| Summary: | The analysis of photosynthetic pigments (chlorophylls and carotenoids) has become a source of essential information on the physiology and ecology of marine microalgae, including the study of natural plankton populations, the ground-truthing of satellite derived biomass estimations, the responses to changes in light environment, etc. The detailed information of the pigment composition of planktonic communities is usually obtained by chromatographic analysis of their pigment extracts, that are prepared in a process that (very briefly) implies the sampling of natural waters from adequate depths in the course of oceanographic campaigns, the filtration of quite large volumes of water onto glass-fiber filters (25 or 47 mm diameter), and then the extraction of the filters with minimum volumes of 2-5 mL of an organic solvent, routinely 90% acetone. But after such a great scientific effort, only a small proportion (less than 10 %) of the extract can be used for the analysis (due to the restrictions imposed by the injection process in liquid-chromatography), thus compromising the sensitivity of the result. This limitation has a special significance in the case of oligotrophic waters where the planktonic biomass is particularly reduced.
It is well known that the addition of a salt into a mixture of water and a water-miscible organic solvent causes a separation of the solvent from the mixture and the formation of a two-phase system. We have employed this approach to concentrate pigment solutions previous to their HPLC analysis: concentrated saline solutions were added to acetonic extracts to generate a biphasic system in which pigments are transferred to the immiscible acetone upper phase in a dispersive liquid-liquid microextraction process.
Model pigment solutions were prepared, adjusting the concentration of 90% acetone extracts of Chrysochromulina throndsenii to mimic extracts coming from the filtration of 2 L seawater with different total chlorophyll contents. In a typical concentration procedure, different volumes of saline solutions were added to 2.5 mL of pigment extract in a screw cap glass tubes with polytetra-fluoroethylene (PTFE) lined cap. The mixture was shaken during 15 seconds by means of a vortex agitator and the two phases were allowed to separate at 25 ºC. After measuring its volume, an aliquot (130 uL) of the upper acetone phase was injected in the HPLC system. We studied the effect of different salts (magnesium chloride, magnesium acetate, ammonium acetate and ammonium formate), their concentrations and the volume ratio to acetonic extracts on the enrichment factor and the stability and recovery of the pigments.
Both magnesium salts assayed were rejected as they promoted alteration products of different pigments. Magnesium chloride solutions produced a carotenoid derivative, probably generated from fucoxanthin, while magnesium acetate led to the formation of epimers of chlorophyll. No alteration product was detected with the ammonium salts assayed.
The volume of immiscible acetone phase (and thus the enrichment factor) depended both on the concentration and the volume of saline solution added to the mixture. Final volumes of concentrated phase of about 400 uL (enrichment factors of 6.25) were chosen due to difficulties in the handling of smaller volumes.
The recoveries of different pigments varied depending on their chemical nature and the degree of enrichment of the extract. Carotenoids and non-polar chlorophylls were recovered above 85 % with 5M saline solutions, while the recovery of acidic clorophylls (c3 and c2) decreased with enrichment factors.
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