Specrtofluorometer

1. spectrofluorometer Presented by: Nour Elrashed Q.C master student Damascus University Faculty of Pharmacy 1

2. Contents : Introduction the principle of spectrofluorometer What can specrofluorometer do? The components of specrofluorometer Fluorescence Spectra Fluorescence and chemical structure Applications of Spectrofluorometer New pharmaceutical studies conducted by spectrofluorometer References 2

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4. History : The term fluorescence comes from the mineral fluorspar (calcium fluoride) when Sir George G. Stokes observed in 1852 that fluorspar would give off visible light (fluoresce) when exposed to electromagnetic radiation in the ultraviolet wavelength. 4

5. the principle of spectrofluorometer : It is an analytical device depends on the fluorescence phenomenon which is a short-lived type of photoluminescence created by electromagnetic excitation. That is, fluorescence is generated when a molecule transmits from its ground state So to one of several vibrational energy levels in the first excited electronic state, S1 , or the second electronic excited state, S2 , both of which are singlet states. Relaxation to the ground state from these excited states occurs by emission of energy through heat and/or photons.   5

6. The difference between the excitation and emission wavelengths is called the Stokes shift . Stokes’ studies of fluorescent substances led to the formulation of Stokes’ Law, which states that the wavelength of fluorescent light is always greater than that of the exciting radiation. Thus, for any fluorescent molecule, the wavelength of emission is always longer than the wavelength of absorption. 6

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8. What is The fluorescence quantum yield ( Φ f )? It is the quantitative expression of the fluorescence efficiency , which is the fraction of excited molecules returning to the ground state by fluorescence. Quantum yields range from 1, when every molecule in an excited state undergoes fluorescence, to 0 when fluorescence does not occur. 8

9. A molecule’s fluorescence quantum yield is influenced by external Variables such as: temperature viscosity of solvent pH Increasing temperature generally decreases Φ f because more frequent collisions between the molecule and the solvent increases external conversion.  Decreasing the solvent’s viscosity decreases Φ f for similar reasons.  For an analyte with acidic or basic functional groups, a change in pH may change the analyte’s structure and, therefore, its fluorescent properties. 9

10. Emission of a photon when the analyte returns from a higher-energy state to a lower-energy state with the opposite spin ( triplet excited state: An excited state in which unpaired electron spins occur.). Average lifetime from 10 -3 to 10 +2 sec Emission of a photon when the analyte returns from a higher-energy state to a lower-energy state with the same spin ( singlet excited state: An excited state in which all electron spins are paired). Average lifetime from 10 -9 to 10 -7 sec Phosphorescence Fluorescence 10

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13. What can specrofluorometer do? It has been used for the direct or indirect quantitative and qualitative analysis by measuring the fluorescent intensity F. It is relatively inexpensive and sensitive (the sensitivity of fluorescence is approximately 1,000 times greater than absorption spectrophotometric methods). 13

14. fluorescent intensity F is dependent on both intrinsic properties of the compound ( fluorescence quantum yield Φ f), and on readily controlled experimental parameters including: intensity of the absorbed light I molar absorption coefficient Ɛ path length of the cell b concentration of the fluorophor in solution c 14

15. At low concentrations of fluorophore , the fluorescence intensity of a sample is essentially linearly proportional to concentration. However, as the concentration increases, a point is reached at which the intensity increase is progressively less linear, and the intensity eventually decreases as concentration increases further. 15

16. fluorescence intensity concentrations 16

17. The most common reason for this is Inner filter effect that, as the absorbance of the sample increases, the light intensity experienced by some of the fluorescent molecules is lower than that experienced by others. When excitation intensity decreases, so does fluorescence emission intensity. It is generally necessary to use concentrations that result in absorbance values of 0.1 or lower to observe concentration dependent emission. 17

18. As the concentration of molecules in a solution increases, probability increases that excited molecules will interact with each other and lose energy through processes other than fluorescent emission. Any process that reduces the probability of fluorescent emission is known as quenching . Other parameters that can cause quenching include: presence of impurities increased temperature reduced viscosity of the solution media 18

19. The components of specrofluorometer : light source ( 75 to 450-W high-pressure xenon arc lamp or Lasers) excitation monochromator sample holder (Quartz/Optical Glass/Plastic Cells) emission monochromator Detector (photomultiplier) Most spectrofluorometers also have a reference sample. The reference is generally a solution of a strongly fluorescent molecule with a broad absorbance spectrum such as rhodamine . The reference is necessary to correct for lamp output , especially when varying the excitation wavelength, and to correct for differences in detector sensitivity . 19

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22. Fluorescence Spectra: Photoluminescence spectra are recorded by measuring the intensity of emitted radiation as a function of either the excitation wavelength or the emission wavelength. The excitation spectra is determined by measuring the emission intensity at a fixed wavelength , while varying the excitation wavelength. It is useful for selecting the best excitation wavelength for a quantitative or qualitative analysis. The emission spectra is determined by measuring the variation in emission intensity wavelength for a fixed excitation wavelength. 22

