Spectroflourimetry

MAT

1. SPECTROFLOURIMETRY Modern Pharmaceutical Analytical Techniques 1

2. CONTENTS Introduction Definition Theory Factors affecting fluorescence Instrumentation Application Conclusion Reference 2

3. INTRODUCTION Absorption of UV-VIS radiation causes transition of electrons from ground state to excited state. As excited state is not stable so, excess energy is released by Collisional deactivation photoluminescence This study or measurement of this emitted radiation is the principle of Flourimetry. Phosphorescence is also related phenomena, which is study of emitted radiation when electron undergoes transition from triplet state to singlet ground state . 3

4. DEFINITION 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 e.g., free radical ↑ or ↓ Triplet state: a state in which unpaired electrons of same spin present ↑ (unpaired and same spin) Singlet excited state: a state in which electrons are unpaired but of opposite spin like ↑ (unpaired and opposite )   4

5. Collisional deactivation: in which entire energy is lost due to collisional deactivation and no radiation is emitted. Fluorescence: a part of energy is lost due to vibrational transitions and remaining energy is emitted as UV-VIS radiation of longer wavelength. Fluorescence is the phenomena of emission of radiation when the is transition from single excited state to singlet ground state. The wavelength of absorbed radiation is called excitation wavelength. The wavelength of emitted radiation is called emission wavelength. 5

6. Phosphorescence: at favourable conditions like low temp. 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 undergoes transition from triplet state to singlet ground state is called as phosphorescence . 6

7. Fig:- Phenomena of fluorescence and phosphorescence. 7

8. THEORY Both fluorescence and phosphorescence are types of photoluminescence (luminescence) L uminescence is described by using a molecular-energy interpretation . Fluorescence of organic molecules means emission of radiant energy during a transition from the lowest excited singlet state to the singlet ground state . Phosphorescence of organic molecules means emission of radiant energy during a transition from the lowest excited triplet state to the singlet ground state .   8

9. In phosphorescence, an intersystem crossing can take place readily from to one of the vibrational levels of state that has very nearly the same energy level (process III). This is followed by non radiative decay (process IV) to the level.   9

10. Fig; Partial energy diagram for a photo luminescent system 10

11. Intersystem crossing process involves a change in the spin of the excited electron and thus a change in spin multiplicity. The triplet state is metastable, and molecules populating it have excess energy. This energy can be lost by Phosphorescence (process V) Oxygen quenching: By collision   11

12. Differences between fluorescence and phosphorescence Phosphorescence may sometimes persist for many seconds after the excitation source is removed. Fluorescence emission is always at shorter wavelength than that of phosphorescence . Fluorescence is usually observed at room temperature in liquid solution, while phosphorescence is observed in rigid medium at very low temperature . Fluorescence life time is usually in the range 10 -7 -10 -9 sec, while phosphorescence lifetime is usually in the range 10 -4 -10 sec. 12

13. Half life time: It is the time required for half of the molecules to emit photons and thus return to the ground states. Effect of molecular structure on luminescence properties 13

14. Fluorescence may be expected generally in; Aromatic molecules that contain conjugated double bonds Polycyclic aromatic compounds (with great number of π electrons) Substituents strongly affect on the fluorescence; substituents such as NH 2 , NHCH 3 , N(CH 3 ) 2 , OH and OCH 3 groups enhance the fluorescence, while electron with drawing group such as NO 2 Cl-, Br-, I- and COOH groups decrease the fluorescence Formation of metal chelates promotes the fluorescence. 14

15. Phosphorescence may be expected generally in Aromatic hydrocarbons Introduction of substituents such as NH 2 , SH, OH to aromatic hydrocarbon enhance the phosphorescence and also aromatic nitro compounds Majority of aromatic aldehydes and ketones show phosphorescence . 15

16. Fluorescence Spectra Instruments that measure the intensity of fluorescence are called fluorimeter. Those that measure the fluorescence intensity at variable wavelengths of excitation and emission, and are able to produce fluorescence spectra are called spectrofluorimeters In the recording the fluorescence spectra, the limitations of light sources and measuring devices assume real significance. These limitations are: Variation of the intensity of available energy with λ. Variation in the response of the detector to light of different wavelengths 16

