2. Fluorimetry.pdf

1. Chapter 2 Fluorescence Spectroscopy 1

2. Key points Principles • Certain molecules, particularly those with a chromophore and a rigid structure, can be excited by UV/visible radiation, and will then emit the radiation absorbed at a longer wavelength. The radiation emitted can then be measured. Applications • Determination of fluorescent drugs in low-dose formulations in the presence of non-fluorescent excipients. • In carrying out limit tests where the impurity is fluorescent or can be simply rendered fluorescent. • Useful for studying the binding of drugs to components in complex formulations • Widely used in bioanalysis for measuring small amounts of drug and for studying drug-protein binding. 2

3. KEYPOINTS (Continued) Strengths • A selective detection method and can be used to quantify a strongly fluorescent compound in the presence of a larger amount of non-fluorescent material. • Can be used to monitor changes in complex molecules such as proteins, which are being used increasingly as drugs. Limitations • The technique only applies to a limited number of molecules. • Fluorescence is subject to interference by UV-absorbing species, heavy ions in solution, and is affected by temperature. 3

4. Introduction • Luminescence spectroscopy is an analytical method derived from the emission of light by molecules which have become electronically excited subsequent to the absorption of visible or ultraviolet radiation. • • Due to its high analytical sensitivity (concentrations of luminescent analytes (1x10-9 moles/L) are routinely deter-mined), – this technique is widely employed in the analysis of drugs and metabolites. 4

5. Introduction… • Luminescence spectroscopy may be divided into two major areas: 1. Fluorescence spectroscopy and 2. Phosphorescence spectroscopy • The differences between the two are based mostly on the time frames on which the phenomena of ﬂuorescence and phosphorescence occur • Phosphorescence decays much more slowly (often taking several seconds) than ﬂuorescence subsequent to excitation. 5

6. Introduction… • Deactivation as fluorescence is a rapid process occurring within 10-6 to 10-9 seconds of excitation. • The average lifetime for phosphorescence ranges from 10–4 to 104 . • Phosphorescence may continue for some time after removing the excitation source. 6

7. Introduction… • After the absorption of U-Vis light, the excited molecular species are extremely short lived and deactivation occur due to – Internal collision (internal conversion) – Cleavage of chemical bonds, initiating photochemical reaction – Re-emission as light (Luminescence) • Molecules on excitation normally possess higher vibrational energy than they had in the ground state. 7

8. Introduction… • In excited molecules which exhibit fluorescence, the spin of π electron and that π* electron, which together constitute a π bond in the chromophore system are in opposite directions, i.e. they are anti parallel (Singlet excited state). 8

9. Introduction… • Some excited molecules, particularly at low temperature, many undergo a slow intersystem crossover to a state (Triplet excited state) in which the spin of the π and π * electrons are unpaired (Parallel) • Return from the Singlet excited state to the ground state results in the emission of Fluorescence. • Return from the Triplet excited state to the ground state results in the emission of phosphorescence. 9

10. Introduction… 10

11. Introduction… TERMS (non radiation conversions)  Internal conversion: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level.  External conversion: A form of radiationless relaxation in which energy is transferred to the solvent or sample matrix  Intersystem crossing: A form of radiationless relaxation in which the analyte moves from a higher electronic energy level to a lower electronic energy level with a different spin state 11

12. Introduction… • The difference in the energy level (E) b/n the excited and the unexcited state during excitation (Absorption), fluorescence and Phosphorescence are in the order of E (Absorption) > E (Fluorescence) > E (Phosphorescence) 12

13. 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. 13

14. Fluorescence… • Fluorescence occurs when a molecule in the lowest vibrational energy level of an excited electronic state returns to a lower energy electronic state by emitting a photon • A quantitative expression of the efficiency of fluorescence is the fluorescent quantum yield (ØF) • It is the fraction of excited molecules returning to the ground state by fluorescence. 14

15. Fluorescence… 15 For low concentrations of the fluorescing species,

16. Fluorescence… • Quantum yields range – from 1:- when every molecule in an excited state undergoes fluorescence, – to 0:- when fluorescence does not occur. • The intensity of fluorescence therefore, increases with an increase in – quantum efficiency, – incident power of the excitation source, – the molar absorptivity and – concentration of the fluorescing species. 16

17. Fluorescence… Excitation Versus Emission Spectra • Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength. • An excitation spectrum is obtained by monitoring emission at a fixed wavelength while varying the excitation wavelength. • In an emission spectrum a fixed wavelength is used to excite the molecules, and the intensity of emitted radiation is monitored as a function of wavelength. 17

18. Fluorescence… • Although a molecule has only a single excitation spectrum, it has two emission spectra, one for fluorescence and one for phosphorescence. 18 Figure: Example of molecular excitation and emission spectra.

