Photolumimiscence spectroscopy

INSTRUMENTATION AND APPLICATIONS

1. FLUORESCENCE SPECTROSCOPY JAYAKRISHNA J MPHIL STUDENT DEPARTMENT OF CHEMISTRY UNIVERSITY OF KERALA KARIAVATTOM CAMPUS 5/30/2019 1 J Cell Sci .  124  (2): 157–160

2. PHENOMENON OF FLUORESCENCE INTRODUCTION TO FLUORESCENCE 1. Luminescence is the emission of light from any substance and occurs from electronically excited states. FLUORESCENCE PHOSPHORESCENCE Spin in the ground and excited states Fluorescence – ground state to singlet state and back. Phosphorescence - ground state to triplet state and back. 5/30/2019 2

3. 5/30/2019 3 JABLONSKI DIAGRAM Scientific foundation for fluorescence was given by Alexander Jablonski Professor Jablonski is regarded as the father of Fluorescence Spectroscopy J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 1-6 Regarded as the starting point for discussing light absorption and emission . Also defined the term ‘anisotropy ’

4. Fluorescence Emission of Photons S 1  → S  radiative transition Timescale of 10 -10  to 10 -7  s Rapid Vibrational Relaxation And Internal Conversion D. C. Harris, Quantitative Chemical Analysis, 7th Ed, W. H. Freeman and Company, New York (2006). Fluorescence occurs at higher wavelength (lower energy) compared to Absorption A Stokes shift is the difference between the absorption and emission peaks of a molecule . 5/30/2019 4 A radiative transition between two electronic states of the same spin multiplicity .

5. Blue glass Filter Church Window! <400nm Quinine Solution Yellow glass of wine Em filter, transmits > 400 nm Adapted from HORIBA JobinYvon Inc., Leading the 21 st Century in Time-Resolved Fluorescence Instrumentation Dr. Adam M. Gilmore Applications Scientist Fluorescence slide share FIRST OBSERVED FROM QUININE BY SIR J.W.HERSHEL IN 1845 3.The first observation of fluorescence was reported by Sir John Frederick William Herschel in 1845 4.He states that superficial colour presented by a homogenous liquid internally colourless

6. 5/30/2019 6 . Fig Adapted Article by:  Hobart M. King , Ph.D., RPG The term fluorescence comes from the MINERAL FLUORSPAR (CALCIUM FLUORIDE) coined by SIR GEORGE G. STOKES. Fluorescent fluorite:  Tumble-polished specimens of fluorite in normal light (top) and under short-wave ultraviolet light (bottom). J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 1-6

7. 5/30/2019 7 CHARACTERISTICS OF FLUORESCENCE EMISSION Stokes Shift Kasha Rule Fluorescence Typically occurs at lower energies or longer wavelengths J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 1-6 Stokes Shift: Difference in energy/wavelength between absorption max and emission max. Kasha’s Rule: Emission predominantly occurs from the lowest excited state (S OR T 1 )

8. 5/30/2019 8 Fluorescence is now a dominant methodology used in Environmental Monitoring, Medical Diagnostics, DNA Sequencing, Forensics, Genetic Analysis, Sensing Applications . 1. High sensitivity 2. Fluorescence intensity, Fluorescence maximum, spectral shape, fluorescence anisotropy, fluorescence lifetime, time-dependence of anisotropy, fluorescence correlation spectroscopy 3. Relatively simple experimentation 4. Lower cost of equipment compared to similar other techniques

9. 5/30/2019 9 FLUORESCENCE SPECTROSCOPY Steady-State Experiments Time Resolved Experiments Excitation Spectra Emission Spectra (Fluorescence Spectra ) D. C. Harris, Quantitative Chemical Analysis, 7th Ed, W. H. Freeman and Company, New York (2006).

