Sunday, February 16, 2014

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.

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.

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 εis the absorptivity of the fluorescent material. C is the concentration of the substance and b is the path length, Isolvent and Isample 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 Isolvent - Isample. The intensity of fluorescence is thus given by:

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 25oC - 30oC.

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 (90o) 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.