19. Fluorescence… Advantages of Fluorescence spectroscopy 1. Sensitivity • Substances that are reasonably fluorescent may be determined at concentration of up to 1000 times less than those required for absorption spectroscopy. • In spectrofluorimetry the detector measures single light intensity which may be amplified electronically many times • In UV/Vis, the detector measures two intensities Io and It and reasonable difference( It=0.05 Io to 0.5 Io) should exist for accurate and precise measurement of absorbance. 19

20. Fluorescence… vSamples containing < 1 mg/dose – Biological samples( blood, urine) containing low concentration of drugs, – Hormones, – Alkaloids and vitamins in formulation or biological samples. 2.Selectivity • Not all substances that absorb in the UV-Visible fluoresce • Wavelength of excitation /emission/ can be easily varied to selectively measure the fluorescence 20

21. Fluorescence… Factors affecting Fluorescence intensity Concentration • In order for a molecule to fluorescence it must first absorb radiation. • If the concentration of the absorbing substance is very high, all the incident light may be absorbed by first layer of solution 21

22. Factors affecting Fluorescence intensity… Concentration…. • Over a wide range of solute concentrations, solute–solute interactions may also account for loss of luminescence intensity with increasing solute concentration. • The fluorescence of such samples will therefore be non uniform and will not be proportional to the concentration of the substance. 22

23. Fluorescence… Factors affecting … Temperature and Viscosity • A molecule’s fluorescence quantum yield is also influenced by external variables such as temperature and solvent. • Increasing temperature generally decreases fluorescence because more frequent collisions between the molecule and the solvent increases external conversion. • Decreasing the solvent ’s viscosity decreases fluorescence for similar reasons. 23

24. Fluorescence… Factors affecting … pH of the solution • The inﬂuences of pH on luminescence spectra are derived from the dissociation of acidic functional groups or the protonation of basic functional groups, associated with the aromatic portions of ﬂuorescing molecules. • Fluorescence intensity from excited states of charged and uncharged species is generally different. (i.e. change in pH alter the ratio of charged and uncharged species) 24

25. Fluorescence… Factors affecting … Oxygen • The presence of oxygen may interfere in two ways – By direct oxidation of fluorescence substances to non- fluorescence products – By Quenching of fluorescence(quenching:- any process which decreases fluorescence intensity) 25 F + hv--------F* F*------F + hv F* + Q-------------F + Q

26. Fluorescence… Factors affecting… The effect of other solutes • Fluorometry is not ideal for the analysis of mixtures due to its un predictability of the effect of one cpd on another. • H a l o g e n s , h e a v y a t o m s , q u e n c h fluorescence( decrease fluorescence efficiency) 26

27. Fluorescence… Molecular structure and fluorescence • In order for a molecule to exhibit fluorescence it is necessary that the excited molecule return to the ground state via a radiative transition from the excited singlet. • It has been found the probability of a radiation transfer from singlet to triplet state is higher for an n-pi* than pi- pi* • Thus the latter will have high probability of fluorescence. 27

28. Fluorescence… Molecular structure… • Fluorescence is generally observed with molecules where the lowest energy absorption is pi-pi* transition, although some n-p* transitions show weak fluorescence. • Most unsubstituted, non-heterocyclic aromatic compounds show favorable fluorescence quantum yields. –Most aromatics fluoresce 28

29. Molecular structure… • Fluorescence also increases for aromatic ring systems and for aromatic molecules with rigid planar structures. 29

