2. FluorescenceAIT
Exercise 2.1
Atomic Molecular
Absorption
Emission
flame AAS
UV/VIS
IR
flame emission
ICP
??Fluorescence
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
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
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
Why is the energy emitted less?
Ground State
Excited State
(a) (b)(c)
Absorption & fluorescence spectra
AnthraceneA – abs.C – fluor.
QuinineB – abs.D – fluor.
• all compounds fluoresce to some extent, most not much
0% F
100% NRR
100% F
0% NRR
naphthalene 10%
chlorophyll 30%
quinine
55%
fluorescein
90%
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)
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• heavy atoms - in solvent or matrix• dissolved oxygen• ligands
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
Effect of excitation wavelength
Exercise 2.3
Excitation Fluorescent
Quinine (B & D) 350 460
Anthracene (A & C) 360 400
Perylene (400)430 (440)470
Instrumentation
Radiation Source
Collimator Excitation
Selector
Sample
Cell
Collimator
Emission
Selector
DetectorReadout
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
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
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
Calibration range
• self-quenching - at high concentrations, the intensity begins to decrease • analyte molecules absorb some of the radiation that others have emitted
Concentration
Inte
ns
ityself-quenching
Exercise 2.4
Why is self-quenching a problem?
• there are two concentrations corresponding to the same intensity value
Concentration
Inte
ns
ity
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
Applications
• pharmaceuticals eg Vitamin 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
Advantages & disadvantages
Compared to UV/VIS
• greater sensitivity • greater linear region • selectivity• cheapness
• limited species availability • one extra step