Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments

TLDR

The study presents a fluorometric method that enables sensitive measurement of extracted chlorophyll a without the errors of conventional acidification techniques. The method optimizes fluorometric optical configurations to maximize sensitivity to chlorophyll a while suppressing responses from chlorophyll b and pheopigments. The method yields only a 10 % overestimate of chlorophyll a at a 1:1 Chl b:Chl a ratio, matches acidification estimates at high pheopigment levels, and provides accurate single‑fluorescence determinations with sufficient sensitivity for samples under 200 ml in oligotrophic waters.

Abstract

A fluorometric method is described which provides sensitive measurements of extracted chlorophyll a free from the errors associated with conventional acidification techniques. Fluorometric optical configurations were optimized to produce maximum sensitivity to Chl a while maintaining desensitized responses from both Chl b and pheopigments. Under the most extreme Chl b :Chl a ratio likely to occur in nature (1 : 1 molar), the new method results in only a 10% overestimate of the true Chl a value, while estimates from older acidification methods are 2.5‐fold low. Under conditions of high pheopigment concentrations (pheo a: Chl a = 1 : 1 molar), the new method provides Chl a estimates that are equivalent to those determined from the acidification technique. The new simple method requires a single fluorescence determination and provides adequate sensitivity for small sample sizes (<200 ml) even in the most oligotrophic marine and freshwater environments.