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.

 

Spectrophotometry - Spectrofluorimetry Part 2

In the last post, we have discussed about the principle, theory and instrumentation of spectrofluorimetry. In this post, we will have a look at various advantages, disadvantages of spectrofluorimetry and also various factors which give rise to fluorescence.

Advantages of Spectrofluorimetry:

High Sensitivity: Spectrofluorimetry gives extremely accurate results even when samples of very low concentrations are used; even in ppm. Substances can be determined at concentrations up to 1000times lower than those required for absorption spectrophotometry. The concentrations which are as low as μg/ml or ng/ml can be determined using spectrofluorimetry. Precision upto 1% can be achieved easily using spectrofluorimeter.

Spectral Selectivity:  As we have seen in the instrumentation that two monochromators are used in spectrofluorimetry where one monochromator selects the activating wavelength while the other one selects the fluorescent wavelength. This arrangement gives great spectral selectivity to spectrofluorimeter.

Disadvantage of Spectrofluorimetry:

Quenching: What is quenching? So, quenching refers to any process which decreases the fluorescence intensity of a given substance.  There is high degree of absorption of fluorescent radiation by the emitting sample itself i.e.; there is quenching by the sample itself. Quenching can also occur because of impurities. Dissolved oxygen is a very effective quencher. And, if the sample contains dissolved oxygen, then nitrogen is bubbled through the sample to remove oxygen.  Thus, this quenching is a major drawback of spectrofluorimetry.

Factors Affecting Fluorescence Intensity:

There are certain factors that give rise or inhibits fluorescence thereby affecting fluorescence. Some of them are as follows:

Conjugation: Aromatic molecules or the molecules having multiple conjugated double bonds with a high degree of resonance stability generally fluoresce. Molecules must have π electrons. Both the groups, (i.e.; the aromatic molecules and the molecules with conjugated double bonds) possess delocalized π electrons and the higher the number of π electrons, the higher will be the fluorescence. For this reason, the polycyclic compounds are more fluorescent than the benzene derivatives.
The substituent groups do affect fluorescence by either increasing it or decreasing it. For example, the electron-donating groups like –NH2, -OH, etc. enhance fluorescence while the electron-withdrawing groups which delocalize π electron like –NO2, -COOH enhance the fluorescence.

Rigidity: The more rigid the structure of compound, the more will be the intensity of fluorescence. Also, the more the sterically uncrowded, the more will be the fluorescence. Also, chelation of aromatic compounds with metal ions promotes rigidity and reduces internal vibrations. Thus, chelation promotes fluorescence. 

Viscosity: If there is an increase in viscosity, there will be decreased collision of molecules thereby increasing fluorescent intensity. 

Temperature: Increase in temperature leads to increased collision between molecules thereby decreasing fluorescent intensity.

I hope these are clear to you. In the next post, we will have a look at the various applications of spectrofluorimetry.

Spectrophotometry - Spectrofluorimetry Part 1

In this post, we will start with the next kind of spectrophotometry which is spectrofluorimetry. Under spectrofluorimetry, we will discuss about the principle, theory and instrumentation for spectrofluorimetry, various factors which give rise to fluorescence, advantages and disadvantages of spectrofluorimetry and lastly the applications.

In this post, we will have a look at the basic principle, the theory and instrumentation for spectrofluorimeter.

Principle:

Spectrofluorimetry, as the name suggests takes the advantage of the fluorescent properties. So, before understanding about spectrofluorimetry, it is necessary to know what is fluorescence. When a molecule after absorbing radiations, emits radiation of a longer wavelength, then this phenomenon is referred to as “fluorescence.” Because of this, the compound absorbing in ultraviolet range might emit radiation in visible range. This is called Stoke’s shift wherein the shift is towards a longer wavelength. Fluorescence is an extremely short-lived phenomenon which lasts for about 10^-7 seconds or less and thus can provide information about events which take less than 10^-7seconds to occur.

After understanding the basic principle of fluorescence, we will now come to the main principle of spectrofluorimetry. As we have learnt in Chemistry, when an atom or molecule absorbs radiation, the energy of the photon absorbed lifts an electron to a higher orbital. Now, the electron needs to come down back to its ground state. It can do so in two different ways. In one way, the electron can directly return to its ground state in a single step where it will emit radiation of the same wavelength that it has absorbed. In another case, the electron can do so in a step-wise manner through intermediate energy levels and in this process, it will obviously emit quanta of radiation to each energy step. Since, each quantum will have a smaller amount of energy; the radiation emitted will have a longer wavelength than the original exciting radiation (since we know that energy is indirectly proportional to wavelength). If this happens, then the emitted light will have many different wavelengths which will correspond to each of the intermediate level which the electron will adopt on its way back to the ground state. Thus, fluorescence spectra are band spectra and they are independent of the wavelength of the radiation absorbed.

Theory:

Fluorimetry can be used as a tool for the determination of very small concentration of substances which exhibit fluorescence. Beer-Lambert law (discussed previously) can also be applied in this case of fluorimetry as:

where ε_f  is the absorptivity of the fluorescent material. C is the concentration of the substance and b is the path length, I_solvent and I_sample represents the values of intensities of the incident radiant energy and transmitted energy respectively.  The intensity of the radiation absorbed can thus be given by I_solvent - I_sample. The intensity of fluorescence is thus given by:

Instrumentation:

The major instrumentation of spectrofluorimeter differs from the spectrophotometer in two major aspects as follows:

Firstly, there are two monochromators (instead of one as is the case of spectrophotometer). These two monochromators are placed before and after the sample holder respectively.

Secondly, the sample-holder has a device to maintain the temperature as the fluorescence is maximum between 25^oC - 30^oC.

The following are the different components of spectrofluorimeter:

a. A continuous source of radiant energy (mercury lamp or xenon arc or tungsten lamp)

b. A monochromator usually a prism (P1), to choose the wavelength with which the sample is to be irradiated. 
c. Sample cell: Sample cells are cylindrical or polyhedral made up of color corrected fused glass and path length normally 10mm to 1cm.

d. A second monochromator (P2) which, placed after the sample, enables the determination of fluorescent spectrum of the sample.

e. A detector which is usually a photomultiplier or photo-voltaic cell or photo-tubes suited for wavelengths greater than 500nm and lastly

f. An amplifier

The fluorescent radiation is emitted in all directions by the sample but in most of the instruments the sample is viewed at the right angles (90^o) to the incident beam as can be seen in the diagram.

This was about the theory, concepts and instrumentation of spectrofluorimeter. In the next post, we will have a look at the various factors giving rise to fluorescence and its advantages and disadvantages.