Fluorescence is important due to its inherent sensitivity which can be several orders of magnitude more sensitive than absorption methods.  Another reason which adds to its importance is the specificity of fluorescence methods since, relatively, small margin of molecules fluoresce.


Origin of Fluorescence


      When radiation of an appropriate wavelength is used to irradiate molecules in a sample, certain electronic transitions take place.  As excited molecules return to the ground state they emit radiation of longer wavelength.  The emitted radiation is referred to as fluorescence.  Figure 1 shows a description of absorption and emission phenomena and energy levels associated with these electronic transitions.



      Figure 1:  Description of fluorescence origin. F, fluorescence; P, phosphorescence; S, singlet; T, triplet; RD, radiationless deactivation; VR, vibrational relaxation; ISC, intersystem crossing; and E, energy.


      Electrons in the ground state (So) absorb energy from incident radiation and are excited to S1 or S2 excited states.  Vibrational relaxation (VR) results in the placement of the electron at the S1, main electronic level.  Vibrational relaxation is a radiationless deactivation process where excess energy is consumed as kinetic energy or heat.  A second collisional deactivation process can take place and the electron returns to the ground state by a radiationless deactivation (RD) process to So.  The other possibility involves direct transition of the electron from the S1 excited state to So ground state and excess energy is emitted as photons at specific wavelengths called fluorescence.


           Electrons in the excited state can follow a third approach by changing their spin and transferring to the triplet state through a process known as intersystem crossing.  The path of the excited electron depends on several factors which will be mentioned shortly.  An electron in the triplet state will surely exhibit a vibrational relaxation to the main triplet energy level.  A second flip in spin is necessary for such an electron to transfer to the ground state by one of two mechanisms.  The first involves emitting a photon at a specific wavelength (phosphorescence) or radiationless deactivation. 

      Some molecules show a different behavior where an electron in the triplet state can experience a flip in spin and return to the first excited singlet state followed by vibrational relaxation then either radiationless deactivation or fluorescence.  Fluorescence of this type can be observed after some time and is referred to as delayed fluorescence.  It should be noticed that the life time of an electron in the excited state is about 10-9 second which is the time needed to observe fluorescence.  Phosphorescence or delayed fluorescence can be observed after some time, usually exceeding 10-4 second.  As all processes of deactivation and phosphorescence are possible, precautions should be considered in order to inforce circumstances that prefer fluorescence route.


Effect of Light Intensity and Concentration


           The first step in a fluorescence process is the absorption of incident radiation in order for electrons to transfer to the excited singlet state.  Therefore, it can be assumed that fluorescence is proportional to amount of incident beam absorbed, or

           F = K (Po - P)                                                                 (1)

      where    F   is fluorescence intensity.  Po and P are intensities of incident and transmitted beams.


      From beer's law



      P/Po =  10-A        or  P =  Po X10-A                                       (2)


      By substitution in equation 1


      F = KPo (1 - 10-A)                                                                (3)


      By expanding the exponential term and assuming that            A < 0.05


      F = K1 Po ebC                                                                    (4)

      At constant Po, e and b

      F = K" C                                                                             (5)


      This means that fluorescence intensity is directly proportional to concentration.  Another important result can be implied from equation 4 where fluorescence is shown to be directly proportional to the intensity of the incident beam.  This suggests that a very intense light source is necessary for fluorescence instrumentation.  Also substances of large e are potential fluorescent molecules and should be sough for better results.


Excitation and Emission Spectra


           It can easily be understood that fluorescence will always occur at wavelengths longer than excitation wavelength.  This can be seen from Figure 1 as the energy of the emitted photon is less than that of the absorbed photon.  Another observation which should be mentioned here is that 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. Figure 2 shows relative locations of an excitation and emission spectra




      Figure 2:  Relative locations of excitation, emission (Fluorescence and phosphorescence) spectra.


           The most important transition that contribute to good fluorescence characteristics is the  p - p* transition since e for this transition is the largest.  The n - p* transition is damaging to fluorescence because it facilitate and increase the possibility of intersystem crossing.




      A schematic of a typical fluorometer is shown in Figure 3 .



      Figure 3:  A schematic diagram of a typical filter fluorometer. L, source; F1, F2 are excitation and emission filters, respectively; S, sample cell; D, detector; and M, mirror.


