1. Fluorescence Spectroscopy Chap 8&5 –T1 Chap 8 –T2 Chap15-R1

2. Introduction: molecular luminescence- a process in which photons or EM radiation are absorbed by molecule, raises them to excited state and, on returning to the ground state, the molecule emit radiation fluorescence, phosphorescence and chemiluminescence. fluorescence differs from phosphorescence in that electronic energy transitions do not involve a change in electron spin, thus fluorescence is short- lived (< 10 -5 sec) as compared to phosphorescence

3. Theory of Fluorescence: Pauli exclusion principle - the spins of two electrons in the same orbit of an atom are opposite to each other ie. paired. Because of spin pairing most molecules exhibits no net magnetic field and are c/a diamagnetic In contrast, free radicals, which contain unpaired electrons, have a magnetic moment and c/a paramagnetic (EPR) ground state for a free radical is a doublet state because the odd electron can assume 2 orientations in a magnetic field

4. molecule with an even number of electrons has all its electrons spin paired and is c/a ‘singlet’ state when one of a pair of electrons of a molecule is excited to a higher energy level, either a singlet or a triplet state is formed. in the excited singlet state, the spin of the promoted electron is still paired with the ground- state electron whether the molecule is in the ground state or excited state, as long as the electrons are paired, the molecule is in a singlet state, S 0, S 1, S 2, …

5. however, in triplet state, the spins of the two electrons have become unpaired and are thus parallel excited triplet state is less energetic and have longer lifetime (10 -4 to several sec) than the corresponding excited singlet state singlet/triplet transition (or the reverse) is a significantly less probable event then the corresponding singlet/singlet transition

6. Energy level diagrams for a photoluminescent molecule.

7. lowest heavy horizontal line - the ground-state energy of the molecule (S 0 ), which is normally a singlet state upper heavy lines - energy levels for the ground vibrational states of 3 excited electronic states: S 1 and S 2 represents first and second excited singlet state and T 1 represents energy of first triplet state. lighter horizontal lines - vibrational energy levels associated with each of the four electronic states excitation process results in conversion of the molecule to any of the several excited vibrational states

8. Deactivation Processes an excited molecule can return to its ground state by a combination of several steps: vibrational relaxation, internal conversion, external conversion, Intersystem crossing, - (wavy arrows) are radiationless processes phosphorescence and fluorescence - involve emission of a photon of radiation (vertical arrows)

9. a)Vibrational Relaxation: In solution, the excess vibrational energy is immediately lost (10 -12 sec) due to collisions between the excited molecule and solvent molecule fluorescence from solution always involves a transition from the lowest vibrational level of an excited state fluorescence emission band for a given transition is displaced towards lower frequencies or longer wavelengths from the absorption band ( a phenomenon c/a Stokes shift)

10. b) Internal Conversion: processes by which a molecule passes to a lower energy electronic state without emission of radiation. These processes are neither well defined nor well understood c) External Conversion: involve interaction and energy transfer between the excited molecule and the solvent or other solute. These processes are also c/a collisional quenching eg. effect of solvent, temperature, viscosity etc

11. Energy level diagrams for a photoluminescent molecule.

12. d) Fluorescence: When the electron returns to any of the vibrational levels of the ground state from the lowest vibrational level of the lowest excited state, radiation of fluorescence occurs. radiation of fluorescence is of lower energy than the exciting radiation because of vibrational relaxation and solvent reorientation, thus appears at longer wavelength

13. e) Intersystem Crossing: spin of an excited electron is reversed and involves vibrational coupling between the excited singlet state S 1, and the triplet state T 1. although singlet-triplet transitions are forbidden, the conversion from the excited singlet to the triplet state occur with some probability, since the energy of the lowest vibrational level of the triplet state is lower than that of the singlet state. most common in molecules that contain heavy atoms such as iodine or bromine ( heavy –atom effect) which favors change in spin

14. f) Phosphorescence: after indirect occupation of the triplet state, the molecule undergoes a vibrational relaxation and solvent reorientation to arrive at the lowest vibrational level of the excited triplet state. from this stage EM radiation can be emitted (phosphorescence), or internal conversion can occur.

15. Factors that affect fluorescence: 1)Quantum Yield (  ) or efficiency: ratio of the number of molecule that fluoresces to the total number of excited molecule for a highly fluorescent molecule (viz. fluorescein) the quantum yield approaches unity 2) Structural Factor: Molecules that are aromatic or contain multiple conjugated double bonds exhibits fluorescence Such substances have delocalized π-electrons that can be readily excited to singlet state

16. Substituents that delocalize the π-electrons enhance fluorescence by increasing transition probability between excited and ground state (ex. –NH 2, -OH, -F) Electron withdrawing groups decreases or quenches fluorescence completely (-Cl, -Br, -I, -NO 2 )

17. 3) Molecular Rigidity: rigid structure fluoresces more reduces the interaction of a molecule with its medium and thus reduces rate of collisional deactivation (internal conversion) Ex quantum efficiencies for fluorene and biphenyl molecules are nearly 1 and 0.2, respectively, due to increased rigidity furnished by the bridging methylene group in fluorene

