MOLECULAR FLUORESCENCE
Transportation safety experts tell people to wear fluorescent tape or articles of clothing at night when walking or riding a bicycle. Some road signs fluoresce at night when automobile headlights shine on them. Many homes and businesses use fluorescent lighting. Most incandescent light bulbs have been replaced by com pact fluorescent lights. Some nightclubs use “black lights” (ultraviolet lights) and fluorescent paint to jazz up the dance floors with wild, fluorescent colors. All of these are examples of the phenomenon of fluorescence. This occurs when a sub stance absorbs energy and then emits it in the form of (usually) visible light. In the fluorescent tape and paint examples above, the dyes in the tape and pigments in the paint absorb ultraviolet or visible light and then emit light of a different wave length. In the case of fluorescent lights, a gas absorbs electrical energy and emits visible light. Most substances do not fluoresce. They absorb light and then emit the same wavelength back. Those substances that fluoresce will always emit light of a longer wavelength (lower energy) than they absorb. Most substances of forensic interest that fluoresce do so when the light absorbed is in the visible or ultraviolet range. Some substances such as certain inks undergo IR fluorescence, where the light absorbed and emitted is in the IR region. (See the next section of this chapter for a discussion on IR spectroscopy.) Fluorescence is measured similarly to UV/visible spectrophotometry. A fluorescence spectrophotometer looks a lot like a UV/visible spectrophotometer with some important differences. Figure 5.9 is the internal diagram of a fluorescence spectrophotometer.
FIGURE 5.9 Diagram of a UV fluorescence spectrophotometer. The source light passes through a monochromator that selects wavelengths of light for excitation of the analyte. The emission monochromator is stationed at right angles to the excitation light. Light emitted by the sample passes through the emission monochromator and is detected by the photomultiplier.
above showed a UV/visible spectrophotometer. The detector of that instrument is in a straight-line path from the source through the sample. In fluorescence spectroscopy, the detector is at a right angle to the source, with the sample at the apex, so that the detector does not see any light that leaves the source and is directly transmitted by the sample. The detector sees only light that is fluoresced by the sample. In addition, in fluorescence spectroscopy, there are two monochromators; one between the source and the sample, so the wavelengths of light that reach the sample can be selected and there is another between the sample and the detector, so that the wavelengths of light that reach the detector can be selected. Using the two monochromators, the specific wavelengths the sample absorbs (excitation) and emits (emission) can be determined. The excitation and emission wavelengths are very characteristic of a particular substance. A surprising variety of evidence types have fluorescent characteristics that are used in their analysis. Certain illicit drugs exhibit ultraviolet fluorescence. The most common example is LSD, which will absorb light strongly at 320 nm and then emit light at 400 nm. Like UV/visible spectrophotometry, fluorescence spectroscopy also obeys Beer’s Law. Thus the concentration of LSD in a sample can be determined from its fluorescence spectrum. Some pigments and dyes used in automobile and decorative paints, some inks, fibers and tape materials will exhibit fluorescence. Some fabrics and cleaning agents contain optical brighteners that exhibit fluorescence. In many of these cases, UV/visible spectrophotometry and fluorescence spectroscopy can both be used to characterize these substances.
