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Analytical Chemiluminescence


Thomas G. Chasteen

Department of Chemistry

Sam Houston State University

Huntsville Texas 77341-2117 U.S.A


Chemiluminescence as an analytical tool has three important strengths:

* sensitivity

* selectivity

* (and often) a wide linear range

The first often means lower detection limits for chemiluminescence when compared to absorption or fluorescence techniques in pursuit of the same analyte. The second, selectivity, is derived from the fact that the analyte of interest generates its signal often in the presence of normally interfering compounds that, in this case, do not themselves produce light when the chemiluminescent reagents are mixed together. And the third is analytically useful because samples of larger concentration ranges can be analyzed without dilution. This advantage is derived from the way that chemiluminescent light is generated and measured: using no source lamp for light production and a phototransducer with an inherent wide range of response for light detection.

The analytical chemiluminescent signal is produced by a chemical reaction (or reactions) and requires no light source for excitation (as in the more common fluorescence and phosphorescence). Its emission appears out of an essentially black background, and therefore the only background signal is that of the photomultiplier tube's (phototranducer's) dark current. Subsequently, light source and detector warm-up and drift and the interference from light scattering present in absorption and fluorescence methods are absent.

Although the above is true, chemiluminescence is still not as widely applicable as absorption, emission, or even fluorescence methods of detection because so few molecules undergo native chemiluminescence with common reagents. That problem is often solved by derivatizing analytes with chemiluminescent tags.

Online papers which utilized chemiluminescence can be seen at this site in the First Internet Conference on Photochemistry and Photobiology.

The following is a brief primer on common chemiluminescence reagents:

Solution phase

Lophine (chemiluminescence discovered in the last quarter of the nineteenth century)
Luminol (or its derivatives)
Oxalate esters
Lucigenin (or its derivatives)
Ruthenium tris-bipyridine

Gas phase

Sodium vapor
Chlorine dioxide

H2O2 or organic peroxides are commonly used as the oxidants in the solution phase chemiluminescent techniques. There are many ways to generate these oxidants or inhibit their generation or catalyze or suppress their action. Also there are a variety of ways to tag otherwise chemiluminescence-less analytes with many of the first set of reagents in the list above. Therefore, solution phase techniques are more common that gas phase analytical gas phase methods. Ergo, HPLC and capillary electrophoresis (and the many associated techniques) are finding wide applicability in this field.

Gas phase chemiluminescence methods have been used with gas chromatography, supercritical fluid chromatography, and many nonchromatographic gas phase detectors in which fast response is required.

A very few analytes of the hundreds or thousands that have been determined with chemiluminescence methods include oxides of nitrogen (NOx), ozone, and hydrogen peroxide in the atmosphere, DNA detection and sequencing, nucleic acids, alkaline phosphatase, and antioxidants in kidney, brain, and plasma. Chemiluminescence has also been used as a probe to determine susceptibility to cancer.

Finally, our
own work has yielded sensitive time course determinations of organo-sulfur, -selenium, -tellurium, and -antimony metabolites in microbial cultures amended with toxic salts of these elements using fluorine-induced chemiluminescence.

And, finally, of course, an excellent reference in this field is the
Journal of Bioluminescence and Chemiluminescence (John Wiley and Sons).