Proceedings of International Symposium on EcoTopia Science 2007, ISETS07 (2007) Quinones Analysis from Environmental Sample using SFE and UPLC Muhammad Faisal 1,2 , Yoichi Atsuta 1 , Hiroyuki Daimon 1 and Koichi Fujie 1 1. Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Japan 2. Department of Chemical Engineering, Syiah Kuala University, NAD, Indonesia Abstract: Supercritical fluids extraction (SFE) with CO 2 and Ultra Performance Liquid Chromatography (UPLC) have been successfully applied to bacterial quinones analysis in sludge sample obtained from methane fermentation process. Experiments were carried out in a SFE system at a pressure of 25MPa, a temperature of 55 o C, and 10% (v/v) methanol for 15 min. The extracted quinones was then analyzed by using Ultra Performance Liquid Chromatography (UPLC) analytical method. Results showed that four ubiquinones and eight menaquinones species were identified in 0.1 g dried sample. The total extracted quinones of 263 μmol/kg-MLSS was identified without pre-fraction of ubiquinones and menaquinones by UPLC. Keywords: Quinones Profile, Solvent Extraction, SFE, UPLC 1. INTRODUCTION The use of quinone profile for analysis of microbial community in various environments including activated sludge, natural aquatic systems, