Download Fluorescence Spectroscopy - Lecture Notes | CHM 3780 and more Study notes Analytical Chemistry in PDF only on Docsity! Fluorescence Spectroscopy - Determination of Quinine Fluorescence Spectroscopy Fluorescence is the emission of light by a molecule, which has absorbed radiant energy; the radiation is emitted at a longer wavelength (lower energy) than the incident absorbed energy. The following figure illustrates absorption transitions from the ground state to various vibrationally excited states of the upper electronic energy on the left. Refer to Figure 15-1, pp. 357 Skoog/Holler/Nieman So represents the singlet ground state (all electrons are paired). S1 refers to the lowest singlet excited state (all electrons are paired). Occasionally, excitation of an electron to a higher energy level can result in a triplet state T1 (unpaired electrons). Electrons in the excited state (with a lifetime of about 10-8 s) will drop to the lowest vibrational energy level due to collisions via vibrational deactivation or relaxation. Electrons can now return to the ground state by internal conversion, emitting heat through vibrational relaxation. This is accomplished by returning to the ground state via various vibrational energy levels. They can also return to various vibrational energy levels of So at a longer wavelength (lower file:///C|/Documents%20and%20Settings/cfjpb/My%20D...website/teaching/ia/iaprojects/molfluorescence.htm (1 of 3) [9/12/2002 2:39:39 PM] Fluorescence Spectroscopy - Determination of Quinine energy) by fluorescence. The longer wavelength is a consequence of loss of energy during vibrational deactivation. In certain cases, electrons cross over to the triplet state T1 via intersystem crossing. When they return to the various vibrational levels of So the light emitted is known as phosphorescence. Phosphorescence, a result of a "forbidden transition", has a longer lifetime (10-4 s ---> hours) than does fluorescence. Saturated molecules and molecules with only one double bond do not exhibit significant fluorescence. Molecules with at least one aromatic ring or multiple conjugated double bonds are prone to having fluorescence spectra in the visible region. Substituents such as -OH, -OCH3, and -NH2 which are electron donating groups can enhance fluorescence. Consult your textbook for a discussion of the relation between fluorescence intensity and concentration. A plot of the fluorescence intensity vs. concentration should be linear (at low concentrations). Reduction in intensity of fluorescence can be due to specific effects of constituents of the solution itself. The term quenching is used to describe any such reduction in intensity. Types of quenching include concentration quenching (a decrease in the fluorescence per unit concentration as the concentration is increased), also referred to as an inner filter effect, collisional quenching and chemical quenching. Concentration quenching results from excessive absorption of either primary or fluorescent radiation by the solution. Collisional quenching may be caused by nonradiative loss of energy from the excited molecules, and the quenching agent (such as oxygen) may facilitate conversion of the molecules from the excited singlet to triplet level. Chemical quenching is due to actual changes in the chemical nature of the fluorescent substance such as conversion of a weak acid to its anion with increasing pH. Aniline is an example. It fluoresces as the molecule between pH 5 and pH 13, below pH 5 it exists as the cation, and above pH 13 it exists as the anion; both do not fluoresce. In order to separate the emitted radiation from the incident beam (the source), fluorescence measurements are made at right angles to the incident beam. This is possible because fluorescence is emitted in all directions, but the incident radiation passes straight through the cell. Think of what would happen if file:///C|/Documents%20and%20Settings/cfjpb/My%20D...website/teaching/ia/iaprojects/molfluorescence.htm (2 of 3) [9/12/2002 2:39:39 PM]