Photosynthetic pigment concentrations are used extensively to estimate phytoplankton biomass.1,2 All green plants contain chlorophyll a, which constitutes about 1% to 2% of the dry weight of planktonic algae. Other pigments in phytoplankton include chlorophylls b and c, xanthophylls, phycobilins, and carotenes. Important chlorophyll degradation products found in the aquatic environment are chlorophyllides, pheophorbides, and pheophytins. The presence or absence of various photosynthetic pigments is used, among other features, to identify major algal groups.
Three methods to determine chlorophyll a in phytoplankton are the spectrophotometric,3–5 fluorometric,6–8 and high-performance liquid chromatographic (HPLC) techniques.9 Fluorometry is more sensitive than spectrophotometry, requires less sample, and can be used for in vivo measurements.10 These optical methods can significantly under- or overestimate chlorophyll a concentrations,11–18 in part because the absorption and fluorescence bands of co-occurring accessory pigments and chlorophyll degradation products overlap.
Pheophorbide a and pheophytin a, two common degradation products of chlorophyll a, can interfere with the determination of chlorophyll a because they absorb light and fluoresce in the same region of the spectrum as chlorophyll a. If these pheopigments are present, significant errors in chlorophyll a values will result. Pheopigments can be measured either via spectrophotometry or fluorometry, but in marine and freshwater environments, the fluorometric method is unreliable when chlorophyll b co-occurs, unless a non-acidification method is used (EPA Method 445.0). On acidifying chlorophyll b, the resulting fluorescence emission of pheophytin b coincides with that of pheophytin a, causing underestimation and overestimation of chlorophyll a and pheopigments, respectively. The nonacidification method has the following advantages: more accurate chlorophyll a data, less labor intensive, fewer resources needed, and very sensitive.
HPLC is a useful method for quantifying photosynthetic pigments,9,13,15,16,19–21 including chlorophyll a, accessory pigments (e.g., chlorophylls b and c), and chlorophyll degradation products (chlorophyllides, pheophorbides, and pheophytins). Pigment distribution is useful for quantitative assessment of phytoplankton community composition and zooplankton grazing activity.22