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24. Fluorescence and chemical structure: Fluorescence is generally observed with molecules where the lowest energy absorption is →* transition, although some n →* transitions show weak fluorescence. Most unsubstituted , nonheterocyclic aromatic compounds show favorable fluorescence quantum yields wich usually increases with the number of rings and their degree of condensation. In addition, substitution to the aromatic ring can have a significant effect on Φ f . For example, the presence of an electron-withdrawing group, such as (NO2), decreases Φ f , whereas adding an electron-donating group, such as (OH), increases Φ f . 24

25. Fluorescence also increases for aromatic ring systems and for aromatic molecules with rigid planar structures. The simple heterocyclics , such as pyridine, furan, thiophene , and pyrrole do not exhibit fluorescence; on the other hand, fused ring structures ordinarily do. 25

26. Spectrofluorometer : Applications of Environmental Significance: To detect environmental pollutants such as polycyclic aromatic hydrocarbons: pyrene benzopyrene organothiophosphorous pesticides carbamate insecticides Geology: Many types of calcite and amber will fluoresce under shortwave UV. Rubies, emeralds, and the Hope Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation. 26

27. Spectrofluorometer : Applications of Analytical chemistry : to detect compounds from HPLC flow TLC plates can be visualized if the compounds or a coloring reagent is fluorescent Biochemistry: used generally as a non-destructive way of tracking or analysis of biological molecules (proteins) Possible direct or indirect analysis aromatic amino acids (phenylalanine- tyrosine-tryptophan) Fingerprints can be visualized with fluorescent compounds such as ninhydrin . 27

28. Spectrofluorometer : Applications of Medicine Blood and other substances are sometimes detected by fluorescent reagents, particularly where their location was not previously known. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign. Pharmacy: Possible direct or indirect analysis drugs such as: vitamins (vitamin A -vitamin B2 -vitamin B6 -vitamin B12 -vitamin E -folic acid) catecholamines (dopamine- norepinephrine ) Other drugs (quinine-salicylic acid–morphine-barbiturates –LSD) 28

29. New pharmaceutical studies conducted by spectrofluorometer : 29

30. Optimization and validation of spectrofluorimetric method for determination of cefadroxile and cefuroxime sodium in pharmaceutical formulations Elbashir AA , Ahmed SM , Aboul-Enein HY . Chemistry Department, Faculty of Science, University of Khartoum, PO Box 321, Khartoum, 11115, Sudan. Abstract A simple, accurate, precise and validated spectrofluorimetric method is proposed for the determination of two cephalosporins , namely, cefadroxile ( cefa ) and cefuroxime sodium ( cefu ) in pharmaceutical formulations. The method is based on a reaction between cephalosporins with 1,2-naphthoquinone-4-sulfonate in alkaline medium, to form fluorescent derivatives that are extracted with chloroform and subsequently measured at 610 and 605 nm after excitation at 470 and 460 nm for cefa and cefu respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over the concentrations of 20-70 ng / mL and 15-40 ng / mL for cefa and cefu , respectively. The detection limits were 4.46 ng / mL and 3.02 ng / mL with a linear regression correlation coefficient of 0.9984 and 0.998, and recoveries ranging 97.50-109.96% and 95.73-98.89% for cefa and cefu , respectively. The effects of pH, temperature, reaction time, 1,2-naphthoquinone-4-sulfonic concentration and extraction solvent on the determination of cefa and cefu , have been examined. The proposed method can be applied for the determination of cefa and cefu in pharmaceutical formulations in quality control laboratories. Copyright © 2013 John Wiley & Sons, Ltd. Luminescence. 2013 Jan 24. doi : 10.1002/bio.2481. [ Epub ahead of print] 30

