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