           This type of fluorometer is a cheap one. In more expensive instruments, gratings can be used instead of filters which give an excellent spectrofluorometer.  The first wavelength selector (F1) transmits the excitation wavelength while the second (F2) transmits the emitted fluorescence at the emission wavelength.


      It should also be noted that the emitted radiation is measured at 90o from the incident beam.  This is because it is necessary to exclude all incident radiation since only emitted radiation is important.  Another reason is that minimum noise will be encountered when measurement is done at 90o in order to decrease scattering, which is minimum at this configuration.


Quantum Efficiency


           The Quantum efficiency (f) is defined as the ratio of the number of fluorescing molecules to the number of excited molecules.



       f =  ___________________

                  Kf + Kisc + KVR + Kx


      Where Kf, Kisc, KVR and Kx are rate constants for fluorescence, intersystem crossing, vibrational relaxation, and any other deactivation process, respectively.  As   f becomes close to 1, the system is considered an efficient fluorescing system.  Conditions should be adjusted to increase the quantum efficiency.



Factors Affecting Fluorescence Efficiency


           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.  The most important factors are summarized below:


      1.    p  System


      As e for p - p* transition is maximum, it is advantageous that a fluorescer contain p  system and preferably an aromatic ring (s).




      2.  Structural Rigidity


      Collisional deactivation is a major fluorescence quenching mechanism.  Therefore, molecules possessing rigid structures are better fluorescers than others which lack rigidity. This explains why fluorene is an excellent fluorescer while biphenyl is a weak one.


      3.  Solvent Polarity


      Polar solvents increase fluorescence efficiency since the energy for  p - p*   transition is lowered and may become less than  n - p*  transition leading to increased absorption, and thus emission.


      4.  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.


      5.  Nonbonding Electrons


      Molecules that contain lone electron pairs (nonbonding electrons) tend to be weaker fluorescers.  This is because n electrons increase intersystem crossing and thus decrease fluorescence.


      6.  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.


      7.  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.


      8.  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.


Performing a Measurement


      The first pieces of information necessary are the excitation and emission wavelengths.  The excitation and emission wavelength selectors should be adjusted to these wavelengths before running a measurement.  If they are unknown the following procedure should be followed:


      a. Excitation Wavelength Selection


      Adjust the emission monochromotor to some certain wavelength and scan the excitation monochromator.  This gives the excitation spectrum.  Choose the wavelength that yield maximum signal and use as the excitation wavelength.


      b. Emission Wavelength Selection


      Adjust the excitation monochromator to the wavelength that gives maximum signal (obtained in a) and scan the emission wavelength.  The resulting spectrum is the emission spectrum and the emission monochromator should be adjusted to the wavelength that yields maximum signal.  This is the emission wavelength.




           Some reactions yield luminescence without external light excitation, instead chemical energy is utilized for the excitation of the luminescent compounds.  As the excited compound returns to ground state, it emits radiation that can be measured using a sensitive photomultiplier tube.  Chemiluminescence is one of the most sensitive spectroscopic analytical methods.  Reports show that detection limits in the nano M levels are routine while femto molar concentrations are possible.  Measurements are also performed against zero background which adds to the good features of this technique.  However, cautious work and addition of exact volumes of reagents are vital.









Experiment 8 : Molecular Fluorescence: A Study of Variables




           Molecular fluorescence is an important analytical technique which is used for the determination of some analytes in the sub micromolar range. Due to 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, in many cases, may be 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.


Chemicals and Reagents


      a. Provided


      1. Solid sample of dichlorofluorescein (DCF).

      2. 0.05 M Buffer solutions covering the range from pH 2-10.

      3. 0.01 M Sodium dodecylsulfate (SDS).

      4. Cetyltrimethylammonium chloride (CTAC), saturated solution diluted 1:1 with distilled water.

      5. 1 M Potassium bromide.

      6. 1 M Potassium iodide.

      7. Solid phenolphthalein.


      b. Need Preparation


      1. 250 mL, of 200 ppm DCF solution.

      2. 25 mL of 25, 50, and 100 ppm DCF, by dilution of solution 1.

      3. 10 ppm DCF solutions in buffers pH 2-10.

      4. You will be asked to perform other preparations through the experimental section.




      1. Fluorometer or spectrofluorometer.

      2. Transmission filters of different colors.

      3. pH Meter.

