Spectrofluorimetry (Chapter 17-7) PDF

Photoluminescence Spectrofluorimetry Chapter 17-7  Luminescence “The emission of light that occurs from electronically excited state (LUMO) upon its return back to the ground state (HOMO).” If the excitation source is light: It is called “Phot...

Photoluminescence Spectrofluorimetry Chapter 17-7  Luminescence “The emission of light that occurs from electronically excited state (LUMO) upon its return back to the ground state (HOMO).” If the excitation source is light: It is called “Photoluminescence” which includes Fluorescence and Phosphorescence. If the excitation source is a chemical reaction: it is called “Chemiluminescence”. 2 Fluorescence or Phosphorescence (Photoluminescence) light Substance Excited Substance* emitted light Chemiluminescence Chemical Substance Excited Substance* Reaction emitted light 3  Spectrometric methods of analysis (UV-Visible regions) M + hν → M* Molecular absorption M* → M + Heat A + hν → A* Atomic absorption A* → A + Heat M + hν → M* Molecular Luminescence M* → M + hν (emission) A + heat → A* Atomic emission A* → A + hν  What happen to the absorbed energy? Relaxation Processes 1. Nonradiative Process A. Vibrational Relaxations: Loss of energy as thermal energy to solvent. B. Internal Conversion: The excited energy of a molecule is transferred from a higher electronic state to a lower electronic state. Transition between states with same spin quantum numbers (e.g., S2-S1) C. External Conversion: The excitation energy is being converted to kinetic energy by collision with other molecules. (Intermolecular energy transfer) D. Intersystem crossing: The excited energy of a molecule is transferred from a higher electronic state to a lower electronic state. Transition between states with different spin quantum numbers (e.g., S1-T1) 2. Radiative Process ❑ Involves emission of excitation energy in the form of electromagnetic radiation. Fluorescence and Phosphorescence https://www.youtube.com/watch?v=5KLBrnauilg Down Conversion processes Jablonski Diagram for FL. and Phosph. Phenomena Mirror image rule!!  Fluorescence Stokes Shift Stokes shift is the difference in wavelength between positions of the band maxima of the absorption and fluorescence spectra of the same electronic transition. The Stokes shift occurs because the molecule loses a small amount of the absorbed energy before re-releasing the rest of the energy as fluorescence. This energy is often lost as thermal energy to solvent molecules due to strong collision of excited molecules with solvent. Stokes Shift is 20 + nm Two λmax Fluorescnece Intensity Fluorescein 495 nm 518 nm molecule Abs. Fl. Ex. Em. 7 Wavelength  Fluorescence and Phosphorescence The difference in the energy level (ΔE) between the excited and unexcited states during absorption, fluorescence and phosphorescence are in the order: ΔE (absorption) > ΔE (fluorescence) > ΔE (phosphorescence) As ΔE α 1/, the order of the max is: absorption < fluorescence < phosphorescence Decrease non-radiative decay…….increase in FL and Phosph. For example, max for absorption, fluorescence and phosphorescence of anthracene are 255nm, 425nm, 680nm respectively. 8  Interpretation of the Energy Diagram Absorption : Ground state to Excited state (10-15 sec) Relaxation (Decay): Excited state to Ground state – Vibrational relaxations: 10-12 sec – Internal Conversion (IC) nonradiative (thermal, collisional) relaxation of electrons through vibrational states (10-12 sec) – Emission Fluorescence (spontaneous emission: 10-9 - 10-6 sec) phosophorescence (10-3 - 10-0 sec) – phosphorescence requires intersystem crossing (10-9 sec) (flip of electron spin) » Ground state singlet » Excited state singlet » Spin flip (now in Triplet state) » intersystem crossing » Need another Spin flip to be allowed to go back to Ground state singlet » Consequently, the molecule "hangs" in the triplet state for a considerably longer period of time Spin Orientations Differences between fluorescence and phosphorescence: Fluorescence Phosphorescence Lifetime (τ) Ceases Persist for many immediately seconds ≈10-9 sec. 10-3 -10 sec.  At shorter  At longer  Temp. At room temp. At low temp. medium In liquid medium In rigid medium 12 See next slide for explanation  The shortest wavelength in the fluorescence spectrum is the longest wavelength in the absorption spectrum Why Mirror image Ex and Em Spectra?  What Do We Measure In Spectrofluorometry technique? Emitted fluorescence (If) = k * Conc. The Intensity of radiation emitted by an analyte after excitation is proportional to I0 (radiant intensity) I (transmitted intensity) concentration (C). F.I = KC Spectrofluorimeter 15  Excitation & emission spectra (Fluorescence spectra) Emission spectrum is measured by holding excitation radiation fixed (ex) at one particular wavelength and scanning through the emitted radiation. Graph: Emission intensity vs. Emission wavelength. Excitation spectrum is measured by varying excitation wavelength and measuring emitted light at one particular wavelength (em): Graph: Emission intensity vs. Excitation wavelength. Effect of molecular structure on photoluminescence (fluorescence) properties: The structural rigidity of molecule increase fluorescence. Experimentally it is found that fluorescence increase in rigid molecules, eg.: phenolphthalein and fluorescein are structurally similar as shown below. However, fluorescein shows higher fluorescence because of its rigidity. WHY? Fluorescein phenolphthalein Fluorescent Non-Fluorescent 17 Assignment 4: Mention differences and draw these instruments or techniques 1- Compare between Spectrophotometer and spectrofluorimeter 2- Compare between Spectrophotometry and spectrofluorimetry. 3- Compare between Fluorescence and Phosphorescence