10. 5/30/2019 10 Fluorometer The Filter Fluorometer The Spectrofluorometer A-1 filter fluorometer Perkin-Elmer 204 A filter fluorometer will use filters while a spectrofluorometer will use grating monochromator . https://www.edinst.com/blog/fluorescence-measurements-introduction/

11. A-1 filter fluorometer 5/30/2019 11 https://www.edinst.com/blog/fluorescence-measurements-introduction/

12. Perkin-Elmer 204 5/30/2019 12 https://www.edinst.com/blog/fluorescence-measurements-introduction/

13. Example of a COMPACT SPECTROFLUOROMETER (FS5) and a more ADVANCED PHOTOLUMINESCENCE SPECTROMETER (FLS1000). 5/30/2019 13 https://www.edinst.com/blog/fluorescence-measurements-introduction/

14. 5/30/2019 14 J. R. Lakowicz , Instrumenation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61

15. 5/30/2019 15 The primary use of a Spectrofluorometer is to record excitation and emission spectra. Excitation and emission spectra are x-y plots of fluorescence intensity vs. wavelength . To obtain accurate spectra, the components must have the following characteristics An ideal Spectrofluorometer J. R. Lakowicz , Instrumentation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Properties of the ideal components of spectrofluorometer

16. 5/30/2019 16 J. R. Lakowicz , Instrumentation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Light Sources, Monochromators, And Photomultiplier Tubes With Such Ideal Characteristics Are Not Available . There is need to correct for the non ideal response of the instrument . Fluorescence Intensity Measurements Are Absolute, Not Relative. In absorption spectrum, the intensity of light transmitted by the sample is measured relative to that of a reference (or blank).

17. https://www.edinst.com/blog/fluorescence-measurements-introduction/ Introduction to Instrumentation The figure above shows the layout of an FLS1000 Photoluminescence Spectrometer 5/30/2019 17

18. 5/30/2019 18 Light Sources Arc and Incandescent Xenon Lamps Pulsed Xenon Lamps High Pressure Mercury Lamps LED Light Sources Laser Diodes J. R. Lakowicz , Instrumentation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 At present the most versatile light source for a steady-state spectrofluorometer is a high-pressure xenon ( Xe ) arc lamp. light output from 250 to 700 nm number of sharp line 450 nm and above 800 nm.

19. 5/30/2019 19 J. R. Lakowicz , Instrumentation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Spectral output of a continuous Xenon arc lamp The wavelength-dependent output of Xe lamps is a major reason for distortion of the excitation spectra of compounds that absorb in the visible and ultraviolet.

20. 5/30/2019 20 J. R. Lakowicz , Instrumentation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Xenon lamps are usually contained within specially designed housings. A xenon lamp that is on should never be observed directly. The extreme brightness will damage the retina, and the ultraviolet light can damage the cornea.

21. 5/30/2019 21 MONOCHROMATORS J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Monochromators are used to disperse polychromatic or white light into the various colors or wavelengths. This dispersion can be accomplished using prisms or diffraction gratings . The monochromators in most spectrofluorometers use diffraction gratings rather than prisms https://www.shimadzu.com/an/uv/support/fundamentals/single_double.html

22. 5/30/2019 22 J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Grating may be PLANAR or CONCAVE. Planar gratings are produced MECHANICALLY and may contain IMPERFECTIONS. Concave gratings are produced by HOLOGRAPHIC or PHOTORESIST methods and have less imperfections . Few Reflecting Surfaces Lower stray light The distance between adjacent grooves and the angle the groove influence both the dispersion and efficiency of a grating.

23. 5/30/2019 23 Efficiencies of two MCs blazed at different angles J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 The transmission efficiency of a grating and monochromator is dependent on wavelength and on the design of the grating. For a mechanically produced plane grating the efficiency at any given wavelength can be maximized by choice of blaze angle, which is determined by the shape and angle of the tool used to produce the grating . By choice of this angle one may obtain maximum diffraction efficiency for a given wavelength region

24. 5/30/2019 24 Second order diffraction of 300 nm light occurs at 600 nm. Hence a peak will appear at 600 . 500 600 550 700 Wavelength The 600 nm peak can be avoided by placing a cut off filter ( eg. <400 cut off) in the emission channel . 500 600 550 700 Wavelength Corrected spectrum Assume that you are recording the emission spectrum of a sample. Excitation WL is 300 nm. Sample emits in the 500-700 nm region

25. 5/30/2019 25 PHOTOMULTIPIER TUBES J. R. Lakowicz , Instrumenation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 Almost all fluorescence spectrometers use PMTs as detectors and it is essential to understand their capabilities and limitations. PMT is a device that converts light intensity into electrical current , the current generated being proportional to the photon intensity .

