2. Fluorescence

1. 2. Fluorescence AIT

2. Exercise 2.1 flame AAS IR flame emission ICP UV/VIS Fluorescence ??

3. Interaction with radiation • absorption to exc. state via heat, electricity, radiation • lifetime in excited state is nanoseconds usually • most molecules return to ground state via collisions • called non-radiative relaxation • no emission of radiation • if in the gas phase, collisions are less likely, so emission possible

4. Luminescence • emission of UV/VIS radiation by exc. state species • photoluminescence – radiation • thermoluminescence – heat (flame emission, ICP) • chemiluminescence – chemical reaction • bioluminescence – within living organisms • triboluminescence – from the fracture of crystals • photoluminescence: • fluorescence – stops immediately after source is removed • phosphorescence – lasts for a number of hours afterwards • fluorescence more useful analytically

5. Fluorescence • emission of radiation, where the energy emitted is less than that absorbed: • UV/VIS – molecules • Xray – atoms • Class Exercise 2.2 • How does this definition of fluorescence translate to the relationship between wavelengths? • emitted is LONGER wavelength than absorbed

6. Excited State (a) (b) (c) Ground State Why is the energy emitted less?

7. Absorption & fluorescence spectra Anthracene A – abs. C – fluor. Quinine B – abs. D – fluor.

8. naphthalene 10% chlorophyll 30% quinine 55% fluorescein 90% 0% F 100% NRR 100% F 0% NRR • all compounds fluoresce to some extent, most not much

9. What makes a molecule fluoresce strongly? • must absorb strongly • not all absorbing species fluoresce strongly • eg permanganate, benzene • no simple guide for inorganic species • eg Ce3+ does, Ce4+ doesn’t • no important ions fluoresce strongly • can be converted into complex that does • organic species require the absorbing part of the structure be rigid Naphthalene (10% F) Phenylbenzene (<1% F)

10. Factors affecting fluorescence Matrix • known as quenching • anything that causes a decrease in intensity is known as a quenching agent • pH • temperature – higher temperatures reduce fluorescent intensity • heavyatoms - in solvent or matrix • dissolved oxygen • ligands

11. Factors affecting fluorescence Spectra • wavelength affects intensity (as always) – need top of peak • need 2 s – excitation & fluorescent • Excitation wavelength • more absorption means more fluorescence • choose wavelength of maximum absorbance (from UV/VIS) • Fluorescent wavelength • if a fluorescence spectrum can be run, choose top of peak (except if <30 nm to abs) • otherwise, choose filter about 40-60 nm > abs

12. Effect of excitation wavelength

13. Exercise 2.3

14. Radiation Source Collimator Excitation  Selector Sample Cell Collimator Readout Detector Emission  Selector Instrumentation

15. Radiation source • the intensity must be much greater than abs. source • need to generate as many excited state species as possible • most common lamps are Xe-arc or Hg-arc Wavelength selectors • two wavelengths must be selected • all wavelengths would cause photodecomposition of the analyte • use of filters increases sensitivity • double-filter instruments cannot record an emission spectrum • transmission spectra of the two filters cannot overlap

16. Sample cell • silica, not quartz (which fluoresces) for the UV • glass for the visible • the cell must be polished all around (all four sides if square) • emission intensity is measured at 90 to the excitation beam • avoids radiation from the lamp hitting the detector (stray light) • fluorescent radiation is generated in all directions • it doesn’t affect its intensity wherever the detector is placed

17. Choosing the analytical wavelengths • absorption & emission wavelengths are about 50 nm apart • simple method for selecting them for filter instruments: • filter closest to max. abs (from UV/VIS) for the excitation selector • if the 2nd selector is a monochromator, then a scan (manual or machine) will find the highest emission • if a filter, then add 40-50 nm onto the λabs and look for the filter closest

18. Calibration range • self-quenching - at high concentrations, the intensity begins to decrease • analyte molecules absorb some of the radiation that others have emitted self-quenching

19. Exercise 2.4 Why is self-quenching a problem? • there are two concentrations corresponding to the same intensity value

20. How to work out which one • a quick, approximate 1:1 dilution with solvent • if intensity decreases, sample is in the linear region • if it increases, it is in the self-quenching region • requires a large dilution to get it into the working range

21. Applications • pharmaceuticals egVitamin A, barbiturates, amphetamines, barbiturates • air pollution monitoring, eg polycyclic aromatic hydrocarbons (PAHs) such as benzo[]pyrene • used in HPLC detectors • very few inorganics fluoresce • can be made to do by complexing with fluorescent ligand • not much point these days

22. Advantages & disadvantages Compared to UV/VIS • greater sensitivity • greater linear region • selectivity • cheapness • limited species availability • one extra step