Tuesday, February 25, 2014

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.