Abstract
Quantification of chlorophyll a (Chl a) is essential to the study of aquatic ecosystems, yet differences in methodology may introduce significant errors to its determination in ethanol extracts. Insufficient acidification slows the conversion of Chl a to pheophytin a leading to an underestimate of Chl a concentration. Furthermore, slight differences in the postacidification reaction time can introduce greater errors in calculated Chl a and impede our ability to make cross-study comparisons. We used known concentrations of pure Chl a from the blue-green algae Anacystis nidulans dissolved in 95% ethanol to evaluate the effect of acid strength and postacidification reaction time on the spectrophotometric determination of Chl a. Increasing acid strength resulted in more rapid stabilization of calculated Chl a concentration. At reaction times less than 120 s estimates of Chl a deviated from known concentrations by as much as 84.8%. The magnitude of error in the calculated Chl a values were dependent on acid strength and reaction time, which allowed us to develop predictive equations to correct Chl a measurements that were insufficiently acidified or read prior to reaction completion. We validated our predictive equations using benthic periphyton samples from northern Alaska and northwestern Vermont, U.S.A. Our results indicate that under-acidified samples with known reaction times can be easily corrected so results from different methods can be standardized. For future analyses we recommend acidifying ethanol-extracted algal samples to 0.008 mol HCl L−1 and allowing samples to react for 30–60 min to ensure accurate and consistent results.
Published Version
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