1. This document describes a procedure to estimate the concentration of quinine sulfate in a sample using fluorescence spectroscopy. 2. Key steps include preparing standard quinine solutions of known concentrations, measuring their fluorescence intensity, plotting a calibration curve, measuring the fluorescence of the unknown sample, and using the calibration curve to determine the sample's concentration. 3. Fluorescence spectroscopy provides a fast, simple, and inexpensive method to determine analyte concentration based on its fluorescent properties when excited by specific wavelengths of light.

1. ESTIMATION OF QUININE SULPHATE BY
FLUORESCENCE SPECTROSCOPY
First Semester 2020-2021
Masters of Pharmacy in Pharmaceutical Chemistry
Reg No:- 201862320110011 of 2020-21
Roll No:- 18620120002
Guru Nanak Institute Of Pharmaceutical Science And
Technology

2. AIM:
To estimate Quinine Sulphate by
Fluoroescence Spectroscopy.

3. THEORY
• Fluorescence: It is caused by the absorption of radiant energy & the re-emission of some of this
energy in the form of light, when there is transition from singlet excited state to singlet ground state.
• Phosphorescence: At favourable conditions like low temperature & absence of oxygen, there is
transition from excited singlet state to triplet state which is called a inter system crossing. The
emission of radition when electrons undergo transition from triplet state to singlet ground state is
called as Phosphorescence.
FIG 1. FLUORESCENCE& PHOSPHORESCENCE
• Fluorescence Spectrometry is a fast, simple & inexpensive method to determine the concentration
of an analyte in solution based on its fluorescent properties.
• In Fluorescence Spectroscopy, a beam with a wavelength varying between 180 to 800 nm passes
through a solution in a cuvette. Then, measurement from an angle the light that is emitted by the
sample.

4. • In Fluorescence Spectrometry, there is a measurement of both an excitation spectrum (the light that
is absorbed by the sample) & an emission spectrum ( the light emitted by the sample) This is done
by means of the quantum yield, which is defined as the fraction of the incident radiation which is re-
emitted as fluorescence.
• The concentration of the analyte is directly proportional with the intensity of the emission.
• When recording an emission spectrum, the intensity is dependent on:
• Excitation of wavelength
• Concentration of the anylte solvent
• Path length of the cuvette
• Self-absorption of the sample
• Factors effecting Fluorescence Intensity:
• Conjugation
• Nature of substituent groups
• Rigidity of structure
• Effect of temperature
• Viscosity
• Oxygen
• Effect of pH
• Photochemical Decomposition

5. WORKING PRINCIPLES OF FLUORESCENCE:
• Only a relatively small number of compounds can fluorescence
• By adding a fluorescent label, some non-fluorescent compounds can be made
fluorescent.
• In general, molecules that show fluorescence have one or more aromatic
groups in its structure.
• A molecule can be excited from its electronic ground state
• In the electronic ground state, the molecule has the lowest possible electronic
energy. Upon excitation (the absorption of a photon) one of the electrons goes
into a higher electronic state and the molecule is excited.
• The molecule will stay in its electronic excited state in the order of pico or
nanoseconds
• Then the electron will fall back to its ground state & will emit a photon of a
longer wavelength than the photon used for excitation.
• In this experiment, the fluorescence mission of Quinine Sulfate in aqueous
solution will be studied as a function of pH. Once there is an establishment of
necessary pH for maximum fluorescence intensity, a calibration curve will be
prepared using a series of standard solutions. The Quinine content of a
commercial preparation will then be determined.

