Fluorimetry By: Bijaya Kumar UpretyLUMINESCENCE SPECTROSCOPY * The emission of radiation from a species after that species has absorbed radiation. FLUORESCENCE UMINESCENCE , <—> ProsPuorEscENce SPECTROSCOPY ee CHEMILUMINESCENCEWhat is luminescence ? ue Luminescence is the emission of photons from electronically excited state. Luminescence is divided into two types, depending upon the nature of the ground and the excited states. In a singlet excited state, the electron in the higher energy orbital has the opposite orientation as the second electron in the lower orbital. These two electrons are said to be paired. Return to the ground state from an excited singlet state does not require an electron to change its spin orientation. In a triplet state these electrons are unpaired, that is, their spins have the same orientation. A change in spin orientation is needed for a triplet state to return to the singlet ground state. Singlet Triplet Le 1s 4 4 + ¥ tin a Ground Excited Excited singlet state singlet state triplet stare (a) (oy w& (b) te) Ss, diamagnetic paramagnetic Ss, TsLUMINESCENCE SPECTROSCOPY > | Absorption first - —<—— Followed by emission in all directions, usually at a lower frequencyTypes of luminescence | | (classification according to the means by which energy is supplied to excite the luminescent molecule) 1) Photoluminescence : Molecules are excited by interaction with photons of radiation. * Fluorescence : Prompt fluorescence : S,> S,+ hv The release of electromagnetic energy is immediate or from the singlet state. Delayed fluorescence : ST,» S;> Sy + hv This results from two intersystem crossings, first from the singlet to the triplet, then from the triplet to the singlet. * Phospholuminescence : T,> S, + hv A delayed release of electromagnetic energy from the triplet state. 2) Chemituminescence : The excitation energy is obtained from the chemical energy of reaction, 3) Bioluminescence : Chemiluminescence from a biological system: firefly, sea pansy, jellyfish, bacteria, protozoa, crustacea. 4) Triboluminescence : A release of energy when ceriain crystals, such as sugar, are broken. 5) Cathodoluminescence : A release of energy produced by exposure to cathode rays 6) Thermoluminescence : When a material existing in high vibrational energy levels emits energy at a temperature below red heai, after being exposed to small amounts of thermal energy.LUMINESCENCE SPECTROSCOPY Collectively, fluorescence and phosphorescence are known as photoluminescence. A third type of luminescence - Chemiluminescence - is based upon emission of light from an excited species formed as a result of a chemical reaction. In favorable cases, luminescence methods are amongst some of the most sensitive and selective of analytical methods available. Detection Limits are as a general rule at ppm levels for absorption spectrophotometry and ppb levels for luminescence methods. Most chemical species are not naturally luminescent. Derivatisation reactions are often available to form luminescent derivatives of non-luminescent compounds. However, this extra step lessens the attractiveness of luminescence methods. Fluorimetry is the most commonly used luminescence method. The terms fluorimetry and fluorometry are used interchangeably in the chemical literature.Introduction * A large number of substance absorb ultraviolet and visible light energy. But, there are some substances which lose excess energy as heat through collisions with neighboring atoms or molecules. * The energy emitted by these substances has a wavelength larger than that of absorbed. This process of emitting radiation with larger wavelength than that of absorbed is known as luminiscence. * Luminiscence is mainly of two types: 1. Fluorescence and 2. Phosphorescence.Concept of singlet and triplet state Singlet and triplet states * Ground state — two electrons per orbital; electrons have opposite spin and are paired * Singlet excited state Electron in higher energy orbital has the opposite spin nee orientation relative to electron in the lower orbital * Triplet excited state The excited valence electron may spontaneously reverse its spin (spin flip). This process is called intersystem crossing. Electrons in both orbitals now have same spin orientation fe | fe enFluorimetry Fluorimetry is an analytical method for the measurement