18. 4) Temperature and solvent: Increasing temperature decreases quantum yield by increasing frequency of collisions, resulting in deactivation be external conversion A decrease in solvent viscosity also increases the external conversion and leads to the same result Solvent containing heavy atoms or substituents (ex. Br, I, NO 2 etc) or other solutes with such atoms in their structure decreases fluorescence by favoring triplet state formation

19. 5)Effect of pH: Changes in the system pH, if it affect the charge status of the chromophore, may influence fluorescence Both the wavelength and the emission intensities are likely to be different for the ionized and nonionized forms of the compound Ex both phenol and anisole fluoresce at pH 7, but at pH 12 phenol is converted to the nonfluorescent anion, whereas anisole remains unchanged Some substances are sensitive to pH and are used as indicators in acid-base titration {ex 1-naphthol-4- sulfonic acid}

20. 6)Effect of dissolved Oxygen: Presence of dissolved oxygen often reduces the intensity of fluorescence in a solution a) Quenches as a result of the paramagnetic properties of molecular oxygen, which promotes intersystem crossing and conversion of excited molecules to the triplet state. b) Oxygen can also oxidizes fluorescing species (Gal-1 example)

21. 7)Effect of concentration: plot of the fluorescence versus concentration of the species is linear at low concentration If the concentration is too high, the curve may have a maximum and then show a negative deviation Reasons: self quenching due to collision between excited molecules; self absorption due to overlapping of the wavelength of emission and absorption peak

22. Emission and excitation spectra An excitation spectrum is obtained by measuring fluorescence intensity at a fixed wavelength while the excitation wavelength is varied Excitation spectrum is essentially identical to an absorbance spectrum Fluorescence emission spectra involve excitation at a fixed wavelength while recording the emission intensity as a function of wavelength Phenanthrene

23. Instrumentation: all fluorescence instruments employ double-beam optics (two optical system) beam from the source first passes through an excitation filter or a monochromator which selects specific wavelength and direct them through the sample fluorescence from the sample is most conveniently observed at right angles to the excitation beam emitted radiation are directed to the photodetector after passing through a second filter or monochromator that isolates the fluorescence for measurement

24. reference beam passes through an attenuator that reduces its power to approximately that of the fluorescence radiation signals from the reference and sample photodetector are then fed into a difference amplifier whose output is displayed

25. Components: The components of spectrofluorometer differ only in detail from those of spectrophotometer Source Filters and monochromators Transducers Cells and cell compartments

26. Application: Sensitivity and selectivity of fluorometric method makes is a very powerful tool 1 determination of inorganic species: a)direct methods : involve formation of a fluorescing chelate and the measurement of its emission most suitable for nontransition-metal ions, since transition metal ions are paramagnetic and this increases the rate of intersystem crossing to the triplet state

27. reagents for cation analysis have aromatic structures with functional groups that permit chelate formation with the metal ion b) Second method is based on the decrease in fluorescence resulting from the quenching action of target species (widely used for anion analysis)

29. 2) determination of organic species: Determination of more than 200 substances (organic and biochemical) have been listed Ex. variety of organic compounds, enzymes and coenzymes, medicinal agents, plant products, steroid and vitamins 3) Application in liquid chromatography: important method for detecting and determining components of a sample as they appear at the end of chromatographic or capillary electrophoresis columns.

30. 4. Fluorescence Lifetime measurements: the time required for the population of the excited state to decrease to 1/e of its original value after the excitation source is turned off (order of 10 -9 to 10 -6 sec: ns to μs) Two different methods are used for such measurements, time-resolved and phase- resolved methods

31. In time-resolved method the sample is excited by a short-duration pulse of radiation and the resulting fluorescence intensity is measured as a function of time after the pulse ceases. For this the equipment employs mode-lock laser that produces pulses of radiation (widths of 70- 100 ps) for excitation and fast-rise-time photomultiplier tubes for detection Fig shows curves from a typical fluorescence lifetime experiment

32. True fluorescence decay signal C is then obtained by deconvolving ( a mathematical process) the contribution of the source to the experimental signal Curve B is the sum of the decay signal from the source and the decaying emission signal from the analyte. curve A-output of the source as a function of time, curve B- shows the fluorescence signal decays.

33. In phase-resolved method sample is excited by a continuous but sinusoidally modulated radiation the emitted fluorescence as a function of time is phase shifted and also partially demodulated to an extent dependent on the lifetime of the species In practice, the demodulation factor and phase shift are measured relative either to a reference solution with known lifetime or a scattering solution that has zero lifetime.

34. Advantages of lifetime measurement Possible to analyze multicomponent mixtures (different decay rates) Provides information about the energy transfer and quenching fluorescence lifetime can function as a molecular stopwatch to observe a variety of molecular events. an antibody may rotate slightly within its molecular environment, a protein can change orientation, a critical binding reaction may occur.

35. Comparison fluorescence and UV-Vis absorption methods: Fluorescence measurements are more selective than absorption methods Fewer substance fluoresces than absorb radiation Both emission and absorption spectra can be obtained Fluorescence lifetime can be measured

36. Fluorescence measurements are more sensitive than absorption methods In absorption spectroscopy, concentration α absorbance ( log of ratio of large quantities) In fluorescence, conc. α fluorescent power measured at right angles to the incident radiation (very small background) Its easier to measure a small signal directly than measuring a small difference between two large signals