Fluorescence and phosphorescence

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1. Fluorescence and phosphorescence Ravish Yadav

2. Fluorescence and phosphorescence Fluorescence and phosphorescence are the deactivation processes by which a molecule gives out the absorbed energy and returns back to the ground state.

3. Definition Emission of light by a substance that has absorbed light or other EMR. Fluorophores: absorption and emission spectrum.

4. Fluorescence and phosphorescence are the luminescent processes. Is associated with the absorption of energy in some form and emission of the absorbed energy in the form of light.

5. Photoluminescence Fluorescence Phosphorescence RESONANCE RADIATION/LUMINESCENCE [SAME] STOKES EFFECT [LONGER]

6. Fluorescence Phosphorescence Excitation of an electron occurs without the change in the spin. Excitation of an electron occurs with the change in the spin. Coming back of an electron from the ESS to the GSS is called as fluorescence Coming back of an electron from the ETS to the GSS is called as Phosphorescence Fluorescence occurs due to singlet singlet transition Phosphorescence occurs due to triplet singlet transition The life time of an excited state is short ranging from 10 -6 to 10- 9 seconds The life time of an excited state is as long as 10 -4 seconds to few seconds or minutes Fluorescence stops immediately as soon as the source of excitation is removed Phosphorescence is seen for a sufficiently longer period, even after removing the source of excitation.

7. Differentiation ETS ESS Less energetic compared to ESS More energetic compared to ETS Molecule is paramagnetic Molecule is diamagnetic Transition of an electron to an ETS does involve the change in spin of electron Transition of an electron to an ESS does not involve the change in spin of electron

8. Various deactivation processes The processes by which excited electrons give out the absorbed energy & come back to the original position i. e to the ground state in most of the cases. Vibrational relaxation Internal conversion Fluorescence External conversion Intersystem crossing Phosphorescence Dissociation Predissociation

9. Deactivation Processes The loss of energy by an electron, while coming back from an excited state (S1) to the ground state (S0), is shown by two arrows. Wavy arrow: radiation less loss of energy, which is usually in the form of heat Straight arrow : indicates the loss of energy which is usually in the form of radiation.[Fluorescence & phosphorescence]

10. Deactivation Processes As excited molecule can return to the ground state (GS) by a combination of deactivation processes. S0: Ground singlet state (GSS) S1: First excited singlet state (FESS) S2: Second excited singlet state (SESS) T1: First excited triplet state (FETS) Thick lines: Electronic state. Thin lines: Vibrational energy levels.

11. Vibrational relaxation When an electron from the GSS (S0) is excited using a narrow range of wavelengths, centered around, it occupies any one of the vibrational energy levels of the FESS.(S1) Direct excitation of an electron from the GSS to the first excited triplet state (FETS,T1) does not occur since it requires a change in the spin of an electron. Deactivation by vibrational relaxation is so fast that the shelf life of an excited state is only 10 -12 seconds or less. It is characterized by the transition of an electron from the higher vibrational energy level to the lower vibrational energy level, within the same electronic state. It involves the loss of energy in the form of heat indicated by wavy arrows.

12. Internal Conversion Instead of excitation of an electron from SO, using a wavelength centered around λ 1, if a shorter or a more energetic wavelength centered around λ 2 is used, an electron can get excited to the second excited singlet state (SESS, S2). In order to come back to the GS, the electron chooses the fastest deactivation process, the vibrational relaxation process, by which it comes down from the higher vibrational energy level of S2 to the ground vibrational energy level V0 of S2. An electron can not directly fall down from S2 to any of the vibrational energy levels of S0 by giving out energy in the form of radiation because it is slower process of deactivation compared to the other available deactivation processes. When one of the vibrational energy levels of the lower excited singlet state (v3 of S1) overlaps with the ground vibrational energy level (vo) of the higher electronic singlet state (S2), an electron prefers to deactivate by the process of internal conversion. Thus a deactivation process involving transition of an electron from a higher singlet state to a lower singlet state, through overlapping vibrational energy levels, without significant loss of energy is termed as internal conversion. The minute loss in the energy is in the form of heat which is radiation less loss of energy.

13. External conversion Like the vibrational relaxation, external conversion is associated with the radiation less loss of energy i.e. the energy is lost by an excited molecule to its surrounding (either to the solvent or the neighboring molecules of the solute itself) in the form of heat. External conversion occurs from the lowest excited singlet (S1) and triplet (T1) states to the ground state. Vibrational relaxation process External conversion Transition of an excited electron from the higher vibrational energy level to the ground vibrational energy level of the same electronic states Transition of an excited electron from the ground vibrational energy level V0 of the higher electronic state to any of the vibrational energy levels of the lower electronic state. Since the transition of an electron is within the electronic state , the amount of energy given out in the form of heat is less. Hence there is slight increase in the temperature of the solvent. The transition of an electron is from one electronic state to the other. So the amount of energy given out in the form of heat is much more compared to that produced during vibrational relaxation and therefore there is marked increase in the temperature of the solvent.

