UV-VISIBLE SPECTROSCOPY THEORY

SPECTROSCOPY:
Spectroscopy is generally the study of the interaction between matter
(atoms or molecules) and Electromagnetic radiation.
The following are the basic types of spectroscopy:
 Emission spectroscopy
 Absorption spectroscopy

UV-VISIBLE SPECTROSCOPY:

Ultraviolet-visible spectroscopy or ultraviolet-visible

spectrophotometry (UV-Vis or UV/Vis) is the absorption spectroscopy in the ultraviolet-visible
spectral region of electromagnetic radiation.
 UV 190-380 nm, VISIBLE 380-780 nm
The absorption of photons (in the UV-VIS region) is due to
the transition in a molecule from the ground to a higher energy state. These higher energy states
are molecular orbitals that are empty in the unexcited state and are called antibonding orbitals.

INSTRUMENTATION:

UN. VISIBLE SPECTROPHOTOMETERS are made up of the following components;

1. Light Sources (UV and visible)
2. Filter or monochromatic
3. Sample containers or sample cells (cuvette)
4. Detector
5. Signal processor and read-out

1. LIGHT SOURCE
 A light source must be stable and provide radiation of constant intensity.
 It must possess sufficient intensity (Photons/sec) to make detections.
 It should be continuous so that its spectrum should provide the desired wavelength
required for analysis
 Deuterium or hydrogen lamps are used as light source Hydrogen or Deuterium lamp
emits electromagnetic radiation in the ultraviolet region of the spectrum and Tungsten
lamps emit radiation in the visible region of the spectrum.
 In a Hydrogen lamp electrodes pair is closed in a glass tube filled with hydrogen gas.
 It is provided with a window of silica or quartz so that radiations can pass.
 When current is passed through these electrodes maintained at high voltage, discharge of
electrons occurs which excites hydrogen molecules which in turn causes emission of UV
radiations.

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  Various other UV radiation sources are as follows:
a) Xenon discharge lamp
b) Mercury arc lamp

 Various Visible radiation sources are as follows:

a) Mercury vapor lamp
b) Carbon one lamp

2. FILTERS OR MONOCHROMATORS:
All monochromators contain the following parts:
 An entrance slit, a collimating lens, a dispersing device (a prism or a grating), a focusing
lens, and an exit slit. Polychromatic radiation (radiation of more than one wavelength)
enters through the entrance slit into the monochromator. The beam is collimated (made
parallel) and then strikes the dispersing element at an angle.
 The beam is split into its component wavelengths by the grating or prism. By moving the
dispersing element or the exit slit, radiation of only a particular wavelength leaves the
monochromator through the exit slit

3. SAMPLE CONTAINERS OR SAMPLE CELLS

A variety of sample cells are available for the UV region. They are selected on the
following basis
a. The path length, shape, and size.
b. The transmission characteristics at the desired wavelength
c. The relative expense

The cell containing the sample must be transparent. Quartz or fused silica cuvettes
are used for UV spectroscopy. Generally, cells of thickness I cm are used. Cells may be
rectangular in shape or cylindrical with flat ends.

4. DETECTORS
Three types of photosensitive devices are used as detectors:
a. Photovoltaic cells or barrier-layer cells
b. Phototubes or photo emissive tubes
c. Photomultiplier tubes.
d. Diode array detector

5. SIGNAL PROCESSING AND OUTPUT DEVICES

The signal from the transducer is amplified and then sent to the recorder to give an output. The
solvent spectrum is subtracted electronically. The output is the plot between the wavelength and
the intensity of absorption and is characteristic of the absorbing species.

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 TYPES OF UV-VISIBLE SPECTROMETER:

A. SINGLE BEAM UV-VISIBLE SPECTROMETERS

They consist of a single beam of light. The same beam of light is used for reading
absorption of sample and reference. The radiation from the source passes through a filter/
monochromator to get a single radiation, which then passes through the sample (or the
reference), and transmitted radiation is detected by the detector, the signal is then recorded. First
cuvette is filled with reference solution and absorbance is recorded, the cuvette is rinsed and
filled with sample solution and absorbance is recorded. The spectrum of the sample is obtained
by subtracting the spectrum of reference from that of the sample solution.