      4. Spectrophotometer.

      5. Glass or quartz cells (standard 1 cm path length).




I. pH Effects on Fluorescence Intensity


      1. Obtain the absorption spectrum of the 50 ppm DCF solution and determine the excitation wavelength from the graph.

      2. Adjust the excitation monochromator of the spectrofluorometer at the maximum wavelength obtained in step 1. Fix the excitation monochromator at this wavelength throughout the rest of this experiment.

      3. Find the emission wavelength by scanning the emission grating (or changing filters) and select the wavelength which yields maximum emission and record the value of the relative fluorescence intensity for each of the solutions that are 10 ppm in DCF in the different buffer solutions from pH 2-10.

      4. Tabulate your results and comment on conclusions.


II. Phenolphthalein versus DCF Fluorescence


      1. Prepare 250 mL of a 100 ppm phenolphthalein solution in the least amount of ethanol and dilute to volume with distilled water.

      2. Find the excitation wavelength of phenolphthalein and record the absorption spectrum.

      3. Prepare a series of phenolphthalein indicator solutions (10 ppm) in buffer solutions, pH 2-10.

      4. Holding the excitation wavelength constant, find the emission wavelength for each of the solutions in step 3.

      5. Draw the structures of DCF and phenolphthalein and compare between the results obtained for each species and relate differences to the difference in the two structures.


III. Effects of Heavy Atoms on Fluorescence


      1. Prepare 50 mL of 10 ppm DCF by dilution of the 200 ppm stock solution, add 1 mL of KI and adjust to volume using the buffer which yielded the maximum fluorescence intensity.

      2. Measure the fluorescence intensity and compare to solutions prepared as in step 1 but without KI.

      3. Repeat steps 1 and 2 but use KBr instead of KI.

      4. Report your conclusions on the effect of iodide and bromide on the fluorescence intensity, explain.


IV. Effect of Surfactants on the Fluorescence Intensity


      1. Prepare 100 mL of a 10 ppm solution of DCF by dilution of the 200 ppm stock solution, add 50 mL of the buffer at optimum pH and adjust to volume with distilled water.

      2. Repeat step 1 but adjust to volume with the SDS solution provided.

      3. Repeat step 1 but adjust to volume with the CTAC solution provided.

      4. Find the fluorescence intensity of each of the previous solutions.

      5. Report your results and give detailed explanations on how the presence of surfactants affects the fluorescence intensity.


Experiment 9: Fluorometric Determination of Salicylic Acid




           Salicylic acid may be determined by fluorometry in acidic solution where efficient fluorescence of this compound is observed. The compound is excited in the UV region while fluorescence occur in the visible region. The intensity of the fluorescence signal may be related to the concentration of salicylic acid. This method can be of significant importance in the determination of the extent of hydrolysis of acetylsalicylic acid (Aspirin) due to aging or artifacts in preparation methodology.


Chemicals and Reagents


      a. Provided


      1. A sample of unknown salicylic acid concentration.

      2. Predried and desiccated salicylic acid (analytical grade).

      3. Glacial acetic acid.

      4. Chloroform.


      b. Need Preparation


      1. 1 % (V/V) Glacial acetic acid, 250 mL.

      2. 0.1 % (W/V) Salicylic acid in solution 1, 25 mL.

      3.  1 mL of solution 2 is diluted to 25 mL using solution 1.




      1. Fluorometer or spectrofluorometer.

      2. Suitable filters (when a fluorometer is used).

      3. UV-Vis Spectrophotometer.




      1. Accurately transfer 0.5, 1, 2, 3, 4, 6, and 8 mL of solution 3 into seven volumetric flasks (25 mL each). Adjust the final volume to the mark using solution 1.

      2. Record the absorption spectrum of salicylic acid in solution 3 and determine the excitation wavelength.

      3. Adjust the fluorometer or spectrofluorometer at the excitation wavelength and scan the emission monochromator (or change filters) and determine the emission wavelength.

      4. Adjust the instrument at best settings of excitation and emission wavelengths and measure the fluorescence signals of solutions prepared in step 1 of this section.

      5. Determine the fluorescence intensity of the unknown, keeping all settings in step 4 constant. If the resulting value is off the range in step 4 make necessary dilutions using the solvent mixture.

      6. Construct a calibration plot between fluorescence intensity and mg salicylic acid / 100 mL.

      7. Report your results as mg salicylic acid /dl from the calibration plot.