![ The quantum yield or quantum efficiency for fluorescence is the ration of the
number of molecules that luminesce to the total number of excited molecules.
the quantum yield (φ) for a compound is determined by the following formula,
Gives a measure of how efficient a fluorophore (i.e., fluorescing molecule) is.
18
The relative rate constants (kx) for the processes which deactivate the lowest excited
singlet states, namely, fluorescence (kf), intersystem crossing (ki), external
conversion (kec), internal conversion (kic), predissociation (kpd), and dissociation
(kd).
Fluorescence…
Quantum Yield, =
total # luminescing molecules
total # of excited molecules
k = rate constant]
k
k k k k k k
f
f i ec ic pd d
[](https://image.slidesharecdn.com/chapter-2-221215165309-076e2e41/85/chapter-2-pptx-18-320.jpg)
chapter -2.pptx
- 1. 1 Chapter 2: Fluorescence Spectrophotometry
- 2. Principle • Certain molecules, particularly those with a chromophore and a rigid structure, can be excited by Uv-Vis radiation, and will then emit the radiation absorbed at a longer wavelength. The radiation emitted can then be measured. 2
- 3. Spectofluorimetry • After the absorption of uv-Vis light, the excited molecular species are extremely short lived and deactivation occur due to • Internal collision • Photochemical reactions • Re-emission as light (Luminescence) • In the first process, (Vibrational deactivation or nonradiative relaxation), the excess energy is released as heat, as A*----------A + heat 3
- 4. Emission molecular spectrophotometric techniques Introduction • Luminescence is the emission of light from any substance and occurs from electronically excited states. Luminescence is divided into three categories, (i) molecular fluorescence (ii) phosphorescence (iii) chemiluminescence • In each, molecules of the analyte are excited to give a species whose emission spectrum provides information for qualitative or quantitative analysis. The methods are known collectively as molecular luminescence procedures. 4
- 5. Spectofluorimetry • Relaxation by a photochemical reaction may involve a decomposition reaction in which A* splits apart (A* --- X + Y) or a reaction between A* and another species (A* + Z ------------- X + Y) • In either case the excess energy is used up in the chemical reaction or released as heat. • In the third mechanism excess energy is released as a photon of electromagnetic radiation. • (A* ------ A + hv) • The release of a photon following thermal excitation is called emission 5
- 6. Spectofluorimetry • Photoluminescence is divided into two categories: fluorescence and phosphorescence. • Absorption of an ultraviolet or visible photon promotes a valence electron from its ground state to an excited state with conservation of the electron’s spin. • For example, a pair of electrons occupying the same electronic ground state have opposite spins and are said to be in a singlet spin state. • Absorbing a photon promotes one of the electrons to a singlet excited state (all electrons are paired) • Emission of a photon from a singlet excited state to a singlet ground state, or between any two energy levels with the same spin, is called fluorescence. 6
- 7. What happens? • Absorption/ Excitation • Relaxation • Radiationless deactivation – Vibrational relaxation, Internal conversion, External Conversion and Intersystem crossing 7 Excited, paired = excited singlet state = fluorescence Excited, unpaired = excited triplet state= phosphorescence The excited triplet state is less energetic than the corresponding excited singlet state.
- 8. • Internal conversion: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level. • External conversion: A form of radiationless relaxation in which energy is transferred to the solvent or sample matrix • Intersystem crossing: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level with a different spin state 8
- 9. 9 Following absorption, a number of vibrational levels of the excited state are populated. Molecules in these higher vibrational levels then relax to the lowest vibrational level of the excited state (vibrational relaxation). From the lowest vibrational level, several processes can cause the molecule to relax to its ground state. The most important pathways are: 1. Collisional deactivation (external conversion) leading to non-radiative relaxation. 2. Intersystem Crossing (10-9s): In this process, if the energy states of the singlet state overlaps those of the triplet state, as illustrated in Figure 1, vibrational coupling can occur between the two states. Molecules in the single excited state can cross over to the triplet excited state. 3. Phosphorescence: This is the relaxation of the molecule from the triplet excited state to the singlet ground state with emission of light. Because this is a classically forbidden transition, the triplet state has a long lifetime and the rate of phosphorescence is slow (10-2 to 100 sec). 4. Fluorescence: Corresponds to the relaxation of the molecule from the singlet excited state to the singlet ground state with emission of light.
- 10. Fluorescence has short lifetime (~10-8 sec) so that in many molecules it can compete favorably with collisional deactivation, intersystem crossing and phosphorescence. The wavelength (and thus the energy) of the light emitted is dependent on the energy gap between the ground state and the singlet excited state. An overall energy balance for the fluorescence process could be written as: where Efluor is the energy of the emitted light, Eabs is the energy of the light absorbed by the molecule during excitation, and Evib is the energy lost by the molecule from vibrational relaxation. The Esolv.relax term arises from the need for the solvent cage of the molecule to reorient itself in the excited state and then again when the molecule relaxes to the ground state. As can be seen from Equation (1), fluorescence energy is always less than the absorption energy for a given molecule. Thus the emitted light is observed at longer wavelengths than the excitation (Stokes Rule).