30. Fluorescence… INSTRUMENTATION Fluorescence spectrophotometers consist of • A light source- to provide the excitation energy, • A device for selecting the excitation wavelength • A sample compartment • A second device for selecting the emission wavelength • Photodetector and • A data acquisition and recording device to determine the intensity of ﬂuorescence at any given wavelength (fig. below) 30

31. Fluorescence… INSTRUMENTATION… 31 Schematic diagram of a spectroﬂuorimeter

32. Fluorescence… INSTRUMENTATION… Light source • Gas discharge lamps are the most commonly used light sources. • These lamps consist of two electrodes in a carrier gas at high pressure, across which a high potential difference is applied. • The carrier gas employed is frequently the inert gas xenon. 32

33. INSTRUMENTATION… Light source… • The high potential difference ionizes the carrier-gas atoms, which then accelerate toward the cathode, generating, through collisions, other excited states which emit radiation upon returning to their ground states. Mercury arc lamps and Xenon arc lamps are in common use • Both lamps emit in the visible and Uv region 33

34. Fluorescence… INSTRUMENTATION… Wavelength Selection Devices • The wavelength selection devices employed in fluorimetry are either filters or grating monochromators. • The filters are either absorption or interference based. • However, the filters are not very useful in scanning instruments. • Monochromators consist of an entrance slit, a dispersion device, and an exit slit. 34

35. INSTRUMENTATION… Wavelength Selection Devices… • The dispersion device is usually a diffraction grating, though a prism may still be used in the older devices. • The gratings are preferred than the prisms, as they are – less expensive, – have uniform resolution, – a linear dispersion throughout the ultraviolet–visible range 35

36. Fluorescence… INSTRUMENTATION… • There are usually two monochromators in ﬂuorescence spectrophotometers, – an excitation monochromator and – an emission monochromator. 36

37. INSTRUMENTATION… Sample Compartment • The sample compartment has its internal surfaces painted a ﬂat black and is covered during measurement to minimize stray light. • The sample compartment is normally positioned so that the excitation and emission monochromators are at right angles to each other, so as to minimize interference from stray excitation light. 37

38. Fluorescence… INSTRUMENTATION… Sample Compartment… • The ﬂuorescence cells are constructed of either quartz or silica, as these materials are able to transmit light of wavelength as low as 200 nm up to well into the near- infrared. • Occasionally, cheaper glass or plastic cells are employed, where the excitation wavelength range is above 330 nm. • The sample cells are normally rectangular, with a horizontal cross-sectional area of 1 cm 2 for room- temperature ﬂuorescence spectroscopy. 38

39. Fluorescence… INSTRUMENTATION… Photodetectors • The most widely used photodetectors are the photomultiplier tubes. These tubes generate an electrical signal upon exposure to light. • For the measurement of low light intensities, photon counting has been employed. • In photon counters the photoelectron pulses at the anode of the photomultiplier tube are counted using a high- speed electronic counter. 39

40. INSTRUMENTATION… Photodetectors… • One pulse accounts for each electron ejected from the photocathode • And the mean pulse count rate is proportional to the light intensity. 40

41. Fluorescence… INSTRUMENTATION… Data Acquisition Devices • The electronic signal obtained from the photodetector is usually electronically ampliﬁed, measured using some sort of galvanometer, and presented in either analog or digital form. • The signal may alternatively be recorded on a strip- chart recorder, to supply a permanent record of the spectrum. 41

42. Fluorescence… Applications • Concentration of drugs and drug metabolites in blood, urine and other biological fluids samples may be extremely low and fluorescence analysis finds wide application in quantitative studies of rates and mechanism of absorption, metabolism and excretion studies( PK studies) • Fluorometric drug analysis can be classified in to three chemical ways. 42

43. Fluorescence… Applications… A) Those drugs that possess intrinsic fluorescence. ØThese require no chemical reactions to create a fluorescent compound. Example Quinine B) Those drugs which can be derivatized to the forms by attaching fluorescent compounds to the drug. Ø Eg. Dansyl chloride can react with aminoacids to give dansyaminoacids which are highly fluorescent 43

44. Fluorescence… Applications… c) Those drugs which needs more extensive molecular change than simple derivative formation. ØEg. Thiamine HCl 44

45. THE END 45

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