Quantitative Applications Using Molecular Luminescence

Quantitative Applications Using Molecular Luminescence

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for the direct or indirect quantitative analysis of analytes in a variety of matrices. A di- rect quantitative analysis is feasible when the analyte’s quantum yield for fluores- cence or phosphorescence is favorable. When the analyte is not fluorescent or phos- phorescent or when the quantum yield for fluorescence or phosphorescence is unfavorable, an indirect analysis may be feasible. One approach to an indirect analysis is to react the analyte with a reagent, forming a product with fluorescent properties. Another approach is to measure a decrease in fluorescence when the an- alyte is added to a solution containing a fluorescent molecule. A decrease in fluores- cence is observed when the reaction between the analyte and the fluorescent species enhances radiationless deactivation, or produces a nonfluorescent product. The ap- plication of fluorescence and phosphorescence to inorganic and organic analytes is considered in this section.

Inorganic Analytes 

Except for a few metal ions, most notably UO ₂+, most inor- ganic ions are not sufficiently fluorescent to allow a direct analysis. Many metal ions can be determined indirectly by reacting with an organic ligand to form a fluorescent, or less commonly, a phosphorescent metal–ligand complex. One ex ample of a chelating ligand is the sodium salt of 2,4,3’-trihydroxyazobenzene-5’ sulfonic acid, also known as alizarin garnet R, which forms a fluorescent metal–ligand complex with Al3+ (Figure 10.47). The analysis is carried out using an excitation wavelength of 470 nm, with fluorescence monitored at 500 nm. Other examples of chelating reagents that form fluorescent metal–ligand com- plexes with metal ions are listed in Table 10.12. A few inorganic nonmetals are determined by their ability to decrease, or quench, the fluorescence of another species. One example is the analysis for F–, which is based on its ability to quench the fluorescence of the Al3+–alizarin garnet R complex.

Organic Analytes 

As noted earlier, organic compounds containing aromatic rings generally are fluorescent, but aromatic heterocycles are often phosphores cent. Many important biochemical, pharmaceutical, and environmental com- pounds are aromatic and, therefore, can be analyzed quantitatively by fluorometry or phosphorometry. 

Several examples are listed in Table 10.13. When an organic ana- lyte is not naturally fluorescent or phosphorescent, it may be possible to incorporate it into a chemical reaction that produces a fluorescent or phosphorescent product. For example, the enzyme creatine phosphokinase can be determined by using it to catalyze the formation of creatine from phosphocreatine. The creatine that is formed reacts with ninhydrin, producing a fluorescent product of unknown structure.

Standardizing the Method 

Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the exci- tation source (A = εbC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emis- sion is given by equation 10.31. Nonlinearity also may be observed at low concen- trations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the pho- toluminescent species, which is sensitive to the sample matrix.