17. . Excitation Spectra: Before a compound can fluoresce, energy must be observed, and with an ideal light source, of constant intensity at different wavelengths, the most intense fluorescence is produced by radiation corresponding in wavelength to that of the absorption peak of the substance. Therefore, if the intensity of the fluorescence is plotted as a function of the wavelength of the radiation used to excite the fluorescence, an activation or excitation spectrum will, result. This will be identical to the absorption spectrum when corrected for instrumental effect, because the fluorescence efficiency is greatly independent of λ . 17

18. Fig; Fluorescence excitation and emission spectra for a solution of quinine 18

19. Emission Spectra (Fluorescence) When a monochromator source of constant light intensity is used to irradiate a sample, the fluorescence may be analysed in a monochromator at constant slit width to give apparent emission spectrum. The true spectrum is obtained by applying a correction for change in detector sensitivity with wavelength and for changes due to fluorescence monochromator i.e., half band width of emergent light and light losses. Fluorescence emission spectra arise because of transition from the first excited state and their shapes are therefore independent of the light used to excite fluorescence. 19

20. FACTORS AFFECTING FLUORESCENCE INTENSITY; Concentration Quantam yield Intensity of incident light Pathlength (b ) Adsorption Oxygen pH Temperature and viscosity Scatter   20

21. Concentration : The fluorescence intensity of a substance is proportional to concentration only when the absorbance in a 1 cm cell is less than 0.02. If the concentration of the fluorescent substance is so great that all incident radiation is absorbed, the equation will be: F = ϕ That is the fluorescence is independent of concentration, and proportional to the intensity of incident radiation only, a property that may be utilized to determine the approximate emission characteristics of a light source   21

22. Diagrammatic representation of the variation of fluorescence intensity with concentration. Region (a): Proportional relationship Region (b): Negative deviation from linearity. Region (c): Fluorescence independent of concentration Region (d): Reabsorption of fluorescence 22

23. Quantum yield of fluorescence ( ϕ ) This is the ratio : ϕ Since some absorbed energy is lost by radiation less pathways , the quantum efficiency is less than 1. Highly fluorescent substances take ϕ value near 1, which shows that most of the absorbed energy is re-emitted as fluorescence. For e.g .; fluorescein in 0.1 M NaOH and quinine in 0.05 M H 2 SO 4 have, ϕ values of 0.85 and 0.54 respectively at 23°C. Non-fluorescent substances have ϕ = 0.   23

24. 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 depends on the intensity of light emitted from the lamp.   24

25. Pathlength (b ) The effective pathlength viewed by the detector depends on both the excitation and emission slit widths. 25

26. Adsorption : The extreme sensitivity of the method requires very dilute solution, 10-100 times, weaker than those employed in absorption spectrophotometry. Adsorption of the fluorescent substance on the container walls may therefore presents serious problems and strong stock solutions must be kept and diluted as required. Quinine is a typical example of a substance which is adsorbed onto cell walls. 26

27. Oxygen The presence of oxygen may interfere in two ways: By direct oxidation of the fluorescent substance to non-fluorescent products . By quenching of fluorescence. 27

28. pH It is to be expected that alteration of the pH of a solution will have a significant effect on fluorescence if the absorption spectrum of the solute is changed. 28

29. Temperature and viscosity Variation in temperature and viscosity will cause variations in the frequency of collision between molecules. Thus, an increase in the temperature or the decrease in the viscosity is likely to decrease the fluorescence by deactivation of the excited molecules by collision. 29

30. Quenchers Quenching is the reduction of the fluorescence intensity by the presence of substances in the sample other than the fluorescent analyte . Static Quenchers : Form a chemical complex with the fluorescent substance and alter its fluorescence characteristics. Certain xanthine derivatives e.g. caffeine, reduce the fluorescence of riboflavin by static quenching. 30

31. Scatter T he excitation and emission monochromators are at the same wavelengths, scattered light of the same wavelength as the incident light will be detected by the photomultiplier arising from colloidal particles in the sample (Tyndall scatter) and from the molecules (Rayleigh scatter) 31

32. . Raman Scatter: Arises from the conversion of some of the incident radiation into vibrational and rotational energy by the solvent molecules. The resultant scattered light is of lower energy and, consequently of longer wavelength . 32

