Fluorescence-Spectroscopy

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PHARM.D-ANALYSIS

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Fluorometry:

Fluorometry SAI KUMAR PHARM.D 1 REVOLUTIONPHARMD.COM

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Emission spectroscopy Emission spectroscopy is a spectroscopic technique which examines the wavelengths of photons emitted by atoms or molecules during their transition from an excited state to a lower energy state. 2 REVOLUTIONPHARMD.COM

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Luminescence Luminescence is the emission of light from any substance, and occurs from electronically excited states. Luminescence is divided into two categories-fluorescence and phosphorescence. The emission rates of fluorescence are typically 10 8 s –1 , so that a typical fluorescence lifetime is near 10 ns. The emission rates of phosphorescence are slow (10 3 to 100 s –1 ), so that phosphorescence lifetimes are typically milliseconds to seconds. Fluorescence is much more widely used for chemical analysis than phosphorescence. 3 REVOLUTIONPHARMD.COM

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The first observation of fluorescence from a quinine solution in sunlight was reported by Sir John Frederick William Herschel in 1845. Quinine The quinine in tonic water is excited by the ultraviolet light from the sun. Upon return to the ground state the quinine emits blue light with a wavelength near 450 nm . 4 REVOLUTIONPHARMD.COM

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Theory of molecular fluorescence Molecular fluorescence is measured by exciting the sample at the absorption wavelength, also called the excitation wavelength, and measuring the emission at a longer wavelength called the emission or fluorescence wavelength. For example, the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH) can absorb radiation at 340 nm . The molecule exhibits fluorescence with an emission maximum at 465 nm . 5 REVOLUTIONPHARMD.COM

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Theory of molecular fluorescence In a non-flurescent molecule when an electron is excited to the electronic excited state, it return back to the ground state by losing the energy it has acquired through conversion of the excess electronic energy into vibrational energy . 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. 6 REVOLUTIONPHARMD.COM

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The energy emitted is of lower energy lower energy than the energy absorbed because the excited electron moves to the lowest energy vibrational state in the excited state in the excited state before returning to the ground state. Thus fluorescence emission is typically shifted by 50-150 nm (Stokes shift) to the longer wavelength in comparison to the wavelength of the radiation used to produce excitation. Theory of molecular fluorescence 7 REVOLUTIONPHARMD.COM

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Relaxation processes Once the molecule is excited to E 1 or E 2 several processes can occur that cause the molecule to lose its excess energy. Two of the most important of these mechanisms, nonradiative relaxation and fluorescence emission are illustrated in Figure b and c. The two most important nonradiative relaxation methods that compete with fluorescence are illustrated in Figure b. 8 REVOLUTIONPHARMD.COM

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Energy-level diagram shows some of the processes that occur during (a) absorption of incident radiation. (b) nonradiative relaxation,and (c) fluorescence emission by a molecular species. Absorption typically occurs in 10 -15 s while vibrational relaxation occurs in the 10 -11 to 10 -10 s time scale. Internal conversion between different electronic states is also very rapid (10 -12 s), while fluorescence lifetimes are typically 10 -10 to 10 -5 s. 10 REVOLUTIONPHARMD.COM

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Vibrational relaxation involves transfer of the excess energy of a vibrationally excited species to molecules of the solvent. This process takes place in less than 10 -15 s and leaves the molecules in the lowest vibrational state of an electronic excited state. Vibrational relaxation depicted by the short wavy arrows between vibrational energy levels. takes place during collisions between excited molecules and molecules of the solvent. 11 REVOLUTIONPHARMD.COM

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Internal conversion is a type of relaxation that involves transfer of the excess energy of a species in the lowest vibrational level of an excited electronic state to solvent molecules and conversion of the excited species to a lower electronic state. 12 REVOLUTIONPHARMD.COM

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Relationship between excitation spectra and fluorescence spectra Because the energy differences between vibrational states is about the same for both ground and excited states, the absorption spectrum, or excitation spectrum, and the fluorescence spectrum for a compound often appear as approximate mirror images of one another with overlap occurring near the origin transition. 13 REVOLUTIONPHARMD.COM