14. Fluorescence From V0 of S1, it can fall down to any of the vibrational energy levels of the GS (S0), either by giving out energy in the form of heat (external conversion) or by giving out energy in the form of radiation (fluorescence). Fluorescence is usually observed at a wavelength which is longer than the wavelength used for excitation. Coming back of an electron from the ESS to the GSS is termed as fluorescence

15. Intersystem crossing Deactivation process is somewhat similar to internal conversion. In case of internal conversion, an electron is transferred from the higher excited singlet state to the lower singlet state (either excited or ground) without substantial loss of energy and it involves singlet singlet transition (does not involve change in the spin of an electron) Deactivation by intersystem crossing occurs if any of the vibrational energy levels of a triplet excited state (v2 of T1) overlaps with the ground vibrational energy level v0 of the lowest excited singlet state (s1) Intersystem crossing involves singlet triplet transition and hence it is associated with the change in the spin of an electron.

16. Phosphorescence Coming back of an electron from the ETS to the GSS is called as phosphorescence.

17. Predissociation When an electron is excited from S0 to S2 by a shorter wavelength λ 2, it can undergo deactivation, first by vibrational relaxation and then by internal conversion. This allows entry of an electron from V0 of S2, into any of the vibrational energy levels of the lower excited electronic singlet state (S1). This internal conversion sometimes results in predissociation. In a predissociation, an electron enters such a vibrational energy level of the lower electronic singlet state (S1), whose energy is equivalent to the energy of one of the bonds in a molecule. Hence as soon as an electron occupies that particular vibrational energy level, it results in the rupture of a particular bond in a molecule. Thus a part of the absorbed energy is lost in the form of heat (during vibrational relaxation from the higher vibrational energy level of S2 to V0 of S2.) and a part of it is used to break a bond in the molecule.

18. Dissociation In case of predissociation, the rupture of a bond results as a effect of the absorption of energy by some chromophore in a molecule, followed by the internal conversion of the electron into the vibrational energy level of the lower excited singlet state, which is associated with the bond energy of one of the bonds in a molecule. In case of dissociation, the absorption of energy by an electron directly takes it to that vibrational energy level whose energy corresponds to one of the bonds in a molecule and results in the rupture of that particular bond.

19. Factors affecting intensity of Fluorescence & Phosphorescence Two major factors deciding whether a molecule will or will not show fluorescence or phosphorescence and if yes then what extent Intrinsic structure of molecule Environment of a molecule The extent to which molecules fluoresce or phosphoresce is expressed quantitatively in terms of quantum yield or quantum efficiency. Ø

20. quantum efficiency Øf = Kf _______________________________ Kf + Kph +Kic +Kec +Kis +Kpd +Kd The quantum efficiency for fluorescence is the ratio of the rate of deactivation of molecules by fluorescence to the rates of all the deactivation processes followed by it.

21. Intrinsic structure of molecule The type of electronic transition involved Saturated compounds involving sigma to sigma star transition Unsaturated compounds involving pi to pi star or n to pi star transition Aromatic, aliphatic and alicyclic compounds The heterocyclic compounds The presence of certain substituent's in the Benzene ring The rigidity

22. Environment of a molecule Temperature of solution Viscosity of the solution Polarity of the solvent PH of the solution Impurities present in the solution Concentration of the solution

23. Summary (Factors) Structure: 1.) Aromatic 2.) Rigid structures exhibit more 3.) Heavy atoms will decrease fluorescence 4.) Fluorescence will increase when molecule is adhered to surface Temperature and Solvent Effects 1.) Lower temperature increases fluorescence 2.) Solvent contains heavy atoms will decrease fluorescence but increase phosphorescence

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25. Instrumentation The basic instrument is a spectrofluorometer. It contains a light source, two monochromators, a sample holder and a detector. There are two monochromators, one for selection of the excitation wavelength, another for analysis of the emitted light. The detector is at 90 degrees to the excitation beam. Upon excitation of the sample molecules, the fluorescence is emitted in all directions and is detected by photocell at right angles to the excitation light beam. 25

26. Instrumentation The lamp source used is a xenon arc lamp that emits radiation in the UV, visible and near-infrared regions. The light is directed by an optical system to the excitation monochromator, which allows either preselection of wavelength or scanning of certain wavelength range. 26

27. Instrumentation The exciting light then passes into the sample chamber which contains fluorescence cuvette A special fluorescent cuvette with four translucent quartz or glass sides is used. When the excited light impinges on the sample cell, molecules in the solution are excited and some will emit light. 27

28. Instrumentation Light emitted at right angles to the incoming beam is analyzed by the emission monochromator. The wavelength analysis of emitted light is carried out by measuring the intensity of fluorescence at preselected wavelength. The analyzer monochromator directs emitted light of the preselected wavelength to the detector. A photomultiplier tube serves as the detector to measure the intensity of the light. The output current from the photomultiplier is fed to some measuring device that indicates the extent of fluorescence. 28

29. SOURCE Mercury lamp : produces intense radiation but gives a line spectrum. Under high pressures, lines are obtained at 366 ,405, 436, 446,577,691 and 773 nm Xenon lamp: more intense radiation and gives a continuous spectrum between 250 and 660nm.