of fluorescence, based upon emission of absorbed radiation by the molecules. When a molecule absorbs incident electromagnetic radiations, it is excited to higher energy level, where it is unstable. Therefore, it returns to the ground state by emitting the absorbed radiations. When the emission occurs directly from singlet excited state to singlet ground state without any transitions and without any change in spin orientation , it is referred as fluorescence. And when the emission occurs within a fraction of second to few days of absorption of radiations due to transition from singlet excited state to triplet state and then to singlet ground state, it is referred as phosphorescence. It involves the change in spin orientation during the transition from excited singlet to triplet and from triplet to singlet ground state. The movement of electrons from excited singlet state to triplet state, ie from unpaired electrons with opposite spin to unpaired electrons with same spin is termed as intersystem crossing. Fluorescence and phosphorescence are combinedly called photoluminescence. The energy emitted by these mechanism has a wavelength larger than that of absorbed.* The basis of fluorimetry is the measurement of fluorescence. Drugs which are intrinsically fluorescent, are determined fluorimetrically. €.g. Quinine sulfate in 0.1 N sulfuric acid; Ergometrine in 1% tartaric acid etc. Singlet S excited states Excited é trite stale Singlet stale Sy Fig. 2. Molecular energy Jevels asscoisted with fluorescence and phosphorescence.Principle of fluorimetry A molecule absorbs incident electromagnetic radiations and get excited. It is unstable in its excited state and tends to return to the ground state by emitting radiation. Fluorescence can be referred as the radiations emitted from an excited molecule in transition from singlet excited state to singlet ground state. In fluorimetry, radiations emitted are of longer wavelength than absorbed radiations. This is because, when the radiations fall on the molecule, vibration occurs (in 10 73 seconds), and has an average life of 10°9 seconds. During the vibration period, loss of energy occurs due to intermolecular collisions and some energy is lost to solvent molecules ( molecules of the solvent used for the dissolution of the sample in fluorimetry). Hence the emitted radiations are of longer wavelength and have less energy. ( since E a c/A)* Orbital changes can be explained as: 1. Fluorescence: Singlet state > Singlet excited state > singlet ground state ™ > 1* (No change in spin) 2. Phosphorescence: Singlet state > Singlet excited state Triplet state (2 unpaired electrons) singlet ground state mt > 1* (No change in spin) > Change in spin + Ineither fluorescence or phosphorescence the frequency of the emitted radiation is less than the frequency of incident radiation. The relationship is ; Vo > Vituor > Vohos Where, v = frequencyFluorescence related to concentration The fluorescence radiant power F is proportional to the absorbed radiant power. F=0(P,-P) where & = fluorescence efficiency, P, = incident power, P = transmitted power The relationship between the absorbed radiant power and concentration can be obtained from Beer's law. PIP, = 10° = 10-8C or, P=P, 10-86 or, F=®P, (1-10) --—---—----(1) This is the fluorescence law. When expanding the exponential terms and assuming ebC to be 0.05 or less, only the first term in the series is significant and equation can be written as F=@P, (inebC) = kbC ——-—-(2)+ where k is a constant equal to ® P,/nc. Thus, when the concentrations are very dilute and not over 2% of the incident radiation is absorbed, there is linear relationship between fluorescent power and concentration. * When ebC is greater than about 1.5, 10° is much less than 1 and fluorescence depends directly on the incident radiation power. F=@P, * From equation 2, we could infer that fluorescence intensity is directly prop ortional to concentration. Another important result can be implied from equation 2 where fluorescence is shown to be directly proportional to the intensity of the incident beam. This suggests that a very intense light sour ce is necessary for fluorescence instrumentation. Also substances of large are potential fluorescent molecules and should be sough for better results,Fluorescence Concentration of