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A simple method to increase sensitivity for RP-HPLC phytoplankton pigment analysis

Mikel Latasa

Limnol. Oceanogr. Methods 12:46-53 (2014) | DOI: 10.4319/lom.2014.12.46

ABSTRACT: HPLC analysis of phytoplankton pigments from natural samples usually requires the filtration of relatively large volumes of water (1-12 L) through filters of 25-47 mm diameter. Pigment extraction from filters of this size is performed with minimum volumes of 2-5 mL extraction solvent, routinely 90% acetone. The difference in polarity between the solvent for extraction and the initial eluent for HPLC analysis of phytoplankton pigments limits the load to c.a. 150 μL sample into the chromatographic system. This implies that less than 10% of the sample is analyzed, with the consequent decrease of pigment detectability. Here, a procedure allowing injections of up to 1440 μL of 90% acetone without pigment loss is presented. The method can be easily implemented in current HPLC systems. The response of the peak area measured against volume injected was linear (r2 > 0.998 for any pigment). The precision for injections between 120 and 1440 μL varied between 0.08 and 4.9% (coefficient of variation, n = 4). This system increases the signal response one order of magnitude, reducing the amount of volume filtered and/or enhancing the detectability and identification of minor pigments.