PRACTICAL 5
AIM: Estimation of riboflavin/quinine sulphate by Fluorimetry.
REFERENCE:
PRINCIPLE: Fluorimetry (or Fuorescence spectroscopy) is an analytical technique that measures the
intensity of fluorescent light emitted by a substance when it absorbs light or electromagnetic
radiation. The principle of Fluorimetry is based on the excitation of electrons in a molecule or atom
to a higher energy state by absorbing light, followed by the emission of light as the electrons return
to their original, lower energy state. Here’s how it works:
1. Excitation: A molecule absorbs photons from an external light source (usually UV or visible
light), causing its electrons to move from a ground state (lower energy level) to an excited
state (higher energy level).
2. Energy Dissipation: The excited molecule can lose some of its energy through non-radiative
processes (such as vibrations or interactions with neighboring molecules). After losing this
excess energy, the molecule will eventually return to its ground state.
3. Fluorescence Emission: As the molecule returns to its ground state, it releases the remaining
energy in the form of emitted photons. This emitted light has a longer wavelength (lower
energy) than the absorbed light because of the energy lost in non-radiative processes. This
phenomenon is known as the Stokes shift.
4. Measurement: A fluorimeter detects and measures the intensity of the emitted fluorescent
light. The intensity of fluorescence is generally proportional to the concentration of the
fluorescent substance, allowing quantification.
Theory: In Fluorimetry, when the light of a suitable frequency falls, it is absorbed by the sample. This
leads to the excitation of electrons from the ground state to the excited electronic state.
The following three phenomena can occur to the excited electron depending upon the molecule
excited:
1. Deactivation without any emission of radiation: Excited state being relatively unstable the
molecules return to the ground state through collisional deactivation without the emission of
radiation.
2. Fluorescence: Excited molecules while returning to the ground state emit visible light photons.
3. Phosphorescence: Molecule in an excited state (relatively stable excited state) goes under
transition and returns to ground state after some time emitting visible radiation.
With the absorption of light, the fluorophores in the samples get excited moving to a state of higher
energy. The excited state being unstable the electron relaxes to its ground state with lower energy
releasing energy in the form of photons. Since the energy is lost during this process, light with lower
energy but longer wavelengths is emitted by the fluorochrome in comparison to the absorbed light
while returning to the ground state. The light emitted is called fluorescence and the detecting
instrument is known as fluorimeter.
Two types of spectrums are observed in fluorescence spectroscopy:
1. Excitation or absorption spectrum
2. Emission spectrum
Components of Fluorimetry:
1. Light Source
2. Mirror
3. Monochromator
4. Sample holder
5. Slits
6. Detector
7. Photomultiplier
8. Output device
Outline of the procedure:
Instrument Setup and Calibration
Turn on the Fluorometer: Allow the lamp or laser source to warm up if required.
Set the excitation and emission wavelengths: Select wavelengths specific to the fluorophore
being analyzed.
Optimize slit widths and sensitivity settings: Adjust settings to balance sensitivity and signal-
to-noise ratio.
Calibrate the instrument: Measure and record fluorescence for standard solutions to create a
calibration curve.
Measurement of Sample Fluorescence
Insert the sample: Place the cuvette or vial with the prepared sample in the sample holder.
Record fluorescence intensity: Measure the fluorescence emission at the pre-set
wavelengths.
Repeat as needed: Measure multiple times or with replicates to ensure accuracy and
precision.
APPLICATIONS:
Fluorimetry has nearly limitless applications. It is difficult to think about the chemical and biological
sciences without this approach today. Only a few examples of application per sector, for some of the
most popular sectors it is employed in, will be discussed in the brief review below.
Bioscience: One of the most common applications of fluorescence spectrophotometers in
biosciences is high-precision DNA and RNA measurement. Current application is single
molecule real-time (SMRT) DNA sequencing. Its ability to manufacture long-read single
molecules with great precision is projected to be essential to the next genetic diagnostic
revolution.
Biochemistry and Molecular Biology: A fluorescence spectrophotometer is critical for
understanding the structure, function, and interactions of biomolecules such as proteins,
nucleic acids, and lipids.
Pharmaceutics: Spectrofluorometric techniques are often employed in pharmaceutical
research to analyze medications. An example is the examination of co-formulated pills given
for cholesterol treatment.
Industry: A fluorescence spectrophotometer is utilized in a variety of industrial contexts to
analyze pollution quickly and non-invasively (Kohli, 2012). For instance, it has been used to
detect contaminating organic chemicals in groundwater following hydraulic fracturing for gas
exploration.
Agricultural: In agriculture, spectroscopic techniques are commonly used to identify distinct
crop kinds. The laser-induced fluorescence emission technique (LIFS) is a good tool for
identifying citrus seedling varieties.
Nanoparticle study: Fluorescence spectrophotometer is an important tool for characterizing
nanoparticles used in medication administration. It aids research into interactions between
nanoparticles and biomolecule.