fluorescing species Theoretical behavior of fluorescence as a function of concentration.z Factors affecting fluorescence Nature of molecules: The molecules which have the tendency to absorb electromagnetic radiations (ie UV-Vis) can only exhibit fluorescence. Unsaturated molecules with pi bonds and good resonance stability can exhibit fluorescence. E.g. alkenes with conjugated double bond. Saturated molecules with sigma bonds donot exhibit fluorescence. E.g. aliphatic and saturated cyclic organic compounds. In general, the greater the absorbancy of a molecule, the more intense its luminiscence. . Affect of functional groups: Functional groups exhibit marked effect on fluorescence oftenly. Electron donating groups improve fluorescence. E.g. amine, hydroxy, and methoxy groups. Electron accepting groups deteriorate or completely destroy fluorescence. E.g. carboxylic, nitro and azo group, halides etc3. High molecular weight compounds: If an electron of an atom with high molecular weight is excited, it exhibits decreased fluorescence. 4. Effect of concentration: * There exists a linear relationship between the concentration of sample and fluorescence upto an absorbance value of 0.02. The linearity holds good to about 5% of cases with absorbances upto 0.05. + In dilute solution, the radiation distribute uniformly throughout the solution and get absorbed uniformly giving high intense fluorescence. + In highly concentrated solution, upper layers of the solution absorb more radiations and less amount of radiatons are transerred to lower layers. Thus , there is no uniform radiations absorption which results in decrease fluorescence. * In highly concentrated solution, intramolecular collisions cause lass of vibrational energy and certain amount of fluorescece which is emitted is reabsorbed resulting in decrease in intensity of fluorescence. This is called as concentration quenching. 5, Effect of oxygen: There is decrease in fluorescence in the presence of oxygen due to * Direct conversion of fluorogenic material into non-fluorogenic material. * Indirectly due to quenching. ‘Quenching refers to the decrease in intensity of fluorescence as a result of decrease in the sensitivity of constituents.6. Affect of pH of the solution * Depending upon the acidity or alkalinity, a substance can be ionized or unionized and hence, can be fluorogenic or non-fluorogenic. e.g. Phenol in neutral and alkaline medium undergoes ionisation and gives weak fluorescence, whereas in acid medium it is unionised and gives intense fluorescence. 7. Affect of Temperature and Viscosity * Alteration in temperature may affect the concentration and viscosity of the sample. * Increase in temperature may result in increase in concentration and decrease in viscosity resulting in intermolecular collisions and deactivation of excited molecule destroying fluorescence. * Some substances may exhibit fluorescence at temperature lower than room temperature or in a viscous solvent or glassy matrix. 8. Affect of impurities: Substances other than the solute molecules are impurities and exhibit fluorescence quenching. E.g. iodide ion is extremely effective quencher.9. Affect of intensity of radiation: * Radiation of adequate intensity must be used to induce fluorescence. * High intensity radiation > causes decrease in fluorescence due to photochemical change. + Light of single wavelength, i.e. monochromatic light should be used, as energy of radiation varies with wavelength. 10. Chemical Quenching: This can occur in two ways ; * The excited molecule transfers its fluorescent intensity to surrounding molecules, ions or impurity by intercollisions, thereby destroying fluorescence. * The unexcited molecule may form a stable complex with quencher molecule inhibiting excitation and fluorescence. 