Course Code:

PHR-322: Pharmaceutical Analysis- ll

Assignment On:
UV-Visible spectrophotometry

Submitted to:
Ms. Mahfuza Afroz Soma
Lecturer,
Department of pharmacy
State University of Bangladesh

Submitted by:
Md. Touhurul Islam Tuhin
ID NO: UG08-31-17-019
Batch: 31
Group: 3

Date of submission: 12/06/2020

 Introduction

Spectroscopy:
It is the branch of science which deals with the interaction of electromagnetic
radiation with matter is called spectroscopy.
Or,
It is the branch of science which deals with the study of interaction of matter
with light.

Electromagnetic Radiation:
Electromagnetic radiation is energy that is propagated through free space or
through a material medium in the form of electromagnetic waves, such as radio
waves, visible light, and gamma rays.
The term also refers to the emission and transmission of such radiant energy.

UV Spectroscopy:
The interaction of electromagnetic radiation with matter when source is UV is
called UV spectroscopy

Spectrophotometer:
Spectrophotometer are spectroscopic instrument that convert radiant
intensities into electrical signal and measure the light passes through the
sample. Spectrophotometer gives readings in transmittance (T) and
absorbance (A)

UV-Visible spectrophotometry:
UV-Visible spectrophotometry is one of the most frequently employed
technique in pharmaceutical analysis. It involves measuring the amount of
ultraviolet or visible radiation absorbed by a substance in solution. Instrument
which measure the ratio, or function of ratio, of the intensity of two beams of
light in the U.V-Visible region are called Ultraviolet-Visible
spectrophotometers.

Qualitative and quantitative pharmaceutical

applications of UV-Visible spectrophotometry
Qualitative applications:
Qualitative analysis through spectrophotometric methods achieves fast and
accurate results using only small sample quantities. This fast and effect
instrumentation has become an essential tool in the pharmaceutical industry
thanks to its adaptability and economic value. Qualitative analysis has proven
highly useful in many major forms of organic compounds and helps to ensure
patient health and safety.

Some qualitative applications include;

1. Detection of conjugation
Conjugation may be between;
a. Between two/more C=C bonds or C≡C bonds.
b. Between C=C or C=O bonds,
c. Between double bonds and aromatic ring.
2. Detection of structure of inorganic complexes
Used to distinguish between cis and trans isomers. Geometrical
isomerism-easily distinguished from visible spectra
3. Detection of functional groups
Detect the presence or absence of a functional group in a
compound. Absence of a band at particular wavelength regarded as
an evidence for absence of particular group.
4. Detection of impurities
The bands due to impurities are very intense. The organic
compounds can be classified into saturated compounds having
little absorption & unsaturated compounds having strong
absorption bands.
5. Determination of pKa value of indicator acid or base.
Let us consider an acid HA. It undergoes dissociation in water to
form H3O+ & A-.
HA + H2O → H3O+ + A-
A- conjugated base. The acid dissociation Ka is defined by the
expression
Ka = [H3O] + [A] → [HA]
On taking logarithm on both sides;
-log Ka = log [H3O+]-log [A-]/[HA]
⇒ pKa = pH –log [A]/[HA]
⇒ pKa = pH+ log [HA]/[A]
The ratio [HA]/[A] can be determined by spectrophotometer from
the graph plotted between absorbance & wavelength at different
pH values.
Quantitative applications:
Used to determine quantity of compounds. Used in Pharmaceutical research,
chemical research, biochemistry, chemical analysis & industrial processing.

Some qualitative applications include;

1. Measurement of unknown concentration by;

a) Calibration curve method: The solution of most of the drug obey beer-
lambert’s law up-to certain limit by making serial solution from a stock
solution & calibration graph is drawn, concentration along x axis and
absorbance along y axis. Eg: beer-lambert’s limit for cetirizine is 2-
16μg/ml
b) Sensitivity: UV absorption analysis is frequently quite sensitive. It
determines compounds of concentration >1ppm. The sensitivity
determines the lowest concentration & that can be determined
quantitatively by this method. Molecular weight determination. Can be
measured spectrophotometicaly by preparing the suitable derivative of
these compounds. E.g. To determine the molecular weight of amine, it is
converted to amine picrate. Then known concentration of amine picrate
is dissolved in a liter of solution & its optical density is measured at
380nm.

