Download Understanding Fluorescence in Chemistry: Importance and Mechanisms - Prof. Gabriele Varani and more Study notes Biochemistry in PDF only on Docsity! University of Washington - Department of Chemistry Chemistry 453 - Spring Quarter 2008 Lecture 27 06/04/08 Text reading: Chapt. 10 p. 561-564 Fluorescence O. Fluorescence Thus far we have only considered absorption processes. If resonant absorption occurs, a system will eventually return to the ground state. There are a number of pathways that can be followed to return to the ground state. Important mechanisms are radiationless transfer (molecular collisions), fluorescent emission, and phosphorescence. Fluorescence is probably the most important form of spectroscopy in biology, think about GFP and all sort of microscopies that are done now using fluorescence as a probe of biological structure or localization. The reason it is so widely used is that it is a very sensitive technique (even single molecules can be detected by fluorescence). Fluorescence is the emission of radiation that causes a transition from an excited state to the ground state, without a change in the electron spin multiplicity. Ground states usually are electronic singlet states in the sense that electrons with opposite spin angular momenta are paired. The ground state has no net spin angular momentum. Suppose a system is irradiated inducing a transition from the electronic n=0 state to the n=1 state. Suppose the system is also in the vibrational ground state (v=0). See diagram. Resonant irradiation induces an absorptive transition from n=0 and v=0 to n=1, v=0,1,2,3, that occurs in about 10-15 sec. Once in the excited state, the molecule can rapidly relax to the vibrational ground state through radiationless transitions in a time on the order of a vibrational period, which is about 10-12 sec. Once in the ground vibrational state, molecules require about 10-9 sec to return to the electronic ground state (fluorescence). Because of the loss of energy in the excited state through radiationless transitions, fluorescence occurs at longer wavelengths than the corresponding absorption. We can collect two types of spectra of fluorescence, the first is excitation spectrum and the second is emission spectrum. The excitation spectrum is similar to the absorption spectrum, though not identical, the emission spectrum is different and of course emission always occurs at higher wavelengths compared to excitation. An important parameter characterizing fluorescent transitions is quantum yield φ defined as the number of quanta of light emitted over the number of quanta of light absorbed. φ varies from 1 (no radiationless pathway, all absorbed light is re-emitted) to 0 (no pathway for emission). Fluorescence sensitivity From Beer’s Law the intensity of transmitted radiation I is related to the intensity of the incident light by: lCII ε−×= 100 Because the frequency of the incident and transmitted light are identical the intensities and the incident and transmitted light are proportional to the number of incident and transmitted photons, N0 and N, respectively, then: lCNN ε−×= 100 and the number of absorbed photons is: ( ) ( )lC lC absorbed eN NNNN ε ε 303.2 0 00 1 101 − − −= −=−= The number of photons emitted in the fluorescent beam is: ( ) ( )( ) lCQN lCQN eQNQNN lCabsorbflr ε ε ε 0 0 303.2 0 303.2 ...303.211 1 ≈ +−−≈ −== − Therefore, in contrast to absorption, in which the transmitted intensity is related to the concentration C by Beer’s Law, the number of photons in the fluorescent beam is linearly related to C. As a consequence, fluorescence is very sensitive, can detect very small amounts of a fluorescent substance. Fluorescence lifetime For the two level system depicted here, we can write rate equations to express how the populations n0 and n1 of the ground and excited states change as a result of induced absorption, stimulated emission, and spontaneous emission. The population of the ground state n0 is depleted by energy
