FACTORS AFFECTING FLUORESCENCE
INTENSITY:
• Presented by:
• Under Guidance of: 05. Yashashree Bakare
Dr. Harshad P. Khade,
06. Punam Bandapatte
Assistant Professor, ADCBP,
Ashta, Pharmaceutical 07. Deepak Bhosale
Chemistry Department. 08. Megha Bhosale
CONTENT
❖FACTORS AFFECTING FLUORESCENCE INTENSITY:
1. Structural rigidity
2. Nature of molecules
3. Nature of substituents
4. Adsorption
5. Light
6. Methods of illumination
7. Oxygen
8. pH
9. Photodecomposition
1. Structural rigidity:
➢ Experiments show that fluorescence is particularly favored in
rigid molecules.
➢ The difference in behavior is a result of the increased rigidity
provided by the bridging methylene group in fluorene.
➢ This rigidity lowers the rate of nonradiative relaxation to the
point where relaxation by fluorescence has time to occur.
➢ The influence of rigidity also explains the increase in
fluorescence of certain organic chelating agents.
2. Nature of molecules:
➢ All molecules cannot show the phenomena of fluorescence
and phosphorescence.
➢ Only such molecules show these phenomena that are able to
absorb UV or visible radiation.
➢ In general, the greater the absorbancy of a molecule, the more
intence its luminescence.
➢ This requirement means that molecules having conjugated
double bonds (𝜋-bonds) are particularly suitable for this study.
3. Nature of substituents:
➢ Substituents often exhibit a marked effect on the fluorescence
and phosphorescence of molecules. There are no rigid rules but a
few generalities may be useful. These are as follows :
i. Electron-donating groups like –NH2 and –OH often enhance
fluorescence.
ii. Electron-withdrawing groups like –COOH, -NO2, -N=N- and
halides decrease or even destroy fluorescence.
iii. If a high-atomic-number atom is introduced into a 𝜋-electron
system, it enhances phosphorescence and decreases
fluorescence.
4. Adsorption:
➢ The extreme sensitivities of the method requires very dilute
solutions, 10-100 times weaker than those employed in
spectrophotometry.
➢ Adsorption of the fluorescent substance on the container walls
may therefore present a serious problem, and strong stock
solutions must be kept and diluted as required.
➢ Ex. Quinine.
5. Light:
➢ Monochromatic light is essentially for the excitation of
fluorescence in quantitative work.
➢ The purity of the irradiating beam especially if obtained via
filters should, therefore, be checked by examination of the light
scattered by a slightly turbid solution.
➢ The trace obtained should show one peak only, corresponding in
wavelength to that expected.
6. Methods of illumination:
➢ The method of illumination described for the Spekker
Fluorimeter is known as the right-angle method.
➢ It has the advantage of giving a smaller blank value for scattered
light and fluorescence from container walls than that given by the
alternative method of frontal illumination.
➢ The frontal method must be adopted for opaque solutions and
for solids.
➢ It can be used for both dilute and concentrated solutions, but
inner-filter effects with consequent distortion of spectra also
occur with this method.
7. Oxygen:
➢ The presence of oxygen may interfere in two ways: by direct
oxidation of the fluorescent substance to non-fluorescent
products, or by quenching of fluorescence.
➢ It is a useful precaution, therefore, to check the de-aerated
solution and compare the result obtained with that from the
oxygen-containing solution.
➢ Anthracence is well known to be susceptible to the presence of
oxygen.
8. pH:
➢ It is to be expected that alteration of the pH of a solution will
have a significant effect on fluorescence if the absorption curve of
the solute is changed.
➢ Many phenols, for example, are fluorescent in both dissociated
and undissociated forms.
➢ Consequently, the fluorescence from a solution of the phenol
will show two peaks.
9. Photodecomposition:
➢ In spectrophotometry, the intensity of the radiation passing
through solutions is weak by photochemical standards although
adequate for measurements: decomposition of solute is, therefore,
not very likely.
➢ Spectrofluorimetry, on the other hand, requires high intensity
illumination for irradiation and the risk of photochemical change
is thereby increased.
➢ Parker and Barnes (1957) have calculated for a hypothetical
case involving quantities to be expected in normal practice.
➢ It may be possible in unfavourable cases to select radiation of a
wavelength which is not strongly absorbed so that the extent of
photochemical change is reduced, whilst at the same time
adequate sensitivity is retained.
SUMMARY
Photodecomposition Structural rigidity
pH Nature of molecules
Factors
Oxygen affecting Nature of substituents
fluorescence
Methods of intensity Adsorption
illumination
Light
❖ Douglas A. Skoog, Donald M. West, F. James Holler, Stanley
R. Crouch, Fundamentals of Analytical Chemistry, CENGAGE
Learning, 9th ed., 2014, P.N.763 and 764.
❖ Gurdeep R. Chatwal, Sham K. Anand, Instrumental Methods
of CHEMICAL ANALYSIS, Himalaya Publishing House PVT.
LTD., 5th ed., 2017, P.N.2.401-2.404.