B. DOUBLE BEAM UV-VISIBLE SPECTROMETERS

In a double beam spectrometer radiation coming from a monochromator is split into
two beams by a beam splitter/chopper. The beams pass simultaneously through sample and
reference cells and transmitted radiations are detected by detectors and the difference in the
signal is amplified and recorded.

BEER-LAMBERT LAW:
The Beer-Lambert law states that the absorbance of a solution is
directly proportional to the concentration of the absorbing species in the solution and the path
length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the
concentration of the absorber in a solution.
A = ƐcI
This formula is the general form of the Beer-Lambert Law, and can be written in terms of
intensities:
A = LOG10 (Io1) = ƐcI
Ɛ  Molar absorptivity (formerly molar extinction coefficient) (L. /mol. cm) and is a measure of
the probability of the electronic transition.
C  The concentration of the sample solution (mol/L)
I  The length of the light path (cm).

APPLICATIONS OF ULTRAVIOLET-VISIBLE SPECTROSCOPY:

1. Detection of Conjugation
It shows a relationship between different groups on the basis of conjugation.
2 or more carbon-carbon double or triple bond.
Between carbon-carbon and carbon-oxygen double bonds.
Between double bonds and an aromatic ring

2. Detection of Impurities:
UV absorption spectroscopy can be used for the determination of impurities in organic
molecules. Additional spectrum peaks can be seen in the spectra due to impurities in the sample
and it can be compared with that of standard

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 3. Structure Elucidation of Organic Compounds
UV spectroscopy is useful in the structure elucidation of organic molecules, the
presence or absence of unsaturation, the presence of hetero atoms. From the location of peaks
and combination of peaks, it can be concluded that whether the compound is saturated or
unsaturated, hetero atoms are present or not etc.

4. Qualitative Analysis
UV absorption spectroscopy can characterize those types of compounds which absorb
UV radiation. Identification is done by comparing the absorption spectrum with the spectra of
known compounds. UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins.

5. Quantitative Analysis
UV absorption spectroscopy can be used for the quantitative determination of DRUGS/
compounds that Absorb LIV radiation by the help of Beer's law.

 A = -log T
 A = log I0 / I
 A = log 1/ T
 A = Ɛbc

Other methods for quantitative analysis are as follows.

a. Calibration curve method.

b. Simultaneous multicomponent method.
c. Difference spectrophotometric method.
d. Derivative spectrophotometric method.

6. Dissociation constants of acids and bases.

pH = pKa + log [A] (HA)
From the above equation, the pKa value can be calculated if the ratio of [A] / [HA] is known at a
particular pH and the ratio of [AV [HA] can be determined spectrophotometrically from the
graph plotted between absorbance and wavelength at different pH values.

7. Chemical kinetics.
Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
passed through the sample solution and the absorbance changes can be observed.

8. As HPLC detector.
A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an
analyte gives an absorbance which is assumed to be proportional to the concentration. For more
accurate results, the instrument's response to the analyte in the unknown should be compared
with the response to a standard; as in the case of calibration curve.

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 9. Molecular weight determination.
Molecular weights of compounds can be obtained spectrophotometrically by preparing
the derivatives of these compounds.
By using formula C-mv/vVXmol, molecular wt. can be determined as m=v/c

HOW TO USE A SPECTROPHOTOMETER:

 Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch
on the exhaust fan and make sure that all the chemicals and reagents required for the
experiment are available. If they are not available, prepare the reagents using the
components for preparation.
 Make sure to clean all your working apparatus with chromic acid and distilled water and
ensure that all the apparatus are free from water droplets while performing the experiment.
 Make sure to calibrate the electronic weigh balance before taking the measurements:
 Ensure that the spectrophotometer is working properly.
 Ensure that you are handling the cuvette with tissue paper. Never touch it with your hand.
 Wipe the cuvette with tissue paper before placing the spectrophotometer.
 Clean all glassware with soap and distilled water. Once the experiment is completed recap
the reagent bottles. Switch off the light and exhaust fan before leaving the lab.
 Discard the used gloves in a waste Bin.

