Comparison of Chlorophyll a Extractions with Ethanol and Dimethyl Sulfoxide/Acetone, and a Concern about Spectrophotometric Phaeopigment Correction

Abstract

Chlorophyll a (Chl a) in water samples from three mesotrophic to eutrophic lakes in north-central Alberta was extracted with one of three solvents (95% ethanol, 90% ethanol, or a 2:3 mixture of dimethyl sulfoxide and 90% acetone (DMSO/acetone)) and analyzed by two techniques (spectrophotometry and high pressure liquid chromatography (HPLC). The dominant phytoplankton were blue-green algae and diatoms. Total Chl a concentrations (i.e. no correction for phaeopigments (Pha)) were not significantly different among solvents (P > 0.5). Total Chl a concentrations from spectrophotometric analyses were significantly higher than those from HPLC analyses (4.2 ± 0.88 and 2.6 ± 0.50 μg∙L−1 respectively, P < 0.05). Pha concentrations derived by spectrophotometry were 64 times higher than those derived by HPLC (1.7 ± 0.52 and 0.025 ± 0.01 μg∙L−1 respectively, P < 0.005). Thus, spectrophotometry appears to dramatically overestimate Pha concentrations and may overestimate total Chl a (i.e. no correction for Pha). Therefore, ethanol and DMSO/acetone are equally suitable for Chl a extraction from natural populations dominated by blue-green algae and/or diatoms, but if information on Pha and/or accessory pigments is required, HPLC analyses are the appropriate route rather than spectrophotometry.