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Spectrofluorometer is an instrument that accurately measures fluorescence from a sample. It is based on molecular emission spectroscopy.
Principle of Spectrofluorometer
Spectrofluorometer is an instrument that works on the principle of fluorescence spectroscopy. It is a rapid and sensitive method for the characterization of molecular components present in a sample. Fluorescence spectroscopy is a type of electromagnetic spectroscopy which analyzes fluorescent molecules. It exploits the phenomenon of electron excitation upon collision with high energy particles like photons and other excited electrons.
In the process of transition of polyatomic fluorescent molecules (fluorophores) from their higher energy level (excited state) to a ground state by lowering their energy level, photons are emitted. Fluorophores play the most important role in fluorescence spectroscopy. Fluorophores are the components in molecules that impart fluorescence. Mostly, fluorophores are the molecules which contain aromatic rings such as tyrosine, tryptophan, fluorescein, etc.
Applications of Spectrofluorometer
Diagnosis of Cancer in Human Tissues
Laser Induced Fluorescence (LIF) has been used for diagnosing cancer in two ways.
- The first method involves the systemic administration of a drug like a hematoporphyrin derivative (HpD) which is selectively retained by the tumor. When photo-excited with the light of an appropriate wavelength the drug localized in the tumor fluoresces. This fluorescence is used for the detection and imaging of the tumor.
- The second method involves photodynamic therapy of the tumor. Photo-excitation leads to the populating of the triplet state via an intersystem crossing. The molecule in an excited triplet state can directly react with bio-molecules or lead to the generation of singlet oxygen which is toxic to the host tissue. The resulting destruction of the host tissue is exploited for photodynamic therapy of the tumor.
Study of Marine Petroleum Pollutants
The main components of any oil are hydrocarbons. Only a few hydrocarbons fluoresce, while the majority of them show no ability to luminescence. At the same time, petroleum strongly absorbs radiation especially the ultraviolet and blue lights. Oils show fluorescence at wavelengths ranging between 270–400 nm. Hence, fluorescence spectroscopy technique is used for the determination of petroleum contaminants in seawater. It also helps in the determination of specific petroleum derivative compounds as well as the identification of pollution sources.
Accurate Determination of Glucose
In biological systems, glucose is considered a major component of animal and plant carbohydrates. Recently, bio-molecule-stabilized gold (Au) nanoclusters were demonstrated as a novel fluorescence probe for the sensitive and selective detection of glucose.
Rapid Monitoring of Freshwater Cyanobacterial Blooms
Chlorophyll analysis and integrated algal class determination by spectrofluorometer provides a realistic estimation of the abundance and dynamics of the cyanobacterial population. These are known to produce hepatotoxic heptapeptides microcystin RR and LR. A recently available submersible fluorescent probe was configured and used to analyze the vertical distribution of the deep-living toxic and filamentous cyanobacterium Planktothrix (Oscillatoria) rubescens among the autochthonous algal communities in Lake Bourget, France.
Study of the In-Vitro Evaluation of Dry Powder Inhalers
Andersen cascade impactor (ACI) was commonly used for the testing of pharmaceutical aerosols. However, this procedure is time consuming. The spectrofluorometric method is considered to be an efficient substitute for the determination of minute quantities of salbutamol after aerosolization from dry powder aerosols. This method is more accurate and less time consuming.
Other Applications
- Extensively used to analyze questioned document analysis mainly in the characterization of inks.
- Analysis of nucleic acids like DNA and proteins.
- It is used to measure inorganic compounds containing metals or ions like beryllium, lithium, aluminum, zinc, etc.
- Analyze organic compounds like steroids, proteins, alkaloids, etc.
- Extensively used in the pharmaceutical industry for the determination of morphine, quinine, indomethacin and vitamins like riboflavin, etc.
Spectrofluorometer is an excellent diagnostic and research tool which is immensely utilized in various fields of science for its high sensitivity and specificity. It is a widely accepted and powerful technique that is used for a variety of environmental, industrial, medical diagnostics, DNA sequencing, forensics, genetic analysis, and biotechnology applications. It is a valuable analytical tool for both quantitative and qualitative analysis.
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