- 11. 5. Internal Conversion: Molecule passes to a lower energy state – vibrational energy levels of the two electronic states overlap and molecules passes from one electronic state to the other. • This is a rapid process (10-12 sec) relative to the average lifetime of the lowest excited singlet state (10-8 sec) and therefore competes effectively with fluorescence in most molecules
- 12. Con’t… • A molecule in the lowest vibrational energy level of an excited triplet electronic state normally relaxes to the ground state by an intersystem crossing to a singlet state or by external conversion., Phosphorescence • Phosphorescence is observed when relaxation occurs by the emission of a photon. • The difference in the energy level (E) b/n the excited and the unexcited state during excitation (Absorption), fluorescence are in the order of • E (Absorption) > E (Fluorescence) > E (Phosphorescence) • Spectrofluorimetry measures, the intensity of light emitted from the system that had absorbed radiant energy 12
- 13. TERMS FROM ENERGY-LEVEL DIAGRAM Term: Absorption Effect: Excite Process: Analyte molecule absorbs photon (very fast ~ 10-14 – 10-15 s); electron is promoted to higher energy state. Slightly different wavelength excitation into different vibrational energy levels. Term: Vibrational Relaxation Effect: Deactivate, Radiationless Process: Collisions of excited state analyte molecules with other molecules loss of excess vibrational energy and relaxation to lower vibrational levels (within the excited electronic state) 13
- 14. Term: Internal conversion Effect: Deactivate, Radiationless Process: Molecule passes to a lower energy state – vibrational energy levels of the two electronic states overlap (see diagram) and molecules passes from one electronic state to the other. Term: Fluorescence Effect: Deactivate, Emission of h Process: Emission of a photon via a singlet to singlet transition (short – lived excited state ~10-7 – 10-9 s). 14
- 15. Term: Intersystem Crossing Effect: Deactivate, Radiationless Process: Spin of electron is reversed leading to change from singlet to triplet state. Occurs more readily if vibrational levels of the two states overlap. Common in molecules with heavy atoms (e.g., I or Br) 15
- 16. Term: External Conversion Effect: Deactivate, Radiationless Process: Collisions of excited state analyte molecules with other molecules molecule relaxes to the ground state without emission of a photon. Term: Phosphorescence Effect: Deactivate, Emission of h Process: Emission of a photon via a triplet to single transition (long–lived excited state ~ 10-4 – 101s) 16
- 17. Fluorimetry… • Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon. • Since molecules return to their ground state by the fastest mechanism, fluorescence is only observed if it is a more efficient means of relaxation than the combination of internal conversion and vibrational relaxation. • A quantitative expression of the efficiency of fluorescence is the fluorescent quantum yield, Ø • which is the fraction of excited molecules returning to the ground state by fluorescence. Ø - 0 – 1 NB 1 (every molecule in an excited state undergoes fluorescence) 0 (Fluorescence doesn’t occur) 17
- 18. The quantum yield or quantum efficiency for fluorescence is the ration of the number of molecules that luminesce to the total number of excited molecules. the quantum yield (φ) for a compound is determined by the following formula, Gives a measure of how efficient a fluorophore (i.e., fluorescing molecule) is. 18 The relative rate constants (kx) for the processes which deactivate the lowest excited singlet states, namely, fluorescence (kf), intersystem crossing (ki), external conversion (kec), internal conversion (kic), predissociation (kpd), and dissociation (kd). Fluorescence… Quantum Yield, = total # luminescing molecules total # of excited molecules k = rate constant] k k k k k k k f f i ec ic pd d [
- 19. Con’t… • If “F” is fluorescence intensity (Intensity of emitted) and “Iab” is the intensity of absorbed radiation, we may define a quantity, Ø (Quantum yield of fluorescence) • Ø=F/Iab, Ø its value are always b/n 0 and 1 • F=Iab* Ø, • Assays are restricted to diluted solution where linear curve can be obtained 19
- 20. Fluorescence and Structure • Low–energy * (aromatic): most intense fluorescence. • Heterocycles do not fluoresce; heterocycles fused to other rings fluoresce. Heteroatom increases ISC then f decreases. • Conjugated double bond structures exhibit fluorescence. • Structural rigidity (e.g., naphthalene or fluorene vs biphenyl). Flexibility increases then f decreases. • If a molecule has a rigid structure, the loss of electronic energy through its conversion into vibrational energy is relatively slow and there is a chance for the electronic energy to be emitted as ultraviolet or visible radiation • Temperature: increase fluorescence intensity with decreasing T (reduce number of deactivating collisions). 20
- 21. 21 The simple heterocyclics, such as pyridine, furan, thiophene, and pyrrole, do not exhibit fluorescence. On the other hand, fused ring structures ordinarily do fluoresce.
