Question

2. a) State and describe any three factors that may affect positively or negatively the analytical

         utility of molecular fluorescence techniques?   

       b) Why does the fluorescence excitation spectrum appear to be a mirror image of fluorescence

              emission spectrum? Why are they not exact mirror images?   

c) What does synchronous fluorescence spectroscopy involve? What kind of spectra is

     obtainable using this technique? Are there any advantages of using synchronous   

     spectrometry in fluorescence techniques?

Answer 1

a) State and describe any three factors that may affect positively or negatively the analytical utility of molecular fluorescence techniques?

Molecular fluorescence is an important analytical technique used for the determination of some analytes in the sub micromolar range. Because of the extreme sensitivity of the technique, interferences, even in very low concentrations, may turn a specific method useless. For example, heavy atoms and oxygen may effectively quench fluorescence. Temperature, viscosity of the solution, molecular structure, and concentration of the fluorescing species are all important factors that should be considered when performing a fluorometric determination. The pH  of the solution is of extreme importance, especially when the structure of the fluorescing species may assume anionic, cationic, or neutral configuration with different luminescence characteristics of each species at different pH values.

There are several factors that contribute to fluorescence efficiency. Some of these factors can be controlled and optimized by the analyst while others are inherent and can be used to explain emission behavior:

• Temperature: As temperature is increased, the translational, rotational and vibrational motions of molecules increase.  This increases the possibilities of collisions and lead to collisional deactivation and quenching of fluorescence.  Therefore, it is always wise to conduct fluorometric measurements at low temperatures.
• Dissolved oxygen and heavy metals: Molecular oxygen is paramagnetic which increases intersystem crossing through triplet - triplet interaction.  Oxygen is thus a good fluorescence quencher and is sometimes determined by its quenching characteristics. Heavy metals also increase intersystem crossing leading to decreased fluorescence.  This is most obvious with paramagnetic heavy metals.
• pH: Usually, fluorescers that contain either acidic or basic moieties have fluorescence quantum yields dependent on pH.  The pH of these substances should be adjusted so that maximum fluorescence is obtained.
• Viscosity: Collisional deactivation can significantly be decreased in viscous systems which result in better quantum efficiencies. Therefore, it is a good practice to add some viscosity modifiers, especially chemical surfactants. Excellent fluorescence efficiencies were obtained when appropriate concentrations of surfactants were used.

b) Why does the fluorescence excitation spectrum appear to be a mirror image of fluorescenceemission spectrum? Why are they not exact mirror images?

Fluorescence will always occur at wavelengths longer than excitation wavelength, as the energy of the emitted photon is less than that of the absorbed photon. Since the emission of radiation is just the reverse of absorption (excitation) it is expected that the emission spectrum should be a mirror image of the excitation spectrum.  This is theoretically correct but it is seldom the case due to instrumental artifacts.

c) What does synchronous fluorescence spectroscopy involve? What kind of spectra is obtainable using this technique? Are there any advantages of using synchronous spectrometry in fluorescence techniques?

Experiments in synchronous fluorescence spectroscopy can be used to compare the detection limits afforded by UV/visible absorption and fluorescence spectroscopy for a given analysis and to show that the latter method is capable of of greater selectivity.

In excitation spectrum the sample is excited with different wavelengths (Ex - not constant) and emission intensity is recorded at one chosen wavelength (Em - constant). Emission spectra measurement is oposite (Ex - const, Em- not constant).

Synchrronous spectra joins in some extent both Excitation and Emission. Emission and excitation monochromators moves together wIth constant wavelengths interval. After measurement one point they move togeather to the next etc.

There are advantages of using synchronous spectrometry in fluorescence techniques because total synchronous fluorescence spectroscopy is more sensitive than molecular fluorescence spectroscopy which requires further sample preparation steps. Studies have been favoring x-ray fluorescence spectrometry, in recent years, since it is quick, requires minimal sample handling and suitable for both quantitative and semi-quantitative analysis.