Fluorometry
- 1. FLUORIMETRY or FLUORESCENCE SPECTROSCOPY Nitesh kumar b. Pharm viii sem Jaipur college of pharmacy
- 2. Fluorescence: It is a phenomenon of emission of radiation when the molecules are excited by radiation at certain wavelength. Fluorimetry: It is measurement of fluorescence intensity at a particular wavelength with the help of a filter fluorimeter or a spectrofluorimeter. Principle: Molecule contains σ electrons, π electrons and nonbonding (n) electron. The electrons may be present in bonding molecular orbital. It is called as highest occupied molecular orbital (HOMO). It has lest energy and more stable. When the molecules absorb radiant energy from a light source, the bonding electrons may be promoted to anti bonding molecular orbital (LUMO). It has more energy and hence less stable.
- 3. Excitation - The process of promotion of electrons from HOMO to LUMO with absorption of energy is called as excitation. Singlet state: a state in which all the electrons in a molecule are paired. (↓↑) Doublet state: a state in which un paired electrons are present. (↓or↑) Triplet state: a state in which unpaired electrons of same spin present. (↑↑ ) Singlet excited state: a state in which electrons are unpaired but of opposite spin like (↑↓) (un paired and opposite spin) HOMO - Highest Occupied Molecular Orbital LOMO - Lowest Occupied Molecular Orbital When light of appropriate wavelength is absorbed by a molecule the electrons are promoted from singlet ground state to singlet excited state. Once the molecule is in this excited state, relaxation can occur via several processes by emission of radiation
- 4. Factors affecting fluorescence intensity: 1. Concentration: Fluorescence intensity is proportional to concentration of substance only when the absorbance is less than 0.02. 2. Quantum yield of fluorescence (ϕ): (ϕ) = number of photons emitted/number of photons absorbed It is always less than 1.0 since some energy is lost by radiation less pathways (Collisional, Intersystem Crossing, Vibrational Relaxation) 3. Intensity of incident light: Increase in the intensity of incident light on the sample fluorescence intensity also increases. 4. Adsorption: Adsorption of sample solution in the container may leads to a serious problem. 5. Oxygen: Oxidation of fluorescent species to a non- fluorescent species, quenches fluorescent substance. 6. pH: Alteration of pH of a solution will have significant effect on fluorescence
- 5. 7. Temperature and viscosity: Temperature increases can increase the collisional de activation, and reduce fluorescent intensity. If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent intensity. 8. Photochemical decomposition: Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non-fluorescent substance. 9. Quenchers: Quenching is the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescent analyte. Quenching is following types: a. Inner fluorescent effect: Absorption of Incident (UV) light or emitted (fluorescent) light by primary and secondary filters leads to decrease in fluorescence intensity.
- 6. b. Self-quenching: At low concentration linearity is observed, at high concentration of the same substance increase in fluorescent intensity is observed. This phenomenon is called self-quenching. c. Collisional quenching: Collisions between the fluorescent substance and halide ions leads to reduction in fluorescence intensity. d. Static quenching: This occurs because of complex formation between the fluorescent molecule and other molecules. Ex: caffeine reduces fluorescence of riboflavin. 10. Scatter: Scatter is mainly due to colloidal particles in solution. Scattering of incident light after passing through the sample leads to decrease in fluorescence intensity. Instrumentation: 1) Source of light: Mercury vapor lamp: Mercury vapor at high pressure give intense lines on continuous background above 350nm.low pressure mercury vapor gives an additional line at 254nm.it is
- 7. Xenon arc lamp: It give more intense radiation than mercury vapor lamp. it is used in spectrofluorimeter. Tungsten lamp: If excitation has to be done in visible region this can be used. It is used in low cost instruments. 2) Filters and Monochromators: Filters: These are nothing but optical filters work on the principle of absorption of unwanted light and transmitting the required wavelength of light. In inexpensive instruments fluorimeter primary filter and secondary filter are present. a. Primary filter: Absorbs visible radiation and transmit UV radiation. b. Secondary filter: Absorbs UV radiation and transmit visible radiation. Monochromators: They convert polychromatic light into monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a source. a. Excitation monochromators: Provides suitable radiation for excitation of molecule.
- 8. b. Emission monochromators: Isolate only the radiation emitted by the fluorescent molecules. 3) Sample cells: These are meant for holding liquid samples. These are made up of quartz and can have various shapes ex: cylindrical or rectangular etc. 4) Detectors: Photometric detectors are used. They are A. Barrier layer /photovoltaic cell: It is employed in inexpensive instruments. For ex: Filter Fluorimeter. It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2O). A semitransparent film of silver is laid on this plate to provide good contact. When external light falls on the oxide layer, the electrons emitted from the oxide layer move into the copper plate. Then oxide layer becomes positive and copper plate becomes negative. Hence an emf develops between the oxide layer and copper plate and behaves like a voltaic cell. So, it is called photovoltaic cell.
- 9. A galvanometer is connected externally between silver film and copper plate and the deflection in the galvanometer shows the current flow through it. The amount of current is found to be proportional to the intensity of incident light. B. Photomultiplier tubes (PMT): These are incorporated in expensive instruments like spectrofluorimeter. Its sensitivity is high due to measuring weak intensity of light. The principle employed in this detector is that, multiplication of photoelectrons by secondary emission of electrons. This is achieved by using a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes are used. Each dyanode is maintained at 75 - 100V higher than the preceding one. At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary emission of electrons and hence an overall factor of 106 is achieved.
- 10. PMT can detect very weak signals, even 200 times weaker than that could be done using photovoltaic cell. Hence it is useful in fluorescence measurements. PMT should be shielded from stray light in order to have accurate results. Instruments: The most common types are: Single beam (filter) fluorimeter Double beam (filter) fluorimeter Spectrofluorimeter (double beam) Single beam (filter) fluorimeter It contains tungsten lamp as a source of light and has an optical system consists of primary filter. The emitted radiations are measured at 900 by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit UV radiation which excites the molecule present in sample cell.
- 11. Instead of 90 if we use 180 geometry as in colorimetry secondary filter has to be highly efficient otherwise both the unabsorbed UV radiation and fluorescent radiation will produce detector response and give false result. Single beam instruments are simple in construction cheaper and easy to operate. Double beam fluorimeter It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinedly on a detector. Spectrofluorimeter: In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator. Incident beam is split into sample and reference beam by using beam splitter.
- 14. Applications: Fluorimetric methods are not useful in qualitative analysis and much used in quantitative analysis. Determination of inorganic substances. Al3+, Li+, Zn2+ Determination of thiamine HCl. Determination of phenytoin. Determination of indoles, phenols, & phenothiazines Determination of napthols, proteins, plant pigments and steroids. Fluorimetry, nowadays can be used in detection of impurities in nanogram level better than absorbance spectrophotometer with special emphasis in determining components of sample at the end of chromatographic or capillary column. Determination of ruthenium ions in presence of other platinum metals. Determination of boron in steel, aluminum in alloys, manganese in steel. Determination of boron in steel by complex formed with benzoin. Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate. Respiratory tract infections