6. INSTRUMENTATION:
• Source of light: There are four types,
1. Mercury Vapour Lamp: - Produce intense line spectrum above 350nm.High pressure lamps gives lines at 366,405,
436, 546,577,691,734nm.Low pressure lamps give additional radiation at 254nm.
2. Xenon Arc Lamp: - Spectrum is continuous over the range between over 250- 600nm, peak intensity about 470nm.
3. Tungsten Lamp: - Intensity of the lamp is low. If excitation is done in the visible region this lamp is used.
4. Tunable Dye Lasers: - Pulsed nitrogen laser as the primary source. Radiation in the range between 360 and 650 nm is
produced.
• Condensing Lens
• Filters & Monochromators:
In inexpensive filter fluorimeter two filters are presnt,
1. Primary filter-absorbs visible light & transmits uv light.
2. Secondary filter-absorbs uv radiations & transmits visible light.
In spectrofluorimeter two mpnochromators are presnt, which have Gratings.
1. Excitation monochromaters-isolates only the radiation which is absorbed by the molecule.
2. Emission monochromaters-isolates only the radiation emitted by the molecule.
• Sample cells & sample holder:
• Cylindrical or Polyhedral (Quadrangular) cells fabricated of silica or colour corrected fussed glass.
• Path length is usually 10mm or 1cm. It need not be made up of quartz, since we are measuring only the emitted
radiation not the absorbed radiation.
• All the surfaces of the sample holder are polished in fluorimetry, because emission measurements are made at 90◦
angle.
• Detectors:
1. Photovoltaic cell
2. Photo tube
3. Photomultiplier tubes – Best and accurate.
• Galvanometer: For output measurement.

7. • TYPES OF INSTRUMENTS:
I. Single beam (filter) fluorimeter
II. Double beam (filter) fluorimeter
III. Spectrofluorimeter Double Beam)
• WORKING OF INSTRUMENTS:
• The light from a mercury-vapour lamp (or other source of ultraviolet light) is passed
through a condensing lens, a primary filter (to permit the light band required for
excitation to pass), a sample container, a secondary filter (selected to absorb the primary
radiant energy but transmit the fluorescent radiation), a receiving photocell placed in a
position at right angles to the incident beam (in order that it may not be affected by the
primary radiation), and a sensitive galvanometer or other device for measuring the
output of the photocell.
FIG 2.ESSENTIAL PARTS OF A SIMPLE FLUORIMETER
•

8. REQUIREMENTS:
• Dilute sulphuric acid, (0.1N):-Add 3 ml of
concentrated sulphuric acid to 100 ml of distilled
water & dilute to one liter in a volumetric flask.
• Stock solution of quinine:-Weigh out accurately
0.100 g quinine and dissolve it in 1 L 0.1N
Sulphuric acid in a graduated flask. Calculate the
Quinine concentration in mg/L
• Ammonium Acetate Solution (10% w/v):-
Dissolve 50 gm in 500 ml of distilled water.

9. PROCEDURE
 Preparation of Standard Quinine Solution:
• Weigh accurately 100 mg of Quinine Sulphate powdered drug.
• Dissolve in 100 ml of 0.1 N H2SO4 (1mg/ml)
• Take 10 ml of above solution & dilute to 100 ml with 0.1 N H2SO4 (100µg/ml)
• Again,Take 10 ml of above solution & dilute to 100 ml with 0.1 N H2SO4 (10µg/ml)
• To get the resulting solution of 0.5,1.0,1.5,2.0, & 2.5 µg/ml concentrations, take 0.5,
1.0,1.5, 2.0 & 2.5 ml of above solutions respectively & dilute to 10 ml with 0.1 N H2SO4
 Preparation of Sample Solution
 Pipette out 1 ml of given unknown sample solution & make up the volume to 10 ml with
0.1 N H2SO4.
 Switch on the instrument, set the excitation & emission filters at the wavelength 365 nm
to 459 nm respectively.
 Set the fluorescence intensity to 0% by using 0.1 N H2SO4 as blank & 100% by using
highest concentration of the standard solution (2.5µg/ml)
 Repeat the same atleast for two times more to minimize the instrumental error
 Measure the percentage fluorescence intensity of different standard & sample solutions
 Plot a graph between concentration vs FI & determine the concentration of unknown
sample by expolating the found FI.

10. Observation table:
 Plot a graph between concentration & %FI
 From graph the contration of unknown solution is found to
be ……..
 Since the sample solution dilutes by 10 times, so the
concentration of Quinine Sulphate is found to be…….
Sr. No Concentration %FI
1 0
2 0.5
3 1.0
4 1.5
5 2.0
6 2.5
7 Unknown

11.  RESULT:
The concentration of given sample of Quinine Sulphate found to be
…………. By fluorimetry.
 CONCLUSION:
Fuorescence spectroscopy is a very sensitive & selective analytical
technique, giving it a major advantage over absorption spectroscopy when used
in the detection & measurement of trace amounts of organic compounds. This
assay method can br utilized for estimation of trace amount of quinine in
different organic samples & compounds.