Assignment-1

Course: Pharmaceutical Analysis (PHA305) Sec-1

Course Instructor: Syeda Neureen Ahmed
Submitted by: Ayesha Rahman (1920560),
Usayatun Mehera (1921143),
Nusrat Jahan Mim (1920495)
Submitted Date:
 Topic: Fluorometry and Polarimetry
Fluorometry: (slideplayer.com, Fluorescence Fluorescent corals by Julianne Louie)
Fluorometry is an analytic method for detecting and measuring fluorescence intensity at a
particular wavelength with the aid of the fluorometer. The fluorometer is the instrument with
which the intensity of the fluoroscene from different objects can be measured. When the
electrons in the sample are excited by the specific wavelength of the light, the electrons move
from lower energy state to higher energy state by emitting fluoresce light of a different
wavelength. Different fluorescent object emit different kinds of light at different wavelengths.

Principle (slideshare.net by V.Shree Vidya)

Molecules consist of bonding electrons such as Sigma and Pi electrons. These electrons have the
least energy. They are more stable so they stay at the ground state. Molecules remain in their
ground vibration level at room temperature. When the molecules get energy in the form of light
with specific frequency, it would absorb light with 10-15 seconds. Due to this absorption, the
molecules move from ground state to the first excited singlet electronic state and the excitation
molecules jump to in any one of the vibrational levels in the first excited electronic state. With
the increase of energy in the form of the light increase the excited level of the electron from
ground state to one, two, three and so on. These excited levels are very unstable for the excited
electrons. They tend to go back to their original energy state with the emission of light when the
source of energy is being removed.
Fluoroscene and Chemical structure (chem.libretexts.org by Department of
Education Open Textbook Pilot Project)

Florescence is the substance that emits the absorbed light as the electron moves from a higher
energy state to a lower energy level and the emitted light has a longer wavelength than the
absorption. Fluorescence is generally observed in compound with rigid structure, organic
chelates agent complexed with metal ion and substances which contain aromatic functional
group with lower energy. Heterocyclic compounds do not show fluorescence properties such as
pyridine, pyrrole furan but if these heterocyclic compounds combine with benzene rings it would
increase fluorescence such as indole, quinoline, and isoquinoline. This is because a substitute on
the benzene increases the wavelength to the maximum and there are corresponding changes to
the florescent. The increase of the atomic number of the compound decreases the florescent.
Unsubstituted aromatic hydrocarbon increases fluorescence as quantum efficiency increases with
the number of rings and degree of condensation but if aromatic rings are substituted by the
carboxylic acid, it does not show fluorescence property.

There are 2 types of fluorescence :

Intrinsic Fluorescence: a substance (molecules, ions, etc.) that emits light strongly in its
original form
Extrinsic Fluoroscene: a substance that emits light strongly through reaction with a
fluorometric reagent.

 Depending on the molecule involved, the electron can move to different energy states:
Ground State-in this state, two electrons are in pairs and opposite spins.

Singlet Excited state-in this state, electrons are in a higher energy orbital with opposite spin
similar to the ground state

Triplet excited state-in this state, electrons are more excited and may have the same spin. This
is known as an intersystem crossing.

Instrumentations (chem.uci.edu by Modern fluorescence spectroscopy, Wehry, E.L., Ed.

Plenum Press)
Sources of light such as 

Xenon arc lamp- intense radiation is formed with the help of the current through creating an
atmosphere of xenon and the range of the spectrum is between 250-600nm with a peak at 470nm
Mercury arc lamp

It produces an intense light spectrum above 350nm and high-pressure lamps show lines at
366,405,536,546,577 and low-pressure lamps show lines at 254nm.

Tungsten Lamp

It is used if excitation is required at visible light and it does not give UV light and the intensity of
the lamp is low.

