Molecular Fluorescence Spectroscopy Chapter 27 Molecular Fluorescence Spectroscopy

27 A Theory of molecular fluorescence Molecular fluorescence is measured by exciting the sample at an absorption wave- length, also called the excitation wavelength, and measuring the emission at a longer wavelength called the emission or fluorescence wavelength. Usually photoluminescence emission is measured at right angles to the incident beam to avoid measuring the incident radiation. The short-lived emission that occurs is called fluorescence, while luminescence that is much longer lasting is called phosphorescence. Fluorescence emission occurs in 10-5 s or less. In contrast, phosphorescence may last for several minutes or even hours.

Once the molecule is excited to E1 or E2, several processes can occur that cause the molecule to lose its excess energy. Two of the most important of these processes, nonradiative relaxation and fluorescence emission. The two most important nonradiative relaxation methods are: Vibrational relaxation involves transfer of the excess energy of a vibrationally excited species to molecules of the solvent. This process takes place in less than 10-15 s and leaves the molecules in the lowest vibrational state of an electronic excited state. Internal conversion is a type of relaxation that involves transfer of the excess energy of a species in the lowest vibrational level of an excited electronic state to solvent molecules and conversion of the excited species to a lower electronic state.

Molecular fluorescence bands are mostly made up of lines that are longer in wavelength, higher in frequency, and thus lower in energy than the band of absorbed radiation responsible for their excitation. This shift to longer wavelength is called the Stoke’s shift. Relationship between Excitation Spectra and Fluorescence Spectra

All absorbing molecules have the potential to fluoresce, but most compounds do not because their structure allows radiationless pathways for relaxation to occur at a greater rate than fluorescence emission. The quantum yield of molecular fluorescence is simply the ratio of the number of molecules that fluoresce to the total number of excited molecules, or the ratio of photons emitted to photons absorbed. Highly fluorescent molecules have quantum efficiencies that approach unity under some conditions.

Quantum efficiency is described by the quantum yield of fluorescence, F. where kF is the first-order rate constant for fluorescence relaxation and knr is the rate constant for radiationless relaxation.

Compounds containing aromatic rings give the most intense and most useful molecular fluorescence emission. While certain aliphatic and alicyclic carbonyl compounds as well as highly conjugate double-bonded structures also fluoresce. The simplest heterocyclics, such as pyridine, furan, thiophene, and pyrrole, do not exhibit molecular fluorescence.

Fused-ring structures containing often do fluoresce. Substitution on an aromatic ring causes shifts in the wavelength of absorption maxima and corresponding changes in the fluorescence bands. In addition, substitution frequently affects the fluorescence efficiency.

The Effect of Structural Rigidity Fluorescence is particularly favored in rigid molecules. Rigidity lowers the rate of nonradiative relaxation to the point where relaxation by fluorescence has time to occur.

Temperature and Solvent Effects In most molecules, the quantum efficiency of fluorescence decreases with increasing temperature. The increased frequency of collision at elevated temperatures increases the probability of collisional relaxation.

27 B Effect of concentration on fluorescence intensity The radiant power of fluorescence emitted F is proportional to the radiant power of the excitation beam absorbed by the system: F = K’(P0 – P) P0 is the radiant power of the beam incident on the solution and P is its power after it passes through a length b of the medium. To relate F to the concentration c of the fluorescing particle, Beer’s law can be written as P/P0 = 10-bc where  is the molar absorptivity of the fluorescing species and bc is the absorbance.

Substitution will result in F = K’P0(1 – 10-bc) Expansion of the exponential term leads to A plot of the fluorescence power emitted versus the concentration of the emitting species should be linear at low concentrations. When c becomes large enough that the absorbance exceeds about 0.05, the relationship becomes nonlinear.

At very high concentrations, F reaches a maximum and may even begin to decrease with increasing concentration because of secondary absorption. This phenomenon occurs because of absorption of the emitted radiation by other analyte molecules.

27 C Fluorescence instrumentation

In fluorescence, the radiant power emitted is directly proportional to the source intensity, but absorbance is essentially independent of source intensity: c = kA = k log (P0/P) 27 D Applications of fluorescence methods 1. Fluorescence has proved to be a valuable tool in oil spill identification. The source of an oil spill can often be identified by comparing the fluorescence emission spectrum of the spill sample to that of a suspected source. 2. Fluorescence methods are used to study chemical equilibria and kinetics in much the same way as absorption spectrophotometry. 3. Quantitative fluorescence methods have been developed for inorganic, organic, and biochemical species.

Inorganic fluorescence methods can be divided into two classes: direct methods and indirect methods. Direct methods are based on the reaction of the analyte with a complexing agent to form a fluorescent complex. Indirect methods depend on the decrease in fluorescence, also called quenching, as a result of interaction of the analyte with a fluorescent reagent. Quenching methods are primarily used for the determination of anions and dissolved oxygen.

Methods for Organic and Biochemical Species

27 E Molecular phosphorescence spectroscopy Phosphorescence is a photoluminescence phenomenon. Ordinary molecules that are not free radicals exist in the ground state with their electron spins paired. A molecular electronic state in which all electron spins are paired is said to be a singlet state. The ground state of a free radical is a doublet state because the odd electron can assume two orientations in a magnetic field. In the triplet state, the spins of the two electrons become unpaired and are thus parallel.

27 F Chemiluminescence methods Chemiluminescence is produced when a chemical reaction yields an electronically excited molecule, which emits light as it returns to the ground state. Chemiluminescence reactions occur in a number of biological systems, where the process is often termed, bioluminescence. Chemiluminescence methods are known for their high sensitivities. Typical detection limits range from parts per million to parts per billion or lower. Applications include the determination of gases, such as oxides of nitrogen, ozone, and sulfur compounds; determination of inorganic species, such as hydrogen peroxide and some metal ions; immunoassay techniques; DNA probe assays; and polymerase chain reaction methods.