11. Affect of structure of the compounds: * Closed ring aromatic compounds exhibit fluorescence e.g. fluorescein, Eosin.Compounds with more than two cyclic structure exhibits fluorescence. E.g. vitamin K, Nucleosides, purines. Rigid molecules like metal complexes have enhanced fluorescence as they inhibit the liberation of excitation energy. The position of functional group (chromophore) which is responsible for absorption affects fluorescence.Instrumentation The basic design of instrumentation for monitoring molecular fluorescence and molecular phosphorescence is similar to that found for other spectroscopies. The most significant differences are discussed in the following sections. Molecular Fluorescence A typical instrumental block diagram for molecular fluorescence is shown in Figure 10.45. In contrast to instruments for absorption spectroscopy, the optical paths for the source and detector are usually positioned at an angle of 90°. Two basic instrumental designs are used for measuring molecular fluorescence. In a fluorometer the excitation and emission wavelengths are selected with absorption or interference filters. The excitation source for a fluorometer is usually a low pressure mercury vapor lamp that provides intense emission lines distributed throughout the ultraviolet and visible region (254, 312, 365, 405, 436, 546, 577, 691, and 773 nm). When a monochromator is used to select the excitation and emission wavelengths, the instrument is called a spectrofluorometer. With a monochromator, the excitation source is usually a high-pressure Xe arc lamp, which has a continuum emission spectrum.Instrumentation for fluorescence spectroscopy “ [ FOTd JS Power Source Excitation monochromator ‘ supply Sample cell Slit Emission monochromator Detector Data processor General layout of fluorescence spectrophotometerao SL bw jv ee pee SP | eee Excitation monochromator ee i a «RJR 2 eal gb ssi isi a eo 2} = He Schematic diagram of a typical spectrofluorometer.1) Light sources a. Gas discharge lamps : Xenon arc lamp High pressure mercury vapor lamp b. Incandescent lamps : Tungsten wire filament lamp c. Laser : tunable dye laser d. X-ray source for X-ray fluorescence 2) Wavelength selection devices a, Filters ; Absorption filters —tinted glass or gelatin containing dyes sandwiched between glass Interference filters —-thin transparent layer of CF, or MgF, sandwiched two parallel, partially refelecting metal films b. Monochramators = Gratings Prism© Represents wave maximum @ Represents wave minimum Transparent spacer ‘one-half wavelength thick —WGE ™—BiDUoC RES Semitransparent silver film Cross-sectional view of an interference filterFluorescence, abritrary units Diverse substance 325 Bandpass of secondary filter 350 375 Wavelength, nm (a) 540 Wavelength, nm (b) Analyte Bandpass primary fil Analyte Diverse substance 580 —— 425 620 Proper choice of primary and secondary filters to avoid interference from another substance: a) excitation spectra (both substances fluoresce over same wavelength —_ region, b) fluorescence spectra (both substances absorb in same wavelength region).3) Sample compartment Cuvettes or cells with area of 1 cm?- Usually made up of; Quarz or fused silica ---200 nm ~ 800 nm Glass or plastic ---- 300 nm ~ 4) Detectors Photomultiplier tubeSelected Examples of Organic Compounds of Biochemical, Pharmaceutical, and Environmental Significance That Show Natural Fluorescence or Phosphorescence Class Compounds aromatic amino acids phenylalanine (F) tyrosine (FD tryptophan (F, vitamins vitamin A EF) vitamin 8, (F) catecholamines sopamine (F) norepeneptcine (FD pharmaceuticals and drugs quinine (FY sallicytic acicl (FP) marphine () barbiturates (Fh tSD «FF codeine {P} calfcine (P) sultaxnilamiete Conversion of acidic to alkaline solution or vice versa. > Conversion of ionic to non-ionic compound or vice versa. > Selection of wavelength of excitation for each drug. > Extraction of any one drug from the mixture and analysing it. (e) Preparation of fluorogenic derivative from non-fluorogenic drug. Some of the examples include; > Complex of atropine with eosin is soluble in chloroform and exhibit fluorescence, > Other non-fluorogenic drugs which can be analysed are morphine and codeine.2. Analysis of inorganic compounds a, Estimation of Uranium in Salts. b. Some non-fluorescent inorganic ions can be made fluorescent by complexing it with non-fluorescent organic reagents. Hence these element can be fluorimetrically analysed. 3. Fluorescent indicators are useful in fluorimetric determination as the color and intensity of fluorescent depends upon the pH of the solution. 4. Hydrogen bonding, geometrical isomerism, polymerization, tautomerism and reaction rates can be studied fluorimetrically. 5. Fluorimetry can be used for both qualitative and quantitative estimation of steroids, proteins, plant pigments etc