Fluorimetry-M0dern pharmaceutical analysis[1].pptx

fluorescence spectroscopy

1. Modern Pharmaceutical Analytical Techniques Fluorimeter

2. Fluorescence Spectroscopy - Principle of Fluorescence - Factors governing fluorescence intensity - instrumentation - applications - advantages

3. What is flourimeter? Instruments that measure the intensity of fluorescence – flourimeter or Fluorometer Instruments that measure the fluorescent intensity at variable wavelengths of excitation and emission - spectroflourimeter

4. What is Fluorescence ? Spontaneous emission of a photon from excited electronic state. due to a chemical reaction. Substances that absorb UV light lose excess energy as heat through collisions with neighboring atoms or molecules. However, a large numbers of important substances are also known which lose only part of this excess energy as heat and emit the remaining energy as electromagnetic radiation of a wavelength longer than that absorbed . This process of emitting radiation is collectively known as luminescence.

5. PHOTOLUMINESCENCE Fluorescence : Does not involve change in electron spin; short lived (less than microsecond). Can be observed at room temperature in solution. 2. Phosphorescence: Involves change in electron spin. Long lived (seconds). Can be observed at low temperature in frozen or solid matrices.

6. Principle

7. From lowest vib. level (excited state) molecule can relax to ground state in various ways: Spontaneous emission of a photon: fluorescence Internal conversion Quenching Intersystem crossing

8. Fate 1: Fluorescence Molecule emits a photon spontaneously Assuming no stimulated emission, i.e. no laser effect. Occurs from lowest vib. state to various vib. excited states of lowest electronic state. Intrinsic rate k F = 1/  R Fluorescence: 10 -8 sec S S 1

9. Fate 2: Internal Conversion (molecule passes to lower energy electronic state without emission of radiation) Non-radiative dissipation of energy. Occurs from collision w/solvent, internal vibration. This rate increases with temperature. (Why? faster motions ). Competes with fluorescence Fluorescence intensity decreases w/temp. Major intrinsic temp. dependence of Fluorescence Hard to monitor Fl. as a function of temp Loss of energy by external conversion : energy transfer from excited molecule to solvent or solute

10. Fate 3: Quenching De-excitation due to collisions with solutes (Q) Bi-molecular (e.g O 2 , Iodine, etc.), Other parts of molecule (Trp quenches Tyr fluor) Good quenchers are very efficient and are just diffusion limited, so at mM [Q], collisions up to 10 8 s -1 . So quenching can be a big factor. O 2 quenching of protein fluorescence

11. Fate 4: Intersystem Crossing Crossover from S to T state (spin forbidded transition) This form can: a) relax to S either by emission of a photon (now at longer wavelengths = lower energy): Phosphorescence b) relax by internal conversion In practice, phosphorescence very low intensity, very long lifetime (seconds or longer) vs. ns for other processes. quenching and I.C. usually decrease phosphorescence to negligible levels except in solids.

12. Intersystem Crossing: 10 -8 sec S S 1 Phosphorescence: up to sec or longer 10 -4 to 10 sec T Direct excitation to triplet state is not shown. Because the transition Has very low probability of occurance. This type of low probability of occurance is called forbidden transition. On excitation two states may be formed Exc ited SS & Excited TS The energy of excited triplet state is less than the excited singlet state

13. STOKES SHIFT -EXCITATION AND EMISSION WAVELENGTHS ARE DIFFERENT Anti stokes florescence If thermal energy is added to an excited state or a compound has many highly populated vibrational energy levels, emission at shorter wavelengths than those of absorption occurs . This is often observed in dilute gases at high temperature. RESONANCE SHIFT EXCITATION AND EMISSION WAVELENGTHS ARE SAME

14. Molecule should have less internal conversion and less triplet state formation to get high fluorescence

15. SINGLET AND TRIPLET STATES Electronic transitions responsible for absorption spectra will give rise to singlet and triplet states and hence to fluorescence and phosphorescence. 1.Ground state: electronic state where all electron spins are paired 2. Singlet State: Electron spins in the ground and excited electronic states are paired. Net spin S is zero. 3.Excited singlet state 4.doublet state: Ground state of free radical is doublet state. 5.Triplet state: atom or molecule that contain two unpaired electrons both having the same spi n Excited Triplet State: Electron Spins in the excited electronic states are not paired. Net spin S = 1.

