HPLC ANALYSIS OF ALGAL PIGMENTS - A COMPARISON EXERCISE AMONG LABORATORIES AND RECOMMENDATIONS FOR IMPROVED ANALYTICAL PERFORMANCE

Pure individual and mixed pigment standards were distributed among Joi nt Global Ocean Flux Study (JGOFS) pigment analysts to estimate the va riability of their spectrophotometric and chromatographic systems. To monitor the integrity of the pigments during the comparison exercise, chlorophyll and carotenoid standards were archived and periodically an alyzed by high-performance liquid chromatography (HPLC). Pigment stand ards stored in the dark under nitrogen at - 20 degrees C were found to be stable for periods of at least one year. Results from three separa te intercalibration exercises document a better agreement for spectrop hotometric analyses than for HPLC. For the spectrophotometric comparis ons, 90% of the pigments analyzed by participant laboratories were wit hin +/- 6% of the mean ''consensus'' values. By contrast, 65 and 85% o f the laboratories agreed to within +/- 10 and +/- 20%, respectively, when chromatographic analyses were compared. Chlorophyll absorption me asurements obtained with a diode array-type spectrophotometer were 6-9 % lower than those obtained with monochromator-type spectrophotometers . These underestimates probably result from chlorophyll fluorescence c ontamination associated with the optical configuration of the diode ar ray spectrophotometer. It was also determined that HPLC methods which are not capable of separating monovinyl chlorophyll a from divinyl chl orophyll a can produce 15-25% overestimates of total chlorophyll a con centration in Prochlorococcus-dominated oceanic waters. A simple dichr omatic approach is described for eliminating this variable source of e rror caused by co-elution of these structurally-related pigments. The use of internal standards and periodic calibration checks with externa l standards is highly recommended for improving analytical performance .