Spectrofluorimetry - Modern pharmaceutical Analytical technique

Spectrofluorimetry principle, instrumentation, fluorescence, phosphorescence, types of luminants, quenching, factors affecting fluorescene, application.
Modern pharmaceutical analytical technique, M.Pharm 1st year, 1st sem.

1. SPECTROFLUORIMETRY Presented by Pavithra J.P.

2. CONTENTS: Introduction Theory of fluorescence Factors affecting fluorescence Quenchers Instrumentation Applications of fluorescence spectrophotometer

3. INTRODUCTION FLUORIMETRY: Absorption of UV/Visible radiation causes transition of electrons from singlet ground state to singlet excited state. As this state is not stable, it emits energy in the form of UV/Visible radiation and returns to singlet ground state. This study or measurement of this emitted radiation is the principle in Fluorimetry. PHOSPHORESCENCE: It is the study of emitted radiation when electrons undergo transition from triplet state to singlet ground state. Principle: Fluorescence is the phenomena of emission of radiation when there is transition from singlet excited state to singlet ground state.

4. The electronic states to understand: Singlet ground state: a state in which all the electrons in a molecule are paired ↑↓ Doublet state: a state in which an unpaired electron is present for Eg : free radical ↑ or ↓ Triplet state: a state in which unpaired electrons of same spin present. ↑ and ↑ Singlet excited state: a state in which electrons are unpaired but of opposite spin like ↑ and ↓

5. To achieve this transition, there are 3 possibilities: 1. Collisional deactivation: in which the entire energy is lost due to collisional deactivation and no radiation is emitted. 2. Fluorescence: A part of energy is lost due to vibrational transitions and the remaining energy is emitted as UV/visible radiation of longer wavelength than the incident light ( Stoke’s Fluorescence). The wavelength of absorbed radiation is called as excitation wavelength and that of emitted radiation is called as emission wavelength .

6. 3. Phosphorescence: At favorable conditions like low temperature and absence of oxygen, there is transition from excited singlet state to triplet state which is called as inter system crossing. The emission of radiation when electrons undergo transition from triplet state to singlet ground state is called as Phosphorescence.

7. TYPES OF LUMINESCENCE: Photoluminescence: In this case, light is produced by exposing the substance to UV radiation. Bioluminescence: In this case, light is produced as a result of biological processes. The light emitted by a glow-worm is because of bioluminescence. Chemiluminescence: The emission of light as a result of chemical reactions is called chemiluminescence. Light sticks producing light as a result of a mixture of chemicals is an example of chemiluminescence. Radioluminescence: The emission of light as a result of interaction of radioactive particles with a compound is called radioluminescence, e.g. luminous paints glow because of the interaction of a compound in it with the radioactive particles, usually provided by tritium (3H) atoms.

8. TYPES OF FLUORESCENCE: 1) Based upon the wavelength of emitted radiation when compared to absorbed radiation: Stoke’s fluorescence: The wavelength of emitted radiation is longer than the absorbed radiation. Eg : as in conventional fluorimetric experiments. Anti- stoke’s fluorescence: The wavelength of emitted radiation is shorter than the absorbed radiation. This type is uncommon and seen in Thermally assisted fluorescence. Resonance fluorescence: When the wavelength of the emitted radiation is equal to the absorbed radiation. Eg : Mercury vapour at 254nm.

9. 2) Based upon the phenomenon: Sensitized fluorescence: When elements like Thallium, Zinc, Cadmium or an alkali metal are added to mercury vapour , these elements are sensitized and thus gives fluorescence. Direct line fluorescence: Where, even after the emission of radiation, the molecules remain in metastable state and finally comes to ground state after loss of energy by vibrational transition. Stepwise fluorescence: This is nothing but the conventional type of fluorescence, where a part of energy is lost by vibrational transition before the emission of fluorescent radiation. Thermally assisted fluorescence: The excitation is partly by electromagnetic radiation and partly by thermal energy.

