chapterFIFTEEN Molecular Luminescence Spectrometry hree related types of optical methods Fluorescence and phosphorescence are alike in that excitation is are considered in this chapter: molecu- brought about by absorption of photons. As a consequence, the two phenomena are often referred to by the more general term lar fluorescence, phosphorescence, and T photoluminescence. As will be shown in this chapter, fluores- chemiluminescence. In each of these methods, cence differs from phosphorescence in that the electronic energy molecules of the analyte are excited to produce transitions responsible for fluorescence do not involve a change a species whose emission spectrum provides in electron spin. Because of this, the excited states involved in 25 information for qualitative or quantitative analysis. fluorescence are short-lived (,10 s). In contrast, a change in The methods are known collectively as molecular electron spin accompanies phosphorescence, and the lifetimes of the excited states are much longer, often on the order of seconds luminescence procedures. or even minutes. In most instances, photoluminescence, be it fluorescence or phosphorescence, occurs at wavelengths longer than that of the excitation radiation. The third type of luminescence, chemiluminescence, is based on the emission of radiation by an excited species formed during a chemical reaction. In some instances, the excited species is the product of a reaction between the analyte and a suitable reagent (usually a strong oxidant such as ozone or hydrogen peroxide). The result is an emission spectrum characteristic of the oxidation product of the analyte or the reagent rather than the analyte itself. In other instances, the analyte is not directly involved in the chemiluminescence reaction. Instead, the ana- lyte inhibits or has a catalytic effect on a chemiluminescence reaction. Measurement of the intensity of photoluminescence or chemiluminescence permits the quantitative determination of a variety of important inorganic and organic species in trace amounts. Currently, the number of fluorometric methods is far greater than the number of applications of phosphorescence and chemiluminescence procedures. One of the most attractive features of luminescence methods is their inherent sensitivity, with detection limits that are often one to three orders of magnitude lower than those encountered in absorption spectroscopy. In fact, for selected species under controlled conditions, single molecules have been detected by fluorescence spectroscopy. Another advantage of photolumines- cence methods is their large linear concentration ranges, which Throughout this chapter, this logo indicates also are often significantly greater than those encountered in absorption methods. Because excited states are quite suscepti- an opportunity for online self-study at www.tinyurl.com/skoogpia7, linking you to ble to being deactivated by collisions and other processes, many interactive tutorials, simulations, and exercises. molecules do not fluoresce or phosphoresce at all. Because of 361 362 Chapter 15 Molecular Luminescence Spectrometry such deactivation processes, quantitative luminescence methods restriction requires that no more than two electrons occupy an are often subject to serious interference effects. For this reason orbital and furthermore the two have opposed spin states. Under luminescence measurements are often combined with such this circumstance, the spins are said to be paired. Because of spin separation techniques as chromatography and electrophoresis. pairing, most molecules exhibit no net magnetic field and are Fluorescence detectors are particularly valuable as detectors for thus said to be diamagnetic—that is, they are neither attracted liquid chromatography (Chapter 28) and capillary electrophore- nor repelled by static magnetic fields. In contrast, free radicals, sis (Chapter 30). which contain unpaired electrons, have a magnetic moment and Generally, luminescence methods are less widely applicable consequently are attracted by a magnetic field. Free radicals are for quantitative analyses than are absorption methods because thus said to be paramagnetic. many more species absorb ultraviolet (UV) and visible radiation Singlet and Triplet Excited States than exhibit photoluminescence when radiation is absorbed in A molecular electronic state in which all electron spins are this region of the spectrum.1 paired is called a singlet state, and no splitting of electronic energy levels occurs when the molecule is exposed to a magnetic field. The ground state for a free radical, on the other hand, is a 15A Theory of fLuoreSCenCe doublet state because there are two possible orientations for the and PhoSPhoreSCenCe odd electron in a magnetic field, and each imparts