy level to an unoccupied level decreases, and the
wavelength of the light absorbed becomes longer.
Effects of Conjugation
 Woodward-Fieser Rules
●
Woodward-
Fieser Rules
to Calculate
Wavelength
of Maximum
Absorption
(Lambda-
max) of
Conjugated
Dienes and
Polyenes.
Woodward-Fieser Rules
Woodward-Fieser Rules
Woodward-Fieser Rules
Woodward-Fieser Rules
Woodward-Fieser Rules
Woodward-Fieser Rules
 Calibration Curve
●
Y = mx + C
●
R2=?
 Calibration Curve
●
Calibration curves are used to understand the instrumental response to an analyte and
predict the concentration in an unknown sample.
●
Generally, a set of standard samples are made at various concentrations with a range
than includes the unknown of interest.
●
Then the instrumental response at each concentration is recorded.
●
For more accuracy and to understand the error, the response at each concentration can
be repeated so an error bar is obtained.
●
The data are then fit with a function so that unknown concentrations can be predicted.
●
Typically the response is linear (Example: A = abc).

JoVE Science Education Database. Analytical Chemistry. Calibration Curves. JoVE, Cambridge, MA, (2020).
 Calibration Curve
●
When making solutions for a calibration curve, each solution can be made separately.
●
However, that can take a lot of starting material and be time consuming.
●
Another method for making many different concentrations of a solution is to use serial
dilutions.
●
With serial dilutions, a concentrated sample is diluted down in a stepwise manner to
make lower concentrations.
●
The next sample is made from the previous dilution, and the dilution factor is often
kept constant.
●
The advantage is that only one initial solution is needed.
●
The disadvantage is that any errors in solution making—pipetting, massing, etc.—get
propagated as more solutions are made. Thus, care must be taken when making the
initial solution.
JoVE Science Education Database. Analytical Chemistry. Calibration Curves. JoVE, Cambridge, MA, (2020).
 Calculation using Calibration Curve
●
Example: Paracetamol
● First, the λmax should be
determined.
●
Scanning of the standard
solution was done in UV
spectrophotometer between
200 nm to 400 nm on
spectrum mode, using
diluents as a blank.
●
Diluent consisted of methanol
- water (50:50, v/v).
Citation: Behera S, Ghanty S, Ahmad F, Santra S, Banerjee S (2012) UV-Visible Spectrophotometric
Method Development and Validation of Assay of Paracetamol Tablet Formulation. J Anal Bioanal Tech
3:151. doi: 10.4172/2155-9872.1000151
 Calculation using Calibration Curve
●
Then, a calibration curve should be constructed using the standard solutions.
●
Linearity of the calibration curve should be checked.

Conc(ppm) Absorbance
0 0
50 0.246
75 0.338
100 0.456
125 0.582
150 0.672
Calculation using Calibration Curve
●
Then, absorbance of the sample
solutions are determined.
●
Concentration is calculated
from the regression equation.

Absorbance Conc.
0.665 ?
0.350 ?
0.552 ?
Calculations using specific absorbance
Calculations
Assay Example
Assay Example
Assay Example
 Key Points
●
Principles
●
Radiation in the wavelength range 200–700 nm is passed
through a solution of a compound.
●
The electrons in the bonds within the molecule become excited
so that they occupy a higher quantum state and in the process
absorb some of the energy passing through the solution.
●
The more loosely held the electrons are within the bonds of the
molecule, the longer the wavelength (lower the energy) of the
radiation absorbed.
 Key Points
●
Applications in pharmaceutical analysis
• A robust, workhorse method for the quantification of drugs in formulations where there
is no interference from excipients.
• Determination of the pKa values of some drugs.
• Determination of partition coefficients and solubilities of drugs.
• Used to determine the release of drugs from formulations with time, e.g. in dissolution
testing.
• Can be used to monitor the reaction kinetics of drug degradation.
• The UV spectrum of a drug is often used as one of a number of pharmacopoeial identity
checks.
 Key Points
●
Strengths
• An easy-to-use, cheap and robust method offering good precision for
making quantitative measurements of drugs in formulations.
• Routine method for determining some of the physico-chemical
properties of drugs, which need to be known for the purposes of
formulation.
• Some of the problems of the basic method can be solved by the use of
derivative spectra.
 Key Points
●
Limitations
• Only moderately selective. The selectivity of the method
depends on the chromophore of the individual drugs, e.g a
coloured drug with an extended chromophore is more
distinctive than a drug with a simple benzene ring
chromophore.
• Not readily applicable to the analysis of mixtures.
 References
●
Pharmaceutical Analysis: A Textbook for Pharmacy Students and
Pharmaceutical Chemists. David G. Watson
●
Spectroscopy: International Edition. Donald L. Pavia and Gary M. Lampman
●
https://www.ssi.shimadzu.com/products/uv-vis-spectrophotometers/faqs/index.
html
●
JoVE Science Education Database. Analytical Chemistry. Ultraviolet-Visible
(UV-Vis) Spectroscopy. JoVE, Cambridge, MA, (2020).
– https://www.jove.com/science-education/10204/ultraviolet-visible-uv-vis-sp
ectroscopy