10. DEACTIVATION PROCESS An excited molecule return to its ground state by a combination of several mechanistic steps is known as deactivation process. It involves, Radiationless pathway - (Vibrational relaxation, Internal conversion, External conversion, Intersystem crossing) Radiation pathway (emission of photons) – (Phosphorescence, Fluorescence) The favored route to the ground state is the one that minimizes the lifetime of the excited state. If deactivation by fluorescence is rapid with respect to the radiationless processes, emission is observed. On the other hand, if a radiationless path has a more favorable rate constant, fluorescence is either absent or less intense.

11. 1) VIBRATIONAL RELAXATION: A molecule may be promoted to any of several vibrational levels during the electronic excitation process. Vibrational relaxation is so efficient that the average lifetime of a Vibrationally excited molecule is 10 -12 or less. The fluorescence band for a given electronic transition is displaced toward lower frequencies or longer wavelengths from the absorption band (Stokes’ shift). 2) INTERNAL CONVERSION: Internal conversion is a crossover between two states of the same multiplicity (singlet-singlet or triplet – triplet). It is particularly efficient when two electronic energy levels are sufficiently close for there to be an overlap in vibrational energy levels. Fluorescence occurs at λ 3 only regardless of whether radiation of wavelength λ 1 or λ 2 was responsible for the excitation.

12. 3) EXTERNAL CONVERSION: External conversion refers to deactivation of excited electronic state to a lower energy state by interaction and energy transfer to solvent molecule or other solutes. Due to the low collision in low temperature and high viscosity solvents leads to enhanced fluorescence, thus it slows down the deactivation process. 4) INTERSYSTEM CROSSING: Intersystem crossing is a process in which there is a crossover between electronic states of different multiplicity (singlet state to the triplet state (S₁→T₁)).The probability of intersystem crossing is enhanced if the vibrational levels of the two states overlap. Intersystem crossing is most common in molecules that contain heavy atoms, such as iodine or bromine (heavy atom effect) and paramagnetic species such as molecular oxygen.

13. 5) PHOSPHORESCENCE: After intersystem crossing to the triplet state, further deactivation can occur either by internal or external conversion or by phosphorescence. A triplet-singlet transition is less probable. The average lifetime of the excited triplet state with respect to emission is large and ranges from 10-4 to 10 s or more. Emission from such a transition may persist for some time after irradiation has ceased.

14. FACTORS AFFECTING FLUORESCENCE: Concentration: It is quite obvious that less number of molecules absorb lesser radiation and so emit lesser radiation. Similarly, more number of molecules absorb more radiation and so emit more radiation. But this has to be quantified by an equation and the concentration limits within which it obeys Beer’s law has to be known. 2. Quantum yield of fluorescence: This is the ratio: Φ=Number of photons emitted / Number of photons absorbed Since some absorbed energy is lost by radiationless pathways, the quantum efficiency is less than 1. Highly fluorescent substances have Φ values near 1, which shows that most of the absorbed energy is re-emitted as fluorescence. Example- fluorescein in 0.1M sodium hydroxide and quinine in 0.05m sulphuric acid have Φ values of 0.85 and 0.54 respectively at 23°. Non-fluorescent substances have Φ  = 0.

15. 3. Intensity of incident light: An increase in the intensity of light incident on the sample produces a proportional increase in the fluorescence intensity. The intensity of incident light and sensitivity of a fluorescence measurement are increased by increasing the width of the excitation slit. The choice of the excitation slit-width is therefore a compromise between sensitivity, selectively and photostability. 4. Adsorption: The extreme sensitivity of the method requires very dilute solutions, 10 -100 times weaker than those employed in absorption spectrophotometry. Adsorption of the fluorescent substance on the container walls may therefore present a serious problem and strong stock solutions must be kept and diluted as required. Example- Quinine which is adsorbed on to cell walls.