16. Factors affecting flourescenece Both molecular structure and chemical environment are influential in determining whether a substance is flourescent or not 1.Molecules which absorb UV or Vis radiation will only show flourescence.( greater the absorbance greater the luminescence. Eg.molecules have aromatic or multiple conjugated double bonds )

17. Non-flourescent

18. FLUORESCENCE AND STRUCTURE Fluorescence from singlet states of -  * have more intensity than those from n-  * transitions as the molar absorptivities for -  * absorptions are much higher than those for n-  * absorptions. 5. Simple heterocycles do not exhibit fluorescence. The n-  * singlet quickly converts to the n-  * triplet and no Fluorescence is observed.

19. FLUORESCENCE AND STRUCTURE (CONTD) 6. Fusion of heterocycles to benzene rings increases the molar absorptivity for n-  * absorptions and shortens the lifetime of the n-  * singlet preventing its conversion to triplet. This increases fluorescence quantum efficiency.

20. Molecular Rigidity 8b)Chelation also can lead to increased fluorescence. Fluorescent intensity of zinc complex is greater than 8 hydroxy quinoline

21. Substition of EDG like NH 2 ,OH, F, OR, NHR in aromatic hydrocarbon enhance flourescence Substitution of EWG like Cl , Br, I, NHCOCH 3 COOH,NO 2 decrease flourescence completely Groups like SO 3 H and NH 4+ does have no effect

22. Aniline in acid is converted to anilium ion and so loses lone pair of elctron and causes less flourescence

23. 7. HEAVY ATOM EFFECT A very significant influence on the fluorescence quantum yield of the benzene ring which is due to -  * singlet states is observed with halogen substitution. The quantum yield decreases with the atomic number of the halogen. This called the heavy atom effect . For atoms of higher atomic number interactions with spin and orbital motion is large and change of spin is more favorable. Thus the probability for intersystem crossing increases with increasing atomic number of the halogen which reduces fluorescence. Substitution of carboxylic acid or carbonyl group on benzene generally inhibits fluorescence due to the n-  * states being lower in energy than the -  * states and these do not fluoresce efficiently.

24. 8.STRUCTURAL RIGIDITY 8a)Fluorescence is favored in molecules with structural Rigidity. The quantum yields for fluorescence for Fluorene and biphenyl are 1 and 0.2 respectively. The increased rigidity of fluorene stabilizes the -  * singlet state leading to higher quantum yield. Phenanthrene & cis stilbene,phenolpthalein & Flourescein

25. 9.QUANTUM YIELD Quantum yield or quantum efficiency ( ) for fluorescence or phosphorescence is the ratio of the number of molecules that luminesce to the total number of molecules that are excited. For a highly fluorescent molecule such as fluorescein  = 1 and for a non-luminescing molecule  = 0. The quantum yield of most of the molecules decreases with increasing temperature . This is because the frequency of collisions increases and hence the probability of deactivation by external conversions also improves. Molecule with high quantum yield have any one or more of the following structural components 1.High molar absorptivity 2.A number of highly conjugated double bonds 3.An EDG 4.A relative high rigid structure or metal complex of molecule

26. TEMPERATURE AND SOLVENT EFFECTS Quantum yield of fluorescence of most molecules decreases with increasing temperature due to collisional deactivation of the singlet state. Fluorescence is decreased by solvent containing heavy atoms such as as those containing halogens. Heavy atoms promote intersystem crossing to the triplet state. This decreases fluorescence quantum yield but increases phosphorescence quantum yield.

27. 10.Solvent effects:Solvents containing heavy atoms (ethyl iodide or ethyl bromide) or other solutes with such atoms in their structure decrease the flourescence. 11. Presence of dissolved oxygen Oxidise the flouroscent subs to non flouroscent decreases fluorescence.(due to photochemical induced oxidation of fluorescent material) 12. Changes in pH influence the degree of ionization, which, in turn, may affect the extent of conjugation or the aromaticity of the compound. pH exhibits marked effect of fluorescence. Eg.Neutral and alkaline solution of aniline shows fluorescence in visible region, but in acidic solution fluorescence disappears. Phenol in acid wont give fluorescence but in alkali it does.