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 EXPERIMENT NO: 07

Object # 07. To Perform The Assay Of Furosemide Tablet (Lasix) By B.P Method
(Absolute Method).

REQUIREMENTS:
Apparatus:
 Weighing Balance,  Beaker
 Tripod Stand  Volumetric flask
 Burette  Pipe
 Conical Flask
Chemicals:
 Sodium Hydroxide  Distilled water
 Furosemide tablets

THEORY:

FUROSEMIDE.
Furosemide is a medication
classified as a loop diuretic. Loop
diuretics are a type of diuretic
(water pill) that work primarily
on the thick ascending limb of
the loop of Henle in the kidney
nephrons. Furosemide is
commonly used to treat
conditions where excess fluid
accumulation in the body needs
to be reduced, such as:
Edema: Furosemide is used to alleviate fluid retention in conditions like congestive
heart failure, kidney disorders, and cirrhosis of the liver. And pulmonary edema. By increasing
urine output, it helps the body eliminate excess salt and water.
Hypertension (High Blood Pressure): In some cases, furosemide may be prescribed
to lower blood pressure by reducing blood volume.
Hypercalcemia: Furosemide can be used to treat high levels of calcium in the blood,
often associated with certain medical conditions.
Furosemide works by inhibiting the reabsorption of sodium and chloride ions from the urine in
the renal tubules. This action prevents the reabsorption of water, resulting in increased urine
production and a decrease in fluid volume within the body.
IUPAC NAME: 4-Chloro-2-[(furan-2-ylmethyl) amino]-5-sulfamoylbenzoic acid
MOLECULAR FORMULA: C12H11CIN2O5S
MOLAR MASS: 330.745g/mol.

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 SONICATION.
Sonication refers to the process of applying sound energy to agitate particles or
discontinuous fibers in a liquid. Ultrasonic frequencies (>20 kHz) are usually used, so the
process is also known as ultra sonication

SERIAL DILUTION.
A serial dilution is the stepwise dilution of a substance in a solution. Serial dilutions
result in the geometric progression of the concentration in a logarithmic fashion, meaning the
concentration decreases by the same quantity in each successive step.

PROCEDURE.

STEP 1: ASSAY PROCEDURE

Prepare 0.IN NaOH.

FLASK A:

 Take 30 tablets and weigh them correctly.

 Transfer the weigh tablets to mortar and pestle and crush them into fine particles.
 Calculate the amount required to contain 02g active in it.
 Dissolve the weigh amount in small quantity of 0.1 N NaOH in 250ml beaker and stir for
 15minutes. Transfer the solution to 500ml volumetric flask.
 Make up the volume with 0.1N NaOH.
 Filter the solution and discard initial 10 to 20ml solution from it.

FLASK B:

 Pipette out 5ml solution from flask A and transfer it to 250ml volumetric flask.
 Make up the volume with 0.1N NaOH.
 From flask B take the absorbance on UV-Visible spectrophotometer at a wavelength of
271mm.

STEP 2: WEIGHING OF ACTIVE INGREDIENT

 Amount of furosemide in one tablet = 40mg or 0.04g
 Weight of 30 tablets =_____________.
 Amount of furosemide in 30 tablets = 30 x 0.04 = 1.2g
 For 1.2g furosemide = 4.708gm.
 Thus, for 0.2g furosemide will be in___________ of tablet.

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 STEP 3: CONCENTRATION CALCULATION.
 Actual Concentration (Conc of Final Dilution).

0.2 g 5 ml
× = -------------
500 ml 250 ml

 Practical Concentration (Observed Conc).

Absorption (a) = _________

Length (b) = _________
Specific Absorbance (A 1% l cm) = 271nm.
a
= _____________ = A 1 % lcm ×b
= Concentration

._______________
STEP 4: PERCETAGE PURITY.

PRACTICAL CONC :
% Purity =
ACTUAL CONC :
× 100

% Purity = ----------------------------- × 100

% Purity =

RESULT:

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DISCUSSION:

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