16. 5. Oxygen: Oxygen decreases the fluorescence intensity in two ways: [ i ] It oxidises fluorescent substance to non-fluorescent substance. [ii] It quenches (decreases) fluorescence, because of the paramagnetic properties of molecular energy, as it has triplet ground state. Eg : Anthracene is susceptible to the presence of oxygen. 6. Effect of pH: The effect of pH depends on the chemical structure of the molecule. Eg. ( i ) Aniline in neutral or alkaline medium gives visible fluorescence but in acidic conditions gives fluorescence in UV region only. (ii) Phenols in acidic condition are undissociated and do not give fluorescence, but in alkaline condition, they are dissociated (ionic) and gives good fluorescent.

17. 7. Photodecomposition: Fluorimetry requires high intensity of radiation is required for excitation. This high intensity radiation may bring irradiation changes or photochemical changes in a substance destroying fluorescence. The light used must be of suitable wavelength so that it is not strong enough to cause photodecomposition. 8. Temperature and viscosity: Increase in temperature leads to increase in collisions of molecules, which results in decrease in Fluorescence intensity and vice versa. Increase in viscosity leads to decreased collisions of molecules, which leads to enhancement of fluorescence intensity and vice versa. 9. Impurities and other substances: Some substances act as a impurity and show fluorescence quenching. Example- Iodine is an effective quencher, organic substances, especially aromatic type in dilute solutions have tendency of adsorption on surface of cell. 

18. QUENCHING OF FLOURESCENCE AND TYPES: Quenching is the decrease in fluorescence intensity due to specific effects of constituents of the solution itself. Various types of quenching are Self quenching or concentration quenching: At low concentrations (µg or ng/ml), linearity is observed. At high concentrations (mg/ml) of the same substance, proportionate increase in fluorescence intensity does not occur. This phenomenon is called as self- quenching or concentration quenching. 2. Chemical quenching: In this type, quenching is due to various factors like change in pH, presence of oxygen, halides or heavy metals. pH: Aniline at pH 5 to 13 gives blue fluorescence when excited at 290nm. But at pH < 5 (exists as cation) and pH > 13 (exists as anion), it does not exhibit fluorescence.

19. b. Oxygen: Presence of oxygen leads to quenching because of its paramagnetic property (triplet ground state). c. Halides and electron withdrawing groups: Halides like chloride, bromide, iodide and electron withdrawing groups like Nitro and carboxylic group leads to quenching (i.e. decrease in fluorescence intensity). d. Heavy metals: Presence of heavy metals also leads to quenching because of collisions and triplet ground state. 3. Static quenching: This occurs because of complex formation. ( eg ) caffeine reduces the fluorescent intensity of riboflavin, by complex formation. 4. Collisional quenching: It is the result of several factors like presence of halides, heavy metals, increased temperature and decrease in viscosity, where number of collisions are increased. Hence quenching takes place.

20. INSTRUMENTATION: It consist of 1) Source of radiation 2) Filter or monochromator 3) A sample holder 4) Detector 5) Readout system In contrast, to ultraviolet-visible instrumentation, two optical systems are necessary. The primary filter or excitation monochromator selects specific bands or wavelengths of radiation from the source and directs them through the sample in the sample cell. The resultant luminescence is isolated by the secondary filter or emission monochromator and directed to the photodetector, which measures the power of the emitted radiation. For the observation of phosphorescence, a repetitive shutter mechanism or electronic delay system is required.

21. SOURCE OF RADIATION 1. High-pressure dc xenon arc lamps are used in nearly all commercial spectrofluorimcters . The xenon lamp emits an intense and relatively stable continuum of radiation that extends from 300 to 1300 nm. Several strong emission lines lie between 800 and 1100 nm. 2. A xenon flash lamp is a compact, low-cost source. The sample is excited by a high- energy flash produced by the discharge of a charged capacitor through a lamp filled with xenon. By making the flash repetitive, ac methods of amplification can be used. 3. Mercury vapour lamp: Mercury vapour at high pressure (8 atmospheres) gives intense lines on a continuous background above 350nm. Lines are seen at 365, 398, 436, 546, 579, 690 and 734nm. Low pressure mercury vapour gives an additional line at 254nm. It is used as source in filter type of fluorimeters.