Tunable dye laser-

It produces radiation between 360-650 nm with the help of pulsed nitrogen as a primary source.
Filters and monochromators-

The filter consists of two types – 

Primary filter- it absorbs visible light and emits UV light
Secondary filter-it absorbs UV radiations and emit visible light

Monochromators-
Excitation monochromator- it gives appropriate radiation for excitation molecules.
Emission monochromators- only isolate the fluorescence radiation.

Sample cell: these are used to hold the sample which is in cylindrical or rectangular shape made
out of silica and glass. In Fluorometry, all the surfaces of the sample cell are polished.

Detectors:
Photomultiplier tube-

This is the best detector as its sensitivity is high as it detects weak light intensity.
Photovoltaic cell

 It has a copper plate coated with a thin layer of cuprous oxide. For good contact, silver is laid
onto this plate. When light falls on them oxide moves from the oxide layer to the copper and thus
oxide becomes positive and copper becomes negative.

The three most common fluorometers are:

Single beam fluorometer-

It is very simple to build, inexpensive, and easy to operate. It has tungsten as a light source and
an optical system as the primary filter. The secondary filter measured emitted radiation at 90
degrees. Secondary filters absorb UV light and emit visible light.

Double beam fluorometer-

Two incident beams passes through the primary filter independently and fall on the sample or
standard solutions. The emitted ray from there passes to the secondary filter.
Spectrofluriometer-

Here, the primary filter is being substituted with the excitation monochromator and the
secondary filter is being replaced with an emission monochromator. With the help of the beam
splitter, the incident beam is split into a sample and reference beam.

Factor affecting the intensity of fluorescence (sciencedirect.com by H. Itagaki)

 Concentration-intensity of the fluorescence is directly proportional to the concentration
when the absorbance is less than o.o2
 Quantum yield of fluorescence-it should be always less than 1
 The intensity of incident light-it is directly proportional to the intensity of fluorescence
 Adsorption-it causes serious damage if the sample solution adsorbs to the container.
 Oxygen-oxidation of fluorescence produces a non-fluorescence substance which
decreases fluorescence intensity
 Ph- changing of ph will have a significant effect on the intensity of fluorescence
 Temperature and viscosity-increase in temperature cause more collusion which reduces
fluorescence intensity. Increased viscosity decreases the fluorescence intensity.
 Photo decomposition-photochemical degradation reduces fluorescence intensity
 Quencher-it reduces fluorescence intensity by the presence of a substance in the sample 
 Scatter-Scattering of incident light through the solution causes a decrease in fluorescence
intensity.

References
 Julianne Louie, “Fluorescence Fluorescent corals.”,
https://slideplayer.com/slide/3837491/

 V.Sree Vidya, Ch.Devdas, “Fluorimetry”,

https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390#:~:text=%EF
%82%A2%20Instruments%3A%2D%20It%20contains,molecule%20present
%20in%20sample%20cell

 Department of Education Open Textbook Pilot Project, the UC Davis Office of

the Provost, the UC Davis Library, the California State University Affordable
 Learning Solutions Program, and
Merlot.https://chem.libretexts.org/Courses/Providence_College/CHM_331_Adva
nced_Analytical_Chemistry_1/10%3A_Molecular_Luminescence_Spectrometry/
10.03%3A_Applications_of_Photoluminescence_Methods/10.3.01%3A_Intrinsic
_and_Extrinsic_Fluorophores

 Modern fluorescence spectroscopy, Wehry, E.L., Ed. Plenum Press, New York
(1976), Vol. 11459 pp.
https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluorescence
%20Spectroscopy.pdf

 H. Itagaki, in “Experimental Methods in Polymer Science,” (2000)

https://books.google.com/books?
hl=en&lr=&id=BRqQuyzyMdYC&oi=fnd&pg=PA155&dq=%E2%80%A2+H.
+Itagaki,+in+%E2%80%9CExperimental+Methods+in+Polymer+Science,
%E2%80%9D+(2000)&ots=xmh8YjD0KF&sig=ukXSxk2XmDVNBgnQmvVn
W4zNg5U