30. Filters & Monochromators Use of two monochromators A primary filter , which selects the wavelength required for the excitation ( λ exe) of a molecule A secondary filter, which selects the wavelength of emission ( λ em) Eg. Interference filters, absorption filters or gratings can be used.

31. Cuvettes / Sample holders The cuvettes are usually made up of quartz and contains the solution of sample to be analyzed. Glass or silica cuvettes can also be used but glass absorbs in the range of 120-350 nm, hence it can not be used if λ exe falls in the uv region.

32. Detectors, Recorder & Printer Detectors are usually placed at right angles to the incident beam. Photomultiplier tube is mostly used in spectrofluorimetric. The intensity of fluorescent can be recorded and printed out using a printer.

33. Excitation and Emission spectra For recording absorption or an excitation spectrum, the substance is excited by using a number of wavelengths and the emission of radiation is measured at a fixed wavelength. The wavelength at which a substance absorbs the maximum is referred to as excitation wavelength ( λ exe) and it should be used for the measurement in order to excite the maximum number of molecules and to get the maximum intensity of emission. The emission spectra are recorded by exciting the molecules at a fixed wavelength and recording the intensity of emission at varying wavelengths. The wavelength at which the molecules give out maximum energy is called as emission wavelength.[ λ em]

34. QUENCHING OF FLUORESCENCE The reduction in or the total loss of intensity of fluorescence is called quenching. Environmental factors responsible for quenching of fluorescence are Temperature of the solution Viscosity of the solution Polarity & PH of the solution Presence of any paramagnetic species in the solution Concentration of the solution The self absorption

35. Temperature of the solution Increase in temperature of the solution increases the number of collisions among the neighboring molecules (solute-solute or solute- solvent). This reduces the intensity of fluorescence and brings about quenching of fluorescence.

36. Viscosity of the solution Decrease in viscosity of the solution also increases the number of collisions among the neighboring molecules, thereby causing quenching of fluorescence.

37. Polarity and pH of the solution Decides the wavelength of maximum absorption of a solute. Any solvent or the change in PH, which brings about the change in the wavelength of maximum absorption of a solute results in the decrease in the intensity of fluorescence.

38. Presence of any paramagnetic species in the solution Like oxygen or the presence of halides or compounds containing heavy atoms in solution also decreases the intensity of fluorescence.

39. Concentration of the solution The use of highly concentrated solution reduces intermolecular distance. This increases the collisions among the molecules. The loss or decrease of fluorescence due to collisions between the excited molecules of solute is called as self quenching. The increase in concentration of a solute increases the extent of self quenching.

40. The self absorption The self absorption occurs when the wavelength of emission overlaps the wavelength of absorption i.e. whatever energy is absorbed by the compound, the same is emitted out by it and is absorbed by the other molecules of the same sample in the solution.

41. Application of Spectrofluorimetry Is used for the analysis only if the compounds are fluorescent in nature. Compounds containing following features usually show a property of fluorescence A conjugated pie system Some extent of rigidity Absence of electron withdrawing groups such as –COOH or NO 2

42. Application of Spectrofluorimetry UV-Visible Spectrometry Spectrofluorimetry Is chosen when the concentration of an analyte is not very low and there is no interference from many substances. Is chosen when an analyte is in low concentration and there are many interfering substances in the sample to be analyzed. The quantitative analysis in spectrofluorimetric is carried out by setting the excitation wavelength at the maximum absorption and setting the emission wavelength at the maximum intensity of emission.

43. Quantitative analysis by Spectrofluorimetry Direct methods: which are used for the compounds that are naturally fluorescent Derivatization methods: which are used for the compounds that are non fluorescent in nature Quenching methods: which are used for the compounds that are able to quench the intensity of fluorescence of some fluorescent species. Fluorescence and chromatography Fluor immunoassays

44. Measurement of Fluorescent Intensity: Fluorimeters or Spectrofluorometers Spectrometers Spectrofluorometers Uses only one monochromator Use of two monochromators First monochromator is used to isolate the excitation wavelength i.e. wavelength required for the excitation of fluorescent molecule. Second monochromator is required to isolate the emission wavelength i.e. wavelength emitted by the molecule while coming back from an excited state to the ground state. Position of detectors: the intensity of the emitted beam is measured along the direction of the path of the incident beam The intensity of the emitted beam is measured at an angle preferably at 90 degree, with respect to the direction of the path of the incident beam The radiation of fluorescence is emitted in all the directions, but the intensity is usually measured at 90 degree to the incident beam ,the intensity of fluorescence is maximum and so accuracy of the measurement is also maximum. In other directions the intensity is reduced due to the increased scattering from the solution and also from the walls of the cuvette or sample holder.