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low concentrations. One major factor adding to the high sensitivity of fluorescence

applications is the spectral selectivity. Due to the Stokes shift, the wavelength of the

emitted light is different from that of the exciting light. Another feature makes use of

the fact that fluorescence is emitted in all directions. By placing the detector perpen-

dicular to the excitation pathway, the background of the incident beam is reduced.

The schematics of a typical spectrofluorimeter are shown in Fig. 12.9. Two mono-

chromators are used, one for tuning the wavelength of the exciting beam and a second

one for analysis of the fluorescence emission. Due to the emitted light always having a

lower energy than the exciting light, the wavelength of the excitation monochromator

is set at a lower wavelength than the emission monochromator. The better fluores-

cence spectrometers in laboratories have a photon-counting detector yielding very

high sensitivity. Temperature control is required for accurate work as the emission

intensity of a fluorophore is dependent on the temperature of the solution.

Two geometries are possible for the measurement, with the 90arrangement most

commonly used. Pre- and post-filter effects can arise owing to absorption of light prior

to reaching the fluorophore and the reduction of emitted radiation. These phenomena

are also calledinner filter effectsand are more evident in solutions with high concen-

trations. As a rough guide, the absorption of a solution to be used for fluorescence

experiments should be less than 0.05. The use of microcuvettes containing less material

can also be useful. Alternatively, the front-face illumination geometry (Fig. 12.9 inset)

can be used which obviates the inner filter effect. Also, while the 90geometry requires

cuvettes with two neighbouring faces being clear (usually, fluorescence cuvettes have

four clear faces), the front-face illumination technique requires only one clear face, as

excitation and emission occur at the same face. However, front-face illumination is less

sensitive than the 90illumination.

Xenon

lamp

Reference detector Sample detector

Photomultiplier

Grating

Slit

Sample

cuvette

Half

mirror

Grating

Slit

Computer

90 ̊

45 ̊

Fig. 12.9Schematics of a spectrofluorimeter with ‘T’ geometry (90). Optical paths are shown as green lines.

Inset: Geometry of front-face illumination.

496 Spectroscopic techniques: I Photometric techniques