Effect of Static extraction time on Extraction Efficiencies using On-line Supercritical Fluid Extraction-High Performance Liquid Chromatography for Lipoquinone Analysis in Activated Sludge

Ni Luh G. R. Juliasih, Lee Chang Yuan, Yoichi Atsuta, Hirotsugu Kamahara, Hiroyuki Daimon


Analysis of microbial community is important for monitoring the performance of biological processes. One of the most simple, quantitative and high reproducible method for analysis of the microbial community is lipoquinone profile method. Lipoquinone is constituent of bacterial plasma membrane that is essential for electron transporter. Lipoquinone could be used as a biomarker to analyze the microbial community because in general one species or genus of bacteria has one dominant type of lipoquinone, thus any changes in the lipoquinone profile would reflect the changes in the microbial community. The method for lipoquinone determination in environmental sample is direct extraction using organic solvent and analysis using chromatography system. Since the method is tedious and uses a large amount of organic solvent, the on-line supercritical fluid extraction-high performance liquid chromatography (on-line SFE-HPLC) has been developed to simplify the method, and was successful determine lipoquinone compounds in activated sludges. The effect of static extraction time on extraction efficiencies of the lipoquinone was investigated in order to eliminate the water pump and methanol pump in the previous system.  The CO2 was used as an extraction solvent.  The 0.1 g of freeze dried activated sludge was placed into a 1 mL stainless steel extraction vessel and methanol was spiked into the sludge as a modifier. The SFE was connected to HPLC by using trapping column as an interface for collecting lipoquinone extracted from the sludge. The static extraction time was conducted by allowed the matrix to immersed in CO2 and methanol. When the static extraction time finished, the dynamic extraction time was carried out. The extracted and trapped lipoquinone then directly transferred to HPLC system for determination. In this study, the effect of static, dynamic extraction time and volume of spiked methanol were optimized using simplified on-line SFEHPLC for lipoquinone analysis. The best results in terms of extraction yield were obtained at 25 MPa, 45°C, 10 min static extraction time with 500 µL methanol spiked, and 25 min dynamic extraction time with 0.9 mL min-1 CO2 flow rate. It was concluded that the developed method could simplify the online SFE-HPLC system of lipoquinone determination which is useful for a rapid and routine analysis of microbial community in activated sludge.

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