2. Charge transfer transitions:

When iodine & benzene are brought together in a 1:1 mole ratio in
heptane, a new absorption band is observed, which is not observable in
the spectra of individual components i.e. benzene & iodine. • According
to mulliken, this new band arises due to the absorption of radiation by a
molecular complex formed between benzene & iodine. This can be
represented as D+A (DA)complex where D is the benzene. DA D+A-
(covalent bond) (ionic bond) The total wave function of the ground
state may be put as;
Ψ0= Ψ(DA)+λΨ(D+A-) λ = measure of charge transfer

3. Tautomeric equilibrium:
Determine the percentage of various keto & enol forms present in a
tautomeric equilibrium. Eg: ethyl acetoacetate CH3COCH2COOCH5
CH3C(OH)=CHCOOC2H5 The keto form has λmax 275nm & ε=16. this
has only the weak n→ Π* band of the isolated carbonyl group. The
enol form has λmax 244nm & ε=16000. from the strength of the
244nm band can measure the proportions of tautomers present in the ethyl
acetoacetate.
4. Determination of structure of chloral:
This compound can have any one of the following structures H CCl 3-C-
OH CCl3-CHO OH (I) (II) When the UV absorption spectrum of this
compound is recorded in hexane, it shows a band at 290nm. On the
other hand, the UV spectrum of chloral in aqueous solution does not
show any band. This confirms that chloral hydrate has structure I
rather than II.

5. Chemical kinetics:
In order to determine the kinetics of a reaction the change in the
concentration of either a reactant or a product with time is measured.
Based on the fact that one of the reactants / products exhibiting
suitable absorption in the UV region is not overlapped by absorption
due to other species present. In this method, two solutions are
entering through X & Y. Then, these are allowed to pass through
reaction chamber B & the flow of the mixed solutions is stoppered by
piston D. The absorbance of any species which absorbs in the UV
region is measured at C with a UV spectrophotometer.
Photomultiplier is used as a detector whose output is displayed on the
screen with a time base.

6. Multicomponent analysis:
Absorbance of the sample is the sum of the absorbance of the
individual components. The selectivity and accuracy of
spectrophotometric analysis of samples containing interfering
substances can be improved by derivative and difference
spectroscopy. Derivative spectroscopy. The change in absorbance
with respect to wavelength is recorded. 1st and 2nd derivative
spectrum is recorded & characteristic peak for individual components
can be identified & quantified, using calibration curve of pure
substance. Difference spectroscopy. Useful to quantify a substance
when interfering species are present.

7. Photometric titrations:
Plot of absorbance as a function of volume of titrant. Usual titrimetric
disadvantages can be overcome by spectrophotometric titrations
using spectrophotometer which determines the end point. Method:
Titration vessel is kept directly in light path of the instrument Titrant
added Absorbance measured Plot of absorbance v/s volume of titrant
 Quantitative analytical procedure of a
Paracetamol using UV-Visible
spectrophotometry
Many drugs are in the form of raw material or in the form of formulation. They
can be assayed by making a suitable solution of drug in a solvent and
measuring the absorbance at specific wavelength. Paracetamol or
acetaminophen is a widely used over-the-counter analgesic (pain reliever) and
antipyretic (fever reducer). It is commonly used for the relief of headaches and
other minor aches and pains and is a major ingredient in numerous cold and flu
remedies. In combination with opioid analgesics, Paracetamol can also be used
in the management of more severe pain such as post-surgical pain and
providing palliative care in advanced cancer patients. However, acute overdose
of Paracetamol can be potentially fatal and its toxicity is the leading cause of
liver failure.

Materials:
 Paracetamol standard was taken.
 Paracetamol tablets containing 500 mg Paracetamol and the inactive
ingredient used in drug matrix were obtained from market.
 Analytical grade methanol and water were obtained

Diluent preparation:
Methanol and water (15:85, v/v) used as a diluent.

Standard preparation:
10 mg drug was dissolved in 15 ml methanol and was shaken well. Then 85 ml
water was added to it to adjust the volume up to 100 ml (100 ppm). From that 5
ml was taken and volume was adjusted up to 50 ml with diluents.

Test preparation:
20 tablets were weighed and powdered. Powdered tablet equivalent to 100 mg
of paracetamol was weighed and taken into 100 ml volumetric flask then 15 ml
of methanol was added and shaken well to dissolve it after that 85 ml of water
was added to adjust the volume up to 100 ml. From that 1 ml of solution was
withdrawn and taken in 100 ml volumetric flask. The volume was adjusted with
diluent up to 100 ml.

Instrumentation:
UV-Visible double beam spectrophotometer with matched quartz cells (1 cm)
Development and optimization of the spectrophotometric method:
Proper wave length selection of the methods depends upon the nature of the
sample and its solubility. To develop a rugged and suitable spectrophotometric
method for the quantitative determination of paracetamol, the analytical
condition was selected after testing the different parameters such as diluents,
buffer, buffer concentration, and other chromatographic conditions.
Our preliminary trials were by using different compositions of diluents
consisting of water with buffer and methanol. By using diluent consisted of
methanol - water (50:50, v/v) best result was obtained and degassed in an
ultrasonic bath.

Selection of wavelength:
Scan standard solution in UV spectrophotometer between 200 nm to 400 nm
on spectrum mode, using diluents as a blank. Paracetamol shows λmax at 243.

Conclusion
The present analytical method was validated and it meets to specific
acceptance criteria. It is concluded that the analytical method was specific,
precise, linear, accurate, robust and having stability indicating characteristics.
The present analytical method can be used for its intended purpose.