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Fluorescence spectra for 1 ppm anthrecene in alcohol: (a) excitation spectrum (b) emission spectrum 14 REVOLUTIONPHARMD.COM

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Fluorescent species It is not entirely possible to predict how strongly fluorescent a molecule will be. For example adrenaline and noradrenaline differ in their structure by only a single methyl group but nor adrenaline exhibits fluorescence nearly 20 times more intensely than adrenaline. Generally, flurescence is associated with an extended chromophore or auxochrome and a rigid structure. 15 REVOLUTIONPHARMD.COM

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- Compounds containing aromatic rings give the most intense and most useful molecular fluorescence emission. - Few aliphatic and alicyclic carbonyl compounds as well as highly conjugate double-bonded structures . The simplest heterocyclics, such as pyridine, furan, thiophene and pyrrole, do not exhibit molecular fluorescence, but fused-ring structures containing these rings often do for example quinoline, isoquinoline, indole. 16 REVOLUTIONPHARMD.COM

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Substitution on an aromatic ring causes shifts in the wavelength of absorption maxima and corresponding changes in the fluorescence peaks. 17 REVOLUTIONPHARMD.COM

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The effect of structural rigidity Experiments show that fluorescence is particularly favored in rigid molecules. For example, under similar measurement conditions, fluorene is more fluorescent than biphenyl. The difference in behavior is a result of the increased rigidity provided by the bridging methylene group in fluorene. This rigidity lowers the rate of nonradiative relaxation. Fluorene Biphenyl 18 REVOLUTIONPHARMD.COM

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The influence of rigidity also explains the increase in fluorescence of certain organic chelating agents when they are complexed with a metal ion. For example, the fluorescence intensity of 8-hydroxyquinoline is much less than that of the zinc complex. 19 REVOLUTIONPHARMD.COM

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Fluorescence spectroscopy Fluorescence spectroscopy (fluorometry or spectrofluorometry), is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. This shift to longer wavelength is called the Stokes shift . Devices that measure fluorescence are called fluorometers or fluorimeters. 21 REVOLUTIONPHARMD.COM

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Instrumentation The light from an excitation source passes through a filter or monochromator, and strikes the sample. A portion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector. 22 REVOLUTIONPHARMD.COM

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Schematic of a fluorometer with 90° geometry utilizing a Xe light source 24 REVOLUTIONPHARMD.COM

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Schematic of a fluorometer with 90° geometry utilizing a Xe light source 90 0 25 REVOLUTIONPHARMD.COM

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Light source Xenon lamps Filters and/or monochromators The most common type of monochromator utilizes a diffraction grating. 26 REVOLUTIONPHARMD.COM

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Application 1. Determination of fluorescent drugs in low-dose formulations in the presence of non-fluorescent excipients. 2. In carrying out the limit tests where the impurity is fluorescent. 3. Useful for studying the binding of drugs to component in complex formulations . 4. Widely used in bioanalysis for measuring small amounts of drug and for studying drug-protein binding . 27 REVOLUTIONPHARMD.COM

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Factors interfering with fluorescence intensity 1. 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 less than 0.02, i.e. solutions of a sample 10-100 weaker than those which would be used for measurement by UV-VIS spectroscopy. 28 REVOLUTIONPHARMD.COM

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2. Heavy atoms in solution quench fluorescence by colliding with excited molecules so that energy is dissipated, e.g. chloride or bromide ions in solution cause collisional quenching. 3. Formation of chemical complex with other molecules in solution can change fluorescence behavior, e.g. the presence of caffeine in solution reduces the fluorescence of riboflavin. 4. In most molecules, fluorescent property decreases with increasing temperature because the increased frequency of collision at elevated temperatures increases the probability of collisional relaxation. A decrease in solvent viscosity leads to the same result. 29 REVOLUTIONPHARMD.COM

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