- 22. 22 Fusion of benzene rings to a hcterocyclic nucleus, however, results in an increase in the molar absorptivity of the absorption band. The lifetime of an excited state is shorter in such structures, and fluorescence is observed for compounds such as quinoline, isoquinoline, and indole.
- 23. 23 Fluorescence is particularly favored in molecules with rigid structures. For example, the quantum efficiencies for fluorene and biphenyl are nearly 1.0 and 0.2, respectively, under similar conditions of measurement. Reason: Presence of rigid methylene group
- 24. • Solvent: increase fluorescence with increased viscosity (decreased likelihood of external conversion – radiationless deactivation) • Heavy atoms such as I, Br, Th increases ISC as a consequence f decreases • pH: Increased resonance structures (protonation or deprotonation) stable excited state and greater quantum yield • pH can also influence emission wavelength (changes in acid dissociation constant with excitation) 24
- 25. 25 The fluorescence of an aromatic compound with acidic or basic ring substituents is usually pH dependent. Both the wavelength and the emission intensity arc likely to be different for the protonated and unprotonated forms of the compound. For example, aniline has several resonance forms but anilinium has only one. That is, The additional resonance forms lead to a more stable first excited state; fluorescence in the ultraviolet region is the consequence.
- 26. Excitation and Emission Spectra • Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength. • Excitation spectrum: Emission wavelength is fixed; excitation wavelength is scanned • Monochromator or filters selected to allow only one of fluorescent light to pass through to the detector. • Excitation wavelength is varied – at each excitation increment fluorescent photons at the fixed emission are collected. • The emission intensity (i.e., the number of fluorescent photons collected) at each increment varies as the excitation comes closer to or goes further from the of maximum absorption this is why an excitation spectrum looks like an absorption spectrum. 26
- 27. • Emission spectrum: Excitation wavelength is fixed; emission wavelength is scanned • Molochromator or filter is selected to allow only one of excitation light to pass onto the sample. • Emission is varied fluorescent photons are collected at each incremental emission . • The emission intensity (i.e., the number of fluorescent photons collected) at each increment varies as the emission is changed. • Spectrum shows at what the fluorescence intensity is a maximum for a given excitation . 27
- 28. Con’t… 28
- 29. Instrumentation • All fluorescence instruments contain five basic • items: 1) Source of light 2) Excitation filter 3) Sample holder 4) Emission filter 5) Detector • Sources • Hg lamp (254 nm) - Xe lamp (300 – 1300 nm) • Filter/monochromator • Isolate excitation - Scan excitation • Isolate emission from excitation - Scan emission • Detector • Usually PMT: very low light levels are measured. 29
- 30. Instrumentation •Fluorimeter is the instrument used to measure fluorescence intensity (F) •The light source must be very intense and very stable because F depends directly on Io. •Mercury arc lamps and Xenon arc lamps are in common use • both lamps emit in the visible and Uv region •The fluorescence light is emitted in all direction by the sample. • Measurement of fluorescence intensity in direction of propagation of the excitation radiation is extremely difficult because it involve measuring the emitted light against a light intensity background of transmitted light. This problem is overcome by observing the F at a right angle to the beam of excitation light 30
- 31. Fluorescence is emitted in all directions but is normally measured at a 90oangle to the excitation radiation. This reduces background noise and in principle the fluorescence is measured on a dark background This is also why the fluorescence has a lower limit of detection than the UV spectroscopy. Since fluorescence intensity is proportional to the intensity of the radiation that excites the molecules of the substance, an increase in radiation intensity provides a proportional increase in fluorescence intensity The excitation wavelength can be chosen from the UV spectrum of the compound. Normally it is preferred to excite the compound at the wavelength where it has its maximum UV absorption but for selectivity reasons another wavelength can be chosen
- 33. Advantages of fluorescence A. Sensitivity • Substances that reasonably fluorescence may be determined at concentration up to 5000 times lower than those required for absorption of spectrophotometer • In Spectrofluorimetric measurement the detector measures single light intensity w/c may be amplified electronically many times without introducing significant noise 33
- 34. Con’t… B. Selectivity • Not all substances that absorb in the uv-vis fluorescence • Wavelength of excitation /emission/ can be easily varied to selectively measure the fluorescence 34
- 35. Factors affecting fluorescence intensity • Concentration • In order for a molecule to fluorescence it must first absorb radiation. If the concentration of the absorbing substance is very high, all the incident light may be absorbed by first layer of solution. • The fluorescence of such samples will therefore be non uniform and will not be proportional to the concentration of the substance. 35