22. 2) FILTERS AND MONOCHROMATORS: In fluorimetry two things are important, i.e. excitation wavelength and emission wavelength . As these two wavelengths are different in most cases, a filter or monochromator is used for the purpose. In an inexpensive instrument like filter fluorimeter - primary filter and secondary filter are present. Primary filter - absorbs visible radiation and transmits UV radiation . Secondary filter - absorbs UV radiation and transmits visible radiation. In Spectroflourimeters , excitation monochromators and emission monochromator are present which have gratings . In Spectroflourimeters , Excitation monochromator - provides a suitable radiation for excitation of molecule (radiation which is absorbed by molecule) Emission monochromator - isolates only the radiation emitted by the fluorescent molecule.

23. 3) SAMPLE HOLDER: There are four arrangements for illuminating and viewing the sample: The right-angle (90°) method 2. The frontal (37°) method 3. The rotating-cell method 4. The straight-through (transmission) The sample cells are cylindrical or polyhedral (quadrangular) like those used in colorimetry. The cells are made up of colour corrected fused glass and path length is normally 10mm or 1cm. It need not be made up of quartz and all the surfaces are polished in fluorimetry, because emission measurements are made at 90° angle.

24. 4) DETECTORS: The intensity of fluorescence is usually low. In order to measure the concentration of sample accurately, the intensity should measured accurately. Hence a large amplification factor is required for its measurements. This necessitates the use of Photomultiplier tube as detector in fluorimetry and in spectrofluorimetry . A Photomultiplier tube is best regarded as a current source. The current is proportional to the light intensity. Detectors are usually placed at right angles to the incident beam. 5)READ OUT SYSTEM: The intensity of fluorescence can be recorded on a recorder and print out using a printer

25. INSTRUMENTS: a. Single beam filter fluorimeter: This is an inexpensive instrument, contains tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits UV radiation which excites the molecules present in sample cell. The emitted radiation (fluorescent radiation) is measured at 90 degree by using a secondary filter and a detector. The secondary filter absorbs UV radiation and transmits visible radiation emitted by the compound. Instead of 90°, if we use 180° geometry as in colorimetry, the secondary filter has to be highly efficient, otherwise both the unabsorbed UV radiation and fluorescent (emitted) radiation will produce detector response and give false results.

26. b . Double beam (filter) 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 either sample or reference solution. Advantage: The sample and reference solution can be analysed simultaneously. Disadvantage: The rapid scanning is not possible due to the use of filters.

27. c. Spectroflourimeter (double beam): The primary filter in double beam (filter) flourimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator. The incident beam is split into sample and reference beam by using beam splitter. Normally the detector is photo multiplier tube. The advantages are: 1). Rapid scanning to get excitation and emission spectrum, 2) More sensitivity and accuracy when compared to filter type instruments.

28. Advantages: High sensitivity (ng/ml to µg/ml). High selectivity (fluorescent substances show specific excitation and emission λ max value). Disadvantages: ➤ All compounds do not fluoresce. ➤ It is not suitable for identification of compounds. ➤ Contaminants can quench fluorescence and mislead the results. ➤ It is susceptible to pH, solvent polarity, temperature, etc.

29. APPLICATIONS: Determination of Organic substances. Plant pigments, steroids, proteins, naphthols , etc can be determined at low concentrations. Generally used to carry out qualitative as well as quantitative analysis for a great aromatic compounds present in cigarette smoking, air pollutant concentrates & automobile exhausts. Determination of inorganic substances. Extensively used in the field of nuclear research for the determination of uranium salts. Determination of vitamin B1 (thiamine) in food samples like meat cereals etc. Determination of Vitamin B2 (riboflavin).

30. This method is generally used to measure the amount of impurities present in the sample. Most important applications are found in the analyses of food products, pharmaceuticals, clinical samples and natural products. Fluorescent indicators: Intensity and colour of the fluorescence of many substances depend upon the pH of solutions. These are called as fluorescent indicators and are generally used in acid base titrations. Eg: Eosin – pH 3.0-4.0 – colourless to green. Fluorescien – pH 4.0-6.0 – colourless to green