26. 5/30/2019 26 PMT Xenon Lamp Excitation Monochromator Emission Monochromator Sample Ex Grating Em Grating Procedure 1) White light source on 2) Shift excitation grating to desired wavelength (excitation wavelength) 3) Light enters sample chamber 4) Light Hits the Sample 5) Emission from the sample enters emission monochromator 6) Set emission grating 7) Detect emitted light at PMT 8) Raster emission grating Measuring Emission Spectra

27. 5/30/2019 27 EFFECT OF CONCENTRATION J. R. Lakowicz , Introduction to Fluorescence, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 At extremely high sample concentrations, the Fluorescence Intensity can actually decrease because most of the light is absorbed in the front layers. Reabsorption of the shorter wavelength.

28. 5/30/2019 28 COMMON ERRORS IN SAMPLE PREPERATION J. R. Lakowicz , Instrumenation for Fluorescence Spectroscopy, Principles of Fluorescence Spectroscopy, 3rd Ed. Springer US (2006) pp 27-61 With the Instrument Stray light Slit Widths Signal/Noise

29. 5/30/2019 29 Fluorescence & Structure : usually aromatic compounds low energy transition quantum yield increases with number of rings and degree of condensation. fluorescence especially favored for rigid structures fluorescence increase for chelating agent bound to metal. quinoline indole fluorene 8-hydroxyquinoline

30. The fluorescence spectrum and intensity of a molecule often depend strongly on the molecule’s environment. 30

31. Temperature: As temperature increases fluorescence decreases. Due to change in temperature viscosity of the medium changes.(less Viscosity) Change in viscosity increases the number of collisions of the molecules of the fluorophore with solvent molecules. No. of collisions increases the probability for deactivation by internal conversion and vibrational relaxation. To overcome this , it is recommended to use thermo stated cell holders. 31

32. pH Relatively small changes in pH can cause considerable changes in the fluorescence intensity and spectral characteristics of fluorescence. The molecules containing acidic or basic functional groups undergoes ionization due to the changes in pH of the medium. It may affect the extent of conjugation or the aromaticity of the molecule which affects its fluorescence. For example, aniline shows fluorescence while in acid solution it does not show fluorescence due to the formation of anilinium ion. Therefore, pH control is essential while working with such molecules and suitable buffers should be employed 32

33. Dissolved Oxygen Oxygen and many transition metals with unpaired electrons are paramagnetic which decrease fluorescence and cause interference in fluorimetric determinations. The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet states in other molecules.(phosphorescence) Presence of dissolved oxygen influences phosphorescence too and causes a large decrease in the phosphorescence intensity. This is actually the oxygen emission and not the phosphorescence. Therefore, it is advisable to make phosphorescence measurement in the absence of dissolved oxygen . 33

34. 5/30/2019 34 Solvent: The changes in the “polarity” or hydrogen bonding ability of the solvent affect the fluorescent behaviour of the analyte. Solvent viscosity and solvents with heavy atoms also affect fluorescence and phosphorescence. A higher fluorescence is observed when the solvents do not contain heavy atoms while phosphorescence increases due to the presence of heavy atoms in the solvent . J. Chem. Educ . 2011, 88, 731–738

35. 5/30/2019 35 APPLICATIONS Naked eye and smartphone applicable detection of toxic mercury ions using fluorescent carbon nanodots doi:10.3906/kim-1701-46 1.Band Gap Determination 2. Impurity level and defect detection 3.Surface Structure and Excited States.

36. 5/30/2019 36 https://www.nobelprize.org/prizes/chemistry/2014/press-release/

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