- 36. • Concentration • Although, its possible that all light absorbed by a molecule may be emitted as fluorescence, its more likely that part of the absorbed energy will be lost in other ways. • If the concentration of a solution prepared for fluorescence measurement is too high, some of the light emitted by the sample as fluorescence will be reabsorbed by other unexcited molecules in solution • For this reason, fluorescence measurements are best made on solutions with an absorbance of less than 0.02 at their maximum, i.e. solutions of a sample 10–100 weaker than those which would be used for measurement by UV spectrophotometry
- 37. Con’t… Oxygen The presence of oxygen may interfere in two ways • By direct oxidation of fluorescence substances to non-fluorescence products • By Quenching of fluorescence • De aerated solution pH Flurophores that contain ionizable groups are affected by the pH • Fluorescence intensity from excited states of charged and uncharged species is generally different. (i.e. change in pH alter the ratio of charged and uncharged species) 37
- 38. Con’t… Temperature and Viscosity • Variation in temperature and viscosity will cause variation in the frequency of collision b/n molecules • Increase To or decrease viscosity ==== decrease fluorescence by deactivation of the excited molecular collision • Higher Temperature === increase thermal motion ==== deactivation through heat • A rise in 1oC results in decrease in intensity of fluorescence by 1% 38
- 39. Con’t… Quenchers • Quenching is the process whereby emission from excited molecule is decreased by energy transfer to another molecule (quencher) • E.g. presence of dissolved oxygen F + hv--------F* F*------F + hv F* + Q ----------- F + Q • Dilution can avoid chemical quenching 39
- 40. Con’t… Molecular Structure • Highlyunsaturated polycyclicaromatic compounds • Structure rigidity and planarity 40
- 41. 41 Methods of fluorescence determination Direct methods - natural fluorescence of the fluorecent sample is measured Indirect (derivatisation) methods - the nonfluorescent compound is converted into a fluorescent derivative by specific reaction or marked with fluorescent dye by attaching dye to the studied substance Quenching methods - analytical signal is the reduction in the intensity of some fluorescent dye due to the quenching action of the measured sample
- 42. Con’t… • Application • Determination of fluorescent drugs in low dosage formulation • Limit if impurity ( Fluorescent or converted fluorescent cpd) • Determination of small amounts of drugs in biological fluids 42
- 43. Applications • Determination of ethinyloestradioal tablets • Measure at 280nm/320 nm • Determination of dissolution rate of Digoxin tablets Digoxin is converted into fluorescence cpd by dehydration in presence of HCl followed by oxidation by H2O2. • Determination of stability studies of peptide drugs in solution (Recombinant fibroblast factor) Tyrosine residue (277 nm /305 nm) Tryptophan (290 nm/350 nm) 43
- 44. Con’t… Determination of aluminum in water for injection as a fluorescent complex • Fluorescence measurements are useful in limit tests where the trace impurity is fluorescent or can be rendered fluorescent by chemical modification. • E.g. aluminum forms a complex 8- hydroxyquinolone followed by quantification the complex. The excitation wavelength is set at 392 nm and the emission is measured at 518nm. This type of fluorescent complex can be used to determine low level of a number of metal complex ions 44
- 45. Phosphorescence and fluorescenee methods tend to be complementary because strongly fluorescing compounds exhibit weak phosphorescence and vice versa. For example, among condensed-ring aromatic hydrocarbons, those containing heavier atoms such as halogens or sulfur often phosphoresce strongly. However, the same compounds in the absence of the heavy atom tend to exhibit fluorescence rather than phosphorescence. Phosphorimetry has been used for determination of a variety of organic and biochemical species. Including such substances as nucleic acids, amino acids. pyrine and pyrimidine, enzymes, petroleum hydrodo carbons, and pesticides. 45 Phosphorescence
- 46. • The method has not, however, found as widespread use as fluorometry, perhaps because of the need for low temperatures and the generally poorer precision of phosphorescence measurements. • On the other hand, the potentially greater selectivity of phosphorescence procedures is attractive. • The reason for this difference in behavior is that efficient phosphorescence requires rapid intersystem crossing to populate the excited triplet state, which in turn reduces the excited singlet concentration and thus the phosphorescence intensity. 46 Phosphorescence …
- 47. Thank You!!! 47
Editor's Notes
- #3: Luminescence is the emission of light from any substance and occurs from electronically excited states. Luminescence is divided into three categories,
- #27: The excitation spectrum provides a convenient means for selecting the best excitation wavelength for a quantitative or qualitative analysis. An excitation spectrum is obtained by monitoring emission at a fixed wavelength while varying the excitation wavelength. In an emission spectrum a fixed wavelength is used to excite the molecules, and the intensity of emitted radiation is monitored as a function of wavelength.