Instrumentation - Molecular Photoluminescence Spectroscopy

Instrumentation

The basic design of instrumentation for monitoring molecular fluores- cence and molecular phosphorescence is similar to that found for other spectroscopies. The most significant differences are discussed in the fol- lowing sections.

Molecular Fluorescence 

A typical instrumental block diagram for molec- ular fluorescence is shown in Figure 10.45. In contrast to instruments for absorption spectroscopy, the optical paths for the source and detector are usually positioned at an angle of 90°.

Two basic instrumental designs are used for measuring molecular fluorescence. In a fluorometer the excitation and emission wavelengths are selected with absorp- tion or interference filters. The excitation source for a fluorometer is usually a low- pressure mercury vapor lamp that provides intense emission lines distributed throughout the ultraviolet and visible region (254, 312, 365, 405, 436, 546, 577, 691, and 773 nm). When a monochromator is used to select the excitation and emission wavelengths, the instrument is called a spectrofluorometer. With a monochroma- tor, the excitation source is usually a high-pressure Xe arc lamp, which has a contin- uum emission spectrum. Either instrumental design is appropriate for quantitative work, although only a spectrofluorometer can be used to record an excitation or emission spectrum.

The sample cells for molecular fluorescence are similar to those for optical molecular absorption. Remote sensing with fiber-optic probes (see Figure 10.30) also can be adapted for use with either a fluorometer or spectrofluorometer. An an- alyte that is fluorescent can be monitored directly. For analytes that are not fluores- cent, a suitable fluorescent probe molecule can be incorporated into the tip of the fiber-optic probe. The analyte’s reaction with the probe molecule leads to an in- crease or decrease in fluorescence.

Molecular Phosphorescence 

Instrumentation for molecular phosphorescence must discriminate between phosphorescence and fluorescence. Since the lifetime for fluorescence is much shorter than that for phosphorescence, discrimination is easily achieved by incorporating a delay between exciting and measuring phospho- rescent emission. A typical instrumental design is shown in Figure 10.46. 

As shown in the inset, the two choppers are rotated out of phase, such that fluorescent emis- sion is blocked from the detector when the excitation source is focused on the sam- ple, and the excitation source is blocked from the sample when measuring the phos- phorescent emission.

Because phosphorescence is such a slow process, provision must be made to prevent deactivation of the excited state by external conversion. Traditionally, this has been accomplished by dissolving the sample in a suitable organic solvent, usu- ally a mixture of ethanol, isopentane, and diethyl ether. The resulting solution is frozen at liquid-N₂ temperatures, forming an optically clear solid. The solid matrix minimizes external conversion due to collisions between the analyte and the sol- vent. External conversion also is minimized by immobilizing the sample on a solid substrate, allowing the measurement of phosphorescence at room temperature. One approach is to place a drop of solution containing the analyte on a small filter paper disk mounted on a sample probe. After drying the sample under a heat lamp, the sample probe is placed in the spectrofluorometer for analysis. Other solid surfaces that have been used include silica gel, alumina, sodium acetate, and sucrose. This approach is particularly useful for the analysis of thin-layer chromatography plates.