Molecular photoluminescence can be used for the routine analysis of trace and ultratrace analytes in macro and meso samples. Detection limits for fluorescence spectroscopy are strongly influenced by the analyte’s quantum yield. For analytes with Φf > 0.5, detection limits in the picomolar range are possible when using a high-quality spectrofluorometer. As an example, the detection limit for qui- nine sulfate, for which Φf is 0.55, is generally between 1 ppb and 1 ppTr (part per trillion). Detection limits for phosphorescence are somewhat poorer than those for fluorescence, with typical values in the nanomolar range for low-temperature phos- phorometry and in the micromolar range for room-temperature phosphorometry using a solid substrate.
The accuracy of a fluorescence method is generally 1–5% when spectral and chemical interferences are insignificant. Accuracy is limited by the same types of problems affecting other spectroscopic methods. In addition, accuracy is affected by interferences influencing the fluorescent quantum yield. The accuracy of phos- phorescence is somewhat greater than that for fluorescence.
When the analyte’s concentration is well above the detection limit, the relative standard deviation for fluorescence is usually 0.5–2%. The limiting instru- mental factor affecting precision is the stability of the excitation source. The preci- sion for phosphorescence is often limited by reproducibility in preparing samples for analysis, with relative standard deviations of 5–10% being common.
From equations 10.32 and 10.33 we can see that the sensitivity of a flu- orescent or phosphorescent method is influenced by a number of parameters. The importance of quantum yield and the effect of temperature and solution composi- tion on Φf and Φp already have been considered.
Besides quantum yield, the sensi- tivity of an analysis can be improved by using an excitation source that has a greater emission intensity (P0) at the desired wavelength and by selecting an excitation wavelength that corresponds to an absorption maximum (ε). Another approach that can be used to increase sensitivity is to increase the volume in the sample from which emission is monitored. Figure 10.48 shows how a 90° rotation of the slits used to focus the excitation source on the sample and to collect emission from the sample can produce a 5–30-fold increase in the signal.
The selectivity of molecular fluorescence and phosphorescence is superior to that of absorption spectrophotometry for two reasons: first, not every compound that absorbs radiation is fluorescent or phosphorescent, and, second, selectivity between an analyte and an interferant is possible if there is a difference in either their excitation or emission spectra. In molecular lumines- cence the total emission intensity is a linear sum of that from each fluorescent or phosphorescent species. The analysis of a sample containing n components, therefore, can be accomplished by measuring the total emission intensity at n wavelengths.
As with other optical spectroscopic methods, fluores- cent and phosphorescent methods provide a rapid means of analysis and are capa- ble of automation. Fluorometers are relatively inexpensive, ranging from several hundred to several thousand dollars, and often are very satisfactory for quantitative work. Spectrofluorometers are more expensive, with some models costing as much as $50,000.
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