Tuesday, March 18, 2014

Spectrophotometry - Spectrofluorimetry Part 3

Till now we have seen about the principle, theory and concepts and instrumentation of spectrofluorimetry; advantages and disadvantages of spectrofluorimetry and various factors giving rise to fluorescence. Now, coming to the last topic in spectrofluorimetry which are its applications. Following are some of the applications of spectrofluorimetry:
Studies on Protein Structure:
A lot of information regarding the structure of the protein can be determined with the help of fluorescent studies. The fluorescence spectra may change depending on the position of amino acids in the protein, the composition of active site, protein denaturation etc.

Qualitative Analysis:
The identity of the compound can be determined by comparing the fluorescence spectra and the absorption spectra of the particular compound.
Compounds which are fluorescent are readily determined with simple instruments and here the solution for examination is normally obtained by dissolving the sample in a suitable solvent.
Certain substances which are in themselves non-fluorescent may be determined as a result of a chemical change. This method can be used for both inorganic and organic compounds. For example, determination of primary and secondary aliphatic amines through the reaction with NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-I,3-diazole which gives yellow fluorescence.

Quantitative Analysis:
This includes the determination of the concentration of various vitamins, hormones, drugs etc. For example, the quantitative analysis includes assay of vitamins like thiamine, riboflavin; hormones such as cortisol, estrogen, serotonin, dopamine and drugs such as lysergic acid and barbiturates.

Intracellular Free Calcium Concentration Assay:
The three probes which allow us to perform the assay for free calcium concentrations are Quin-2, Quin-2 AM and Flura-2. These probes are permeable and they enter into the plasma membrane.

Fluorescent Microscopy:
When spectrofluorimeter is combined with a microscope, it allows the determination of subcellular location of fluorescent compounds or of materials which can bind to the fluorescent dyes. This technique is important especially in the field of pharmacology and immunology. Thus, an antibody can detect the fluorescent labelled antigen present on the surface of the cell. For example, this technique allows the visualization of nucleic acids within subcellular organelles with the help of acridine orange dye.

Assay of Membrane Potential:
There occur changes in membrane potential which regulate entry of ion into the cells. These membrane potential changes can be monitored by using some of the fluorescent probes.

Studies on Membrane Structure:
There are various fluorescent probes such as ANS (anilinonapthalene 8-sulphonate) and N-methyl-2-anilino-6-naphthalene sulphonate (MNS). They both contain charged and hydrophobic areas and therefore are situated at the water-lipid interface of the membrane. The fluorescent properties of the molecule vary with its mobility and also with the polarity of the environment. Studies with the ANS probe has shown that structural changes occur in mitochondrial membrane during oxidative phosphorylation. These probes have also helped in giving information about the structural features of the plasma membrane. 

Thus, by this post, we complete the topic of spectrofluorimetry. In the next post, we will start with another type of spectrophotometry.