28. Why is spectrofluorometry potentially more sensitive than spectrophotometry? The key difference is that spectrofluorometry measures the intensity of fluorescence, while spectrophotometry (absorption spectroscopy) measures the ratio of the intensities of two beams (incident and passed beams). The fluorescence signal is directly proportional to the intensity of the excitation source. F∝Io We can increase the intensity of the excitation source (use a laser!) and gain a subsequent increase in the fluorescence signal—potentially leading to greater sensitivity. Spectrophotometry, on the other hand, is an absorption technique. Absorption depends on the ratio of incident to passed light: A=logIo/It so simply increasing I also increases It.

29. High sensitivity Conc upto1000 times lower than reqd for absorption spectrophotometer can be detected. Flu-PMT measures single light intensity that can be amplified many times without noise UV-VIS-PMT measures two intensities Io & It with reasonable diff. At low absorbance, the small diff approached to noise of signal and cannot be measured with reasonable precision.

30. Selectivity Fluorometry is more selective than UV/Vis absorption spectrometry for two reasons. First, many molecules absorb strongly in the UV or visible range but do not exhibit detectable fluorescence. Second, two wavelengths (excitation and emission) are available in fluorometry, but only one wavelength is available in absorptiometry. If two sample constituents with similar absorption spectra f luoresce at different wavelengths, they may be distinguished from one another by appropriate choice of emission wavelength . Similarly, two compounds that have similar fluorescence spectra but absorb strongly at different wavelengths may be distinguished by proper choice of excitation waveleng th (selective excitation).

31. Quenching Quenching is the process of reduction in the intensity of fluorescence. It can be of three types 1.collisional quenching or dynamic quenching 2.static quenching 3.chemical quenching Fl int decreases as the conc of quencher increases

32. Collisional quenching involving collisions with other molecules that result in the loss of excitation energy as heat instead of as emitted light. This process is always present to some extent in solution samples; species that are particularly efficient in inducing the process are referred to as collisional quenchers (e.g. iodide ions, molecular oxygen, nitroxide radical). It occurs when fluorophore and another molecule diffuse in solution and collide with each other . Here these two molecules donot form complex. Static quenching . Interaction of the fluorophore with the quencher forms a stable non-fluorescent complex . (dark complex) Since this complex typically has a different absorption spectrum from the fluorophore, presence of an absorption change is diagnostic of this type of quenching (by comparison, collisional quenching is a transient excited state interaction and so does not affect the absorption spectrum). Eg complex formation of caffeine with riboflavin reduces fluorescence Chemical quenching: fluorophore is chemcially converted to nonflurophore

33. Trp fluorescence (excitation at 280 nm) fluorescence without quencher

34. Self quenching Excessive absorption of fl subs itself Linear curve for low conc NonLinear curve for high conc

35. Instrumentation Light source Filters/monochromators Sample holder Detector Recorder

36. 1.Light source Fluorometer -- Low pressure mercury vapour lamp This source produces intense lines at certain wavelengths. One of these lines will usually be suitable for excitation of a fluorescent sample. Spectrofluorometers, which need a continuous radiation source, are often equipped with a 75-450 W high-pressure xenon arc lamp . Lasers are sometimes used as excitation sources. A tunable dye laser, using a pulsed nitrogen laser as the primary source can produce monochromatic radiation between 360 and 650 nm. Since the radiation produced is monochromatic, there is no need for an excitation monochromator. Deuterium or hydrogen arc lamp are not used because they donot produce more intense beam required for fluorimetry .

37. 1. SOURCE OF LIGHT: Mercury vapour lamps Xenon arc lamps Tungsten lamps Lasers LED’s

38. THE ELEMENTS OF A FLUORESCENT LAMP A fluorescent lamp contains the following basic elements: Bulb  Electrodes  Gases Base  Phosphors  Mercury

39. Mercury vapour lamp The fluorescent lamp produces light by the passage of an electric current flowing through a vapor of mercury. Electron emitted from electrode collides with mercury atom. Impact produces ultraviolet rays Phosphor converts ultraviolet to visible light. This process is known as “fluorescence,” hence the name fluorescent lamp.