33. INSTRUMENTATION When both the excitation and emission spectra are to be recorded, two monochromator are essential, one for the light source (excitation monochromator) and one for the fluorescence (emission monochromator). The light source must provided a high level of UV and Visible radiation and a compact high pressure Zenon arc lamp is used. The production of ozone by the photochemical conversion of atmospheric oxygen in the lamp compartment presents a toxic hazard unless the ozone is thermally decomposed or removed by adsorption onto charcoal. As many experiments will almost certainly entail the measurement of very weak fluorescence. The detector must be a highly sensitive photomultiplier tube of low dark current. If the main interest lies in the fluorescence emission spectra, one monochromator may be dispensed with a suitable light source 33

34. and filter used instead. The rather poor luminosity associated with the monochromator even with a xenon arc lamp is replaced by the much more intense light from a source such as a mercury vapour lamp, from which a suitable activation beam is isolated by means of the filter. This arrangement partially overcome, one of the difficulties inherent in spectrofluorimetry, i.e., that so much of the available light is lost. 34

35. Fig; simple fluorometer 35

36. ADVANTAGES OF SPECTROFLUORIMETRY High sensitivity Selectivity Quantitative Aspects 36

37. Quantitative Aspects: Many of the quantitative aspects of spectrofluorimetry may be understood by reference to the fundamental equation for the intensity of the fluorescence emitted. This equation may be derived from that of the Beer-Lambert law: A = Log / = abC Or / = ∴ = x   37

38. but fluorescence (F) = ( - ) ϕ where ϕ is the quantum yield of the fluorescence At very low absorbance (< 0.02), the equation will be F = 2.3 abC ϕ For a fixed set of instrumental ( and b) and sample (a and ϕ ) parameters, the fluorescence is proportional to the concentration. F= K C where K = 2.3 a b ϕ   38

39. APPLICATIONS OF SPECTROFLUORIMETRY Compounds which are fluorescent are readily determined with simple instruments as the solution for examination is normally obtained by dissolution of the sample in a suitable solvent (table 1) Compound pH Λ max Excitation emission Min. concn required ( μ g/ml) Adrenaline 1 295 335 0.1 Allyl morphine 1 285 355 0.1 Amylobarbitone 14 265 410 0.1 Chloroquine 11 335 400 0.05 Chlorpromazine 11 350 480 0.1 Cinchonidine 1 315 445 0.01 39

40. Single substances which are in themselves, non-fluorescent may be determined as a result of chemical change. e.g .; Determination of primary amines, amino acids, peptides etc. through: 40

41. Reaction with fluorescamine reagent 41

42. Determination of primary and secondary aliphatic amines through: a- reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole ( NBD-CI ) give yellow fluorescence 42

43. b- reaction with l-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl, chloride) 43

44. Thiamine HCI in pharmaceutical preparations such as tablets and elixirs and in food stuffs such as flour is relatively easily determined by oxidation to highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol and hence is easily extracted from the reaction mixture for measurements 44

45. For mixture of two components, it may be possible to select the exciting radiation of appropriate wavelengths, such that only one compound fluoresces at any time. Even if there is not possible, measurements of the fluorescence at two wavelengths may be sufficient to determine the composition of the mixture. 45

46. CONCLUSION Fluorescence is most sensitive analytical techniques. Detection studies will increase the development of fluorescence field. Flurometric method are much useful in qualitative analysis as compared to quantitative analysis. 46

47. REFERENCES Douglas a, S koog , Holler, PRINCIPLE OF INSTRUMENTAL ANALYSIS , 5 th edition , S aunders college, west W ashington square, P hilidhepia D r Ravishankar , A TEXTBOOK OF PHARAMACEUTICAL ANALYSIS, 3 rd edition, Rx pub., 57, west car street, Tiruneveli-627006 G r C hatwal , S.K. A nand, INSTRUMENTAL METHOD OF CHEMICAL ANALYSIS , H imalaya pub. house pvt ltd, R amdoot', Dr. B haleraomarg, girgaon , Mumbai - 400 004, M aharashtra, I ndia 47

48. THANK YOU 48