Flow scintillation counting of C-14-labeled microalgal photosynthetic pigments

Photopigment radiolabeling, a useful method for measuring the in situ carbon-specific growth rates of microalgae, is based on the determination of synthesis rates of chemosystematic (i.e. specific for microalgal phylogenetic groups) chlorophylls and carotenoids using photosynthetically assimilated C-14 as a radiotracer. The reliability of this method depends on accurate measurements of the C-14-specific activity of individual photopigments. Typically, photopigments are separated by high-performance liquid chromatography (HPLC) with fraction collection of individual peaks, followed by further purification and standard scintillation counting. To simplify analyses, we evaluated in-line now scintillation counting to determine its applicability and reliability for measuring the activity of radiolabeled photopigments. Incubations were conducted using both pure cultures and natural phytoplankton samples. The radiochemical purity of photopigments was determined by extract acidification (10% HCl) to transform chlorophylls into degradation products. Purity was also checked by comparing absorbance spectra with purified standards. Although C-14-labeled colorless compounds are a common feature in radiograms, these compounds do not co-elute with photopigments using our HPLC protocol. Flow scintillation counting, coupled with a highly selective HPLC protocol, provides an efficient, reliable and feasible alternative to fraction collection/repurification methods for measuring the C-14-specific activity of microalgal photosynthetic pigments.