31. cefadroxile 1,2-naphthoquinone-4-sulfonate cefuroxime sodium 31

32. A highly sensitive fluorimetric method for determination of lenalidomide in its bulk form and capsules via derivatization with fluorescamine Darwish IA , Khalil NY , Bakheit AH , Alzoman NZ . Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,, P,O, Box 2457, Riyadh, 11451, Saudi Arabia. [email protected]. ABSTRACT: BACKGROUND: Lenalidomide (LND) is a potent novel thalidomide analog which demonstrated remarkable clinical activity in treatment of multiple myeloma disease via a multiple-pathways mechanism. The strong evidences-based clinical success of LND in patients has led to its recent approval by US-FDA under the trade name of Revlimid ® capsules by Celgene Corporation. Fluorimetry is a convenient technique for pharmaceutical quality control, however there was a fluorimetric method for determination of LND in its bulk and capsules. RESULTS: A novel highly sensitive and simple fluorimetric method has been developed and validated for the determination of lenalidmide (LND) in its bulk and dosage forms (capsules). The method was based on nucleophilic substitution reaction of LND with fluorescamine (FLC) in aqueous medium to form a highly fluorescent derivative that was measured at 494 nm after excitation at 381 nm . The factors affecting the reaction were carefully studied and optimized. The kinetics of the reaction was investigated, and the reaction mechanism was postulated. Under the optimized conditions, linear relationship with good correlation coefficient (0.9999) was found between the fluorescence intensity and LND concentration in the range of 25-300 ng / mL. The limits of detection and quantitation for the method were 2.9 and 8.7 ng / mL , respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 1.4%. The proposed method was successfully applied to the determination of LND in its bulk form and pharmaceutical capsules with good accuracy; the recovery values were 97.8-101.4 ± 1.08-2.75%. CONCLUSIONS: The proposed method is selective and involved simple procedures. In conclusion, the method is practical and valuable for routine application in quality control laboratories for determination of LND. Chem Cent J. 2012 Oct 16;6(1):118. doi : 10.1186/1752-153X-6-118. 32

33. lenalidomide fluorescamine 33

34. Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations, human plasma and urine through derivatization with dansyl chloride Ulu ST . Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, Turkey. [email protected] Abstract A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1-dimethylaminonaphthalene-5-sulphonyl chloride ( dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm . The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the ranges 50-500 and 20-300  ng / mL for plasma and urine, respectively, detection limits of 1.81 and 0.54  ng / mL and quantification limits of 5.43 and 1.62  ng / mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t- and F-tests. Luminescence. 2012 Sep-Oct;27(5):426-30. doi : 10.1002/bio.1367. Epub 2011 Oct 28. 34

35. dansyl chloride tizanidine 35

36. Spectrofluorimetric determination of paroxetine hcl in pharmaceuticals via derivatization with 4- chloro-7- nitrobenzo-2-oxa-1,3-diazole ( nbd-cl ) Walsh M , Belal F , El- Enany N , Elmansi H . Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura , Mansoura , Egypt. Abstract: A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the antidepressant paroxetine HCl (PXT) in its dosage forms. The method was based on coupling reaction of PXT with 4-chloro-7-nitrobenzo-2- oxa-1,3-diazole (NBD- Cl ) in an alkaline medium (pH 8) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm . The factors affecting the formation and stability of the reaction product were carefully studied and optimized. The fluorescence-concentration plot is rectilinear over the range 0.2-6 μg / mL with LOD of 0.08 μg / mL and LOQ of 0.24 μg / mL respectively. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. The mean percentage recoveries for paxetin and xandol tablets were 101.27 ± 1.79 and 101.33 ± 1.19 respectively. A proposal of the reaction pathway was postulated. J Fluoresc . 2011 Jan;21(1):105-12. doi : 10.1007/s10895-010-0693-2. Epub 2010 Jul 1. 36

37. paroxetine 4-chloro-7-nitrobenzo-2- oxa-1,3-diazole 37

38. High-performance liquid chromatographic and spectrofluorometric determination of α lpha - tocopherol in a natural plant: Ferula hermonis ( Zalooh root) Emile Aoun a , Jamil Rima b , , Ghiwa Chidiac b , Khalil Hanna c a Lebanese Agricultural Research Institute (LARI), Fanar , P.O. Box 90 1965, Jdeidet El- Metn , Lebanon b Laboratory of Molecular Chemistry, Faculty of Science II, The Lebanese University, Fanar , P.O. Box 26110217 Fanar-Matn , Lebanon Abstract: A high-performance liquid chromatographic (HPLC) method for the determination of α - tocopherol in a natural plant ( Ferula hermonis –Zalooh roots) is reported. The method includes saponification of samples and extraction of α- tocopherol with a mixture of acetonitrile and methanol (1:1 v/v). However, the presence of α- tocopherol in Zalooh is confirmed with HPLC-UV and fluorescence detection. A spectrofluorometric and the internal addition standard methods are also used to quantify the α- tocopherol in the plant. An internal standard method is based on a known concentration of α- tocopherol that is added in every sample that is analyzed. Alpha- tocopherol levels as determined in samples by HPLC with UV and fluorescence detection did not differ significantly from the levels determined by Shimadzu spectrofluorometer . However, the amount of tocopherol determined by both techniques in Zalooh roots was relatively very high. Standards were checked for linearity giving correlation coefficients that were higher than 0.99 in the concentration range of 1 and 6  μmol  L −1 . Volume 18, Issue 7 , November 2005, Pages 607–615 38

39. α- tocopherol 39

40. References : www.pubmed.com www.sciencedirect.com Harvey.D , 2000 , Modern Analytic Chemistry. Fluorescence and Fluorescence Applications- Integrated DNA Technologies-2010 Brandt.M , 2010, Spectrofluorometers and Fluorescence Phenomena 40

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