Correction of Baseline Irregularities in the Gradient Elution of Phytoplankton Pigments by HPLC Using Methanol/Water/Ethyl Acetate

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A practical solution is found for an unexplained baseline irregularity occurring during the HPLC analysis of pigments extracted from phytoplankton. Pigments are eluted with 90% methanol/9.4% water/0.6% ethyl acetate for 10 min followed by the addition of ethyl acetate at the rate of 3%/min for 20 min. For two different spectrophotometric detectors, the initial addition of 0.6% ethyl acetate to the mobile phase is necessary to prevent an apparent absorbance change of up to 0.2 AUFS during gradient elution. This irregularity, which does not occur for another detector used, appears to be independent of solvent refractive index, detector bandpass, and transient mixing phenomena. Detector flow cell design is suggested as a possible cause.