22] Profiling and quantitation of carotenoids by high-performance liquid chromatography and photodiode array detection

Abstract

This chapter discusses the potential of high performance liquid chromatography (HPLC)–photodiode array detection for carotenoid profiling in a variety of biological materials. Proper peak identification is of paramount importance for the profiling of carotenoids in biological materials. In daily HPLC practice, this is done by comparing retention times of unknown peaks with those of authentic reference substances. Alternatively, fractions can be collected from the HPLC column and compounds characterized off-line by spectrometric techniques. The most sophisticated scanning absorption detector presently available is the photodiode array detector. This versatile instrument permits the on-line recording of the complete absorption spectrum of a chromatographic peak in less than 1 second. It owes this unique performance to a reverse-optics configuration, the presence of an array of photodiodes each dedicated at a particular band of wavelengths, and its connection to a powerful computer for data handling. As carotenoids possess highly characteristic absorption spectra and as small structural modifications induce detectable changes in their spectra, they can be considered as model components for (tentative) identification by the combined technique of HPLC–photodiode array detection. Typical applications include the determination of carotenoids in fruits and vegetables, flowers, orange juice, algae, palm oil, and bacteria.