slightly dif- ferent energies to the system. Fluorescence occurs in simple as well as in complex gaseous, When one of a pair of electrons of a molecule is excited liquid, and solid chemical systems. The simplest kind of to a higher energy level, a singlet or a triplet state is formed. In fluorescence is that exhibited by dilute atomic vapors, which was the excited singlet state, the spin of the promoted electron is described in Chapter 9. For example, the 3s electrons of vapor- still paired with the ground-state electron. In the triplet state, ized sodium atoms can be excited to the 3p state by absorption of however, the spins of the two electrons have become unpaired radiation of wavelengths 589.6 and 589.0 nm (see Figure 8-1a). 28 and are thus parallel. These states can be represented as in After about 10 s, the electrons return to the ground state and Figure 15-1, where the arrows represent the direction of spin. in so doing emit radiation of the same two wavelengths in all The nomenclature of singlet, doublet, and triplet derives from directions. This type of fluorescence, in which the absorbed spectroscopic multiplicity considerations, which need not con- radiation is reemitted without a change in frequency, is known cern us here. Note that the excited triplet state is less energetic as resonance radiation or resonance fluorescence. than the corresponding excited singlet state. Many molecular species also exhibit resonance fluores- The properties of a molecule in the excited triplet state cence. Much more often, however, molecular fluorescence (or differ significantly from those of the excited singlet state. phosphorescence) bands occur at wavelengths longer than the For example, a molecule is paramagnetic in the triplet state absorption band. This shift toward longer wavelengths is termed and diamagnetic in the singlet. More important, however, the Stokes shift (see Section 6C-6). is that a singlet-to-triplet transition (or the reverse), which 15A-1 Excited States Producing Fluorescence and Phosphorescence The characteristics of fluorescence and phosphorescence spectra can be rationalized by means of the molecular orbital consider- ations described in Section 14B-1. However, an understanding of the difference between the two photoluminescence phenomena requires a review of electron spin and the differences between singlet and triplet excited states. Electron Spin The Pauli exclusion principle states that no two electrons in Ground Excited Excited singlet state singlet state triplet state an atom can have the same set of four quantum numbers. This (a) (b) (c) FIGURE 15-1 electronic spin states of molecules. In (a) the 1 For further discussion of the theory and applications of fluorescence, ground electronic state is shown. In the lowest energy, or ground, phosphorescence, and luminescence, see D. M. Jameson, Introduction to state, the spins are always paired, and the state is said to be a Fluorescence, Boca Raton, FL: Taylor & Francis, 2014; J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., New York: Springer, 2006; Molecular Lumi- singlet state. In (b) and (c), excited electronic states are shown. nescence Spectroscopy, S. Schulman, ed., New York: Wiley, Part 1, 1985; Part 2, If the spins remain paired in the excited state, the molecule is 1988; Part 3, 1993; E. L. Wehry, in Physical Methods of Chemistry, 2nd ed., Volume in an excited singlet state (b). If the spins become unpaired, the VIII, Chap. 3, B. W. Rossiter and R. C. Baetzold, eds., New York: Wiley, 1993. molecule is in an excited triplet state (c). 15a Theory of fluorescence and Phosphorescence 363 also involves a change in electron spin, is a significantly less first electronic triplet state. As is normally the case, the energy probable event than the corresponding singlet-to-singlet of the first excited triplet state is lower than the energy of the transition. As a result, the average lifetime of an excited trip- corresponding singlet state. 24 let state may range from 10 to several seconds, compared Numerous vibrational energy levels are associated with 28 with an average lifetime of ,10 s for an excited singlet state. each of the four electronic states, as suggested by the lighter hor- Furthermore, radiation-induced excitation of a ground-state izontal lines. As shown in Figure 15-2, absorption transitions molecule to an excited triplet state has a low probability of can occur from the ground singlet electronic state (S0) to various occurring, and absorption bands due to this process are several vibrational levels of the excited singlet electronic states (S1 and orders of magnitude less intense than the analogous singlet- S2). Note that direct excitation to the triplet state is not shown. to-singlet absorption. We shall see, however, that an