Fluorescence Spectroscopy 0.0 / 5 ? PharmacySpectroscopyUniversityNone Created by: RscottqubCreated on: 28-11-19 16:49 Fluorescence ability to absorb light of specific wavelength and then ability to emit the light at a longer WL after a brief interval 1 of 26 3 steps to fluorescence 1. Excitation 2. Vibration relaxation and internal conversion 3. Emission 2 of 26 Excitation Excitation of electrons by incoming photon to higher excited electronic state. Most electrons at room temp occupy lowest level of ground state 3 of 26 Vibrational relaxation and internal conversion Relaxation of excited electrons to the lowest level of the the 1st excited state . slightly slower than excitation 4 of 26 Vibrational relaxation Transfer of electrons from higher vibrational level to another within the same state 5 of 26 Internal conversion transfer of electrons from higher vibrational level to lower level between different states 6 of 26 Emission the emission of a longer WL photon - return of excited electron to ANY level of the ground state . Much slower than step 1/2 7 of 26 Jablonski diagram - see diagram in notes there are a no of ways of returning back to the ground state - intersystem crossing is possible but is rare as it is v slow 8 of 26 intersystem crossing transition between states of different spin. ie. excited triplet--> ground singlet 9 of 26 Phosphorescence radiative transition from excited triplet to ground singlet state 10 of 26 We produce a flourescent spectrum by.. exciting a fluorophore with a specific WL 11 of 26 Fluorophore molecule which will exhibit fluorescence - contain multiple C=C conjugations 12 of 26 Mirror image rule Vib energy is similar to ground and excited states -spectrum looks like mirror image 13 of 26 exception to mirror image rule excitation of high energy photons - to higher levels that will then will quickly lose energy as it relaxes back down to ground state 14 of 26 Instrumentation- monochromator select specific WL, filters out all other WL of light 15 of 26 Why apparatus set up at right angles to increase sensitivity of measurement , collecting at 90 degrees reduces interference 16 of 26 The ideal flurophore ... high extinction coeff. (measure of light absorb capacity ) high fluorescence quantum yield. Large stokes shift 17 of 26 Stokes shift Difference between peak excitation WL and peak emission WL 18 of 26 Interfering factors (5) 1. Quenching 2. Concentration 3. Temp 4. pH 5. inner filter effects 19 of 26 Quenching Decreases activity due to interaction between excited flurophore and surroundings 20 of 26 Concentration higher solute concentration means intensity of excitation light is not uniform. Also emitted light is reabsorbed by the other molecules 21 of 26 Temp Quantim efficiency is decreased as temp increases . Higher no of collisions decreases solvent viscosity 22 of 26 pH WL and emission id different for protonated an unprotected form of the fluorophore 23 of 26 Inner Filter effects decreased quantum yield due to reabsorb of emitted light or absorption of incident light by contaminants 24 of 26 Advantages highly sensitive , specific 25 of 26 Cons limited to fluorescent molecules , temp dependent , possible interference e 26 of 26
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