Principle Of Fluorometry 13.pdf

[Sharolyn Uriegas]

Principle of Fluorometry

Fluorometry is a technique that measures the intensity of fluorescence emitted by a sample when it is exposed to a specific wavelength of light. Fluorescence is the phenomenon of emission of radiation when the molecules are excited by radiation at a certain wavelength. Fluorometry can be used to analyze various biological, chemical, and environmental samples that contain fluorescent molecules or can be labeled with fluorescent probes.

Principle Of Fluorometry 13.pdf

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Origin of Fluorescence

Fluorescence occurs when a molecule absorbs a photon of light and undergoes an electronic transition from the ground state to an excited state. The excited state is usually a singlet state, in which all the electrons are paired but have opposite spins. The excited state is unstable and can relax back to the ground state by various mechanisms, such as collisional deactivation, internal conversion, intersystem crossing, and fluorescence emission. Collisional deactivation and internal conversion are non-radiative processes that involve the transfer of energy to the surrounding molecules or vibrations. Intersystem crossing is a process that involves a spin flip of an electron, resulting in a transition from a singlet state to a triplet state, which has two unpaired electrons with parallel spins. Fluorescence emission is a radiative process that involves the emission of a photon of light as the molecule returns to the ground state or a lower energy singlet state. The energy difference between the absorbed and emitted photons is called the Stokes shift and is usually in the range of 10-100 nm.

Factors Affecting Fluorescence

The intensity and wavelength of fluorescence emission depend on several factors, such as:

• The quantum yield of fluorescence, which is the ratio of the number of photons emitted to the number of photons absorbed. It is always less than 1.0 since some energy is lost by non-radiative pathways.

• The concentration of the fluorescent species, which is proportional to the fluorescence intensity only when the absorbance is less than 0.02.

• The intensity of incident light, which is proportional to the fluorescence intensity.

• The absorption spectrum of the fluorescent species, which determines the wavelength of excitation light that can induce fluorescence.

• The emission spectrum of the fluorescent species, which determines the wavelength range of fluorescence emission.

• The oxygen concentration, which can quench fluorescence by collisional deactivation or intersystem crossing.

• The pH, which can affect the protonation or deprotonation of fluorescent molecules and alter their absorption and emission properties.

• The temperature and viscosity, which can affect the rate of collisional deactivation and intersystem crossing.

• The photodecomposition, which can reduce the fluorescence intensity by breaking down the fluorescent molecules.

• The quenchers, which are molecules that can interact with the fluorescent species and reduce their fluorescence intensity by various mechanisms, such as static quenching, dynamic quenching, energy transfer quenching, or chemical quenching.

• The scatter, which is the reflection or refraction of light by particles or impurities in the sample that can interfere with the fluorescence measurement.

Instrumentation for Fluorometry

A typical fluorometer consists of four main components: a source, a wavelength selector, a sample holder, and a detector. The source provides a beam of light that can excite the sample. The wavelength selector filters out unwanted wavelengths and selects a specific wavelength or range of wavelengths for excitation or emission. The sample holder contains the sample solution or solid that emits fluorescence. The detector measures the intensity of fluorescence emission at a specific wavelength or range of wavelengths. There are two types of fluorometers: filter fluorometers and spectrofluorometers. Filter fluorometers use filters to select wavelengths for excitation and emission. They are simple, inexpensive, and suitable for routine analysis. Spectrofluorometers use monochromators to select wavelengths for excitation and emission. They are more complex, expensive, and versatile than filter fluorometers. They can measure fluorescence spectra by scanning different wavelengths for excitation or emission.

Applications of Fluorometry

Fluorometry has many applications in various fields, such as:

• Biochemistry and molecular biology: Fluorometry can be used to measure biomolecules such as proteins, nucleic acids, enzymes, hormones, vitamins, and drugs that are naturally fluorescent or can be labeled with fluorescent probes. Fluorometry can also be used to study the structure, function, and interactions of biomolecules by monitoring changes in fluorescence intensity, wavelength, lifetime, polarization, or anisotropy.

• Microbiology and immunology: Fluorometry can be used to detect and quantify microorganisms such as bacteria, viruses, fungi, and parasites that can be stained or tagged with fluorescent dyes or antibodies. Fluorometry can also be used to measure the immune response of cells or tissues by using fluorescent markers or indicators.

• Clinical chemistry and medicine: Fluorometry can be used to diagnose diseases and monitor the health status of patients by measuring the levels of fluorescent biomarkers or metabolites in biological fluids such as blood, urine, saliva, or cerebrospinal fluid. Fluorometry can also be used to assess the efficacy and toxicity of drugs by measuring their fluorescence in vivo or in vitro.

• Environmental science and engineering: Fluorometry can be used to monitor the quality and safety of water, air, soil, and food by measuring the presence and concentration of fluorescent pollutants or contaminants such as pesticides, heavy metals, organic compounds, or microorganisms. Fluorometry can also be used to detect and quantify the biodegradation or bioremediation of pollutants by measuring the fluorescence of microbial enzymes or products.

References:

• [FLUORIMETRY - Centurion University]

• [UNIT 5 FLUORIMETRY AND PHOSPHORIMETRY - COPBELA]

• [Principles of fluorimetry by ANN PPT - SlideShare]

• [Fluorescence spectroscopy and its applications: A Review]

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