FLUORIMETRY DEFINITION/BACKGROUND

Fluorimetry is the quantitative study of the fluorescence of fluorescent molecules with the
help of fluorimeter or spectrofluorimeter.
Fluorescence
It is a phenomenon of emission of radiation when the molecules are excited by radiation at
certain wavelength. The phenomenon of fluorescence was discovered and published by Sir John
Fredrick William Herschel in the mid-1800s. He observed that, when illuminated with white light,
a solution of quinine emitted a strange blue light perpendicular to the direction of the illumination,
even though it remained colourless when observed facing the light source.

Physical basis of fluorescence

Photons of a given wavelength are absorbed by the fluorophore and excite some of its
electrons. The system remains in this excited state for only a few nanoseconds and then relaxes
into its ground state. (Note that light travels 30 centimetres in a single nanosecond.)
When returning from the excited state to the ground state, the electron may emit a photon.
This is known as fluorescent emission. The wavelength of the absorbed photon is always shorter
than that of the emitted photon (i.e. the energy of the emitted light is lower than that of the absorbed
one). Fluorophores
Fluorophores are characterised by specific fluorescence spectra, namely their excitation
(absorption) spectrum and emission spectrum. The excitation spectrum is recorded by measuring
the intensity of emission at a given wavelength, while the wavelength of excitation is continuously
changed. The emission spectrum is recorded by measuring the intensity of emitted light as a
function of its wavelength, while the wavelength of the exciting light is constant.

USES OF THE INSTRUMENT

1. Precise quantitation of biological molecules
The biological molecules include nucleic acids and proteins in microliter (μl) samples.
Fluorimeter can measure the concentration of double- and single-stranded DNA (dsDNA and
ssDNA), ribonucleic acid (RNA), and proteins in solution with demonstrable applications in a
range of biochemical fields.

2. Determination of Vitamin A
The method is sensitive and relatively rapid. The vitamin can be quantitatively estimated
in a blood sample of 0.1 ml, obtained from a fingertip puncture or in a tissue sample containing
less than 1 μg of the compound. The normal values of Vitamin A in blood and different tissue
samples are determined by the application of this method.

3. Liquid Chromatography
It is an important method in determining compounds as they appear at the end of
chromatogram or capillary electrophoresis column.
4. Organic Analysis
Qualitative and quantitative analysis of organic aromatic compounds present in cigarette
smoke, air pollutants, automobile exhaust, etc.

5. Fluorescent indicators
Mainly used in acid - base titration
● Eosin: colorless to green
● Fluorescein: colorless to green
● Quinine Sulfate: blue to violet
● Acridine: green to violet

6. Determination of inorganic substances

● Aluminum
● Boron
● Ruthenium

APPLICATION IN INDUSTRY
1. Food Industry
-Food-relevant fluorescent compounds include aromatic amino acids, vitamins and cofactors,
nucleic acids, porphyrins, polyphenols and alkaloids. Fluorescence coupled with multivariate
regression data analysis has been used for quantitative determination of chemical, physical and
sensory food properties. In fact, fluorescence has become an analytical tool for process control in
the food industry.
•Edible Oils
- Important applications for fluorescence in edible oils studies include: authentication of
virgin olive oils, discrimination between their different quality grades and geographical
origins, and detection of adulteration with low-grade olive oils or other vegetable oils.

•Dairy Products
-Fluorescence was investigated in dairy products, as an indicator of the protein structure.
Fluorescence was used for discrimination of the cheese types, ripening stages and
geographic origins. Intrinsic milk fluorescence was used to evaluate the heat treatment of
milk.

•Alcoholic Drinks
-Fluorescence was proposed for monitoring bitterness and determining the aromatic amino
acids and riboflavin content in beer. It allows classification of wines according to variety,
typicality and manufacturer.

2. Fabric and Paper Industry

-Fluorescent compounds are often used to enhance the appearance of fabric and paper, causing a
"whitening" effect. A white surface treated with a fluorescent brightening agent or an optical
brightener can emit more visible light than that which shines on it, making it appear brighter.
3. Pharmaceutical Industry
-Fluorimetry is particularly heavily used in the pharmaceutical industry where it has almost
completely replaced radiochemical labelling.
-Fluorescence detection is used for dissolution testing of tablets and products in the pharmaceutical
industry when the use of UV absorption is not appropriate.

REFERENCES

Basil, B. (September 2014). Fluorometry. Retrieved from:

https://www.slideshare.net/brunobasil/fluorometery.

Bio-rad. VersaFlour Fluorometer. Retrieved from:

http://www.bio-rad.com/en-ph/product/versafluor-fluorometer?fbclid=IwAR08nf-XyyrnA
Hsep8QK2O6B7GZ5Mr1bfBxWS7fnu896xYfnbiId4QBjZ-I&ID=b100e565-eb31-4303-83
05-9db89ddcf920

Denovix. The working principle of a fluorometer. Retrieved from:

https://www.denovix.com/blog/the-working-principle-of-a-fluorometer/.

Drujan, B.D, Castillon, R. and Guerrero E. (April 1968). Application of fluorometry in the
determination of Vitamin A. Retrieved from:
https://www.sciencedirect.com/science/article/pii/0003269768900079.

Fluorescence spectroscopy. Retrieved from

https://www.sciencedirect.com/topics/chemistry/fluorescence-spectroscopy.

Fluorimetry. Retrieved from:

http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch
04s07.html?fbclid=IwAR1UD3LLTO4RGW6REecDKNb85ERhsRYgEuUW51_aLmx5M
zqMXEFKc51Fjsw.

Sikorskaa, E. & Khmelinskii, I. (2016). Glowing colours of foods: application of fluorescence

and chemometrics in food studies. Retrieved from
https://www.spectroscopyasia.com/article/glowing-colours-foods-application-
fluorescenceand-chemometrics-food-studies.

Vidya, V.S. Fluorimetry. Retrieved from:

https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390?fbclid=IwAR1lDVUq6
MhUvYTEB1f5vEzxXWooh7gFkD_GVMprzotr82-KAev7780Mv0o.