Flow scintillation counting of 14 C-labeled microalgal photosynthetic pigments

Photopigment radiolabcling, 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 14C as a radiotracer. The reliability of this method depends on accurate measurements of the 14C-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 flow scintillation counting to determine its applicability and reliability for measuring the activity of radio-labeled photopigments. Incubations were conducted using both pure cultures and natural phyto-plankton samples. The radiochemical purity of photopigments was determined by extract acidification (10% HC1) to transform chlorophylls into degradation products. Purity was also checked by comparing absorbance spectra with purified standards. Although 14C-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 14C-specific activity of microalgal photosynthetic pigments.