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 εf is
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:
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 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.
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.
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.
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