40. THE ELEMENTS OF A FLUORESCENT LAMP THE BASE The base provides the means of holding the lamp firmly in the lamp holders or sockets and providing the electrical connections for the lamp/ballast circuit. The basic types are: Bipin – Used on preheat and rapid start lamps.

41. THE ELEMENTS OF A FLUORESCENT LAMP THE ELECTRODES Coiled tungsten wires coated with an emission material When heated, emit electrons Electrons bombard mercury atoms producing ultraviolet rays. THE PHOSHPORS Phosphors are the coated powders on the inside of the bulb that convert the ultraviolet rays to visible light. There are two basic types: Halophosphates Trichromatics or Triband Phosphors

42. 2.Filters/monochromators Fluorometers use two filters to restrict excitation and emission beam wavelengths. Spectrofluorometers have two diffraction grating monochromators; one allowing choice of excitation wavelength and the other allowing fluorescence emission spectra to be scanned. Filters and monochromators Fluorometers use either interference or absorption filters. Spectrofluorometers are usually fitted with grating monochromators.

43. Why two filters/two monochromators are used in filter and spectroflourometers? First one for allowing UV radiation (which absorbs visible radiation) Second one allows flourscent radiation and absorbs UV radiation

44. Sample holder Glass upto 320 nm Below 320 nm quartz

45. Detectors Fluorescence signals are usually of low intensity,hence photomultiplier tubes are used as detectors. Diode-array detectors are sometimes used.

46. Two types Filter fluorometer Spectrofluorometer Single beam spectroflurimeter Double beam spectrofluorimeter

47. Single beam fluorometer

48. Double beam fluorometer Often, fluorescence spectrometers use double-beam optics to compensate for power fluctuations in the source. The fluorescent emission is measured at right angles to the incident beam. Emitted radiation passes through a second filter or monochromator to isolate the fluorescent peak for measurement. The reference beam passes through an attenuator to reduce its power to that of the fluorescent radiation.

49. Advantages Sensitive (1000 times lower conc can be detcted than absorp spectrscopy) Selective & specific – no interference -only flur spec Wide conc detection range than absorp spectrscopy Simple & fast low cost Disadvantage :only fluroscent subst can be detected

50. Applications of Flourimeter two main reasons extensive use of fluorimeter high level sensitivity and wide dynamic range instrumentation reqd is convenient modest cost 1.Molecules with intrinsic flourescence 2.Molecules that are not having flourescence converted to flourescent molecules by tagging 3. Molecules that are not having flourescence can be converted to flourescent molecules by structural change

51. Other examples: riboflavin, fluorescein

52. 70

53. 2.Molecules that are not having flourescence converted to flourescent molecules metal ions as fluorescent organic complexes Al,Zn,Mg,Ga flourescent strongly with oxine complex Al also forms flourescent complex with eriochrome black Al & F alizarin garnet Zr & Sn flavonol Berylium flourescent complex with quinizarin B,Ga,Si,Zr benzoin Uranium forms fluorescent complex with a mixture of sodium carbonate & sod fluoride

54. 2.Molecules that are not having flourescence converted to flourescent molecules by tagging Dansyl chloride - 5(dimethylamino) naphthalene -1-sulphonyl chloride + non fluorescent primary aliphatic and aromatic amines = blue or blue green flourescent compounds (sulphonamide adducts) Eg. Amino acids, compounds contaning free amino groups

55. Fluorescent tags 73

56. ANS, dansyl chloride, fluorescein are used for protein studies. Ethidium, proflavine and acridines are used for nucleic acid characterization. Ethidium bromide has enhanced fluorescence when bound to double stranded DNA but not single stranded DNA. 74

57. Molecules – structural change Determination of thiamine – thiochrome USP method potassium ferricyanide – oxidising agent Reagents & intermediates –non flourescent Diadv:excess of oxidant used (for completion of reaction) may decompose thiochrome may cause error in analysis(this can be overcome by using Hg 2+ ion in basic media)

58. End of Lecture