Development of a fluorescence-based microplate method for the determination of volatile fatty acids in anaerobically...

This paper presents a simple, accurate and multi-sample method for the determination of volatile fatty acids (VFAs) thanks to a 96-well microplate technique. A procedure using an activating reagent of the carboxylic function (water-soluble carbodiimide EDC) and a fluorescent amino labeling reagent (N-(1-naphthyl)ethylenediamine, EDAN) allows the formation of an isoindole derivative that needs to be separated from initial fluorescent amine for efficient VFAs determination. Isolation of these fluorescent VFA-derivatives was carried out by use of the fluorescent quenching of EDAN with o-phthaldialdehyde (OPA). Quenching was most efficient at pH around 7 and by heating at 40°C within the microplate reader. This optimized procedure has been applied to various carboxylic acids and other organic compounds, demonstrating that VFA exhibit the highest fluorescence responses with homogeneous results for the main ones (acetic, propionic and butyric acid, all mass concentration expressed as acetic acid equivalents). This protocol was calibrated against acetic acid and determination of VFA was thus possible in the range 3.9-2,000 mg L(-1) (acetic acid equivalents). Subsequent application to real samples (sewage sludges or anaerobically digested samples) and comparison to gas chromatography analyses gave accurate results, proving the great potential of our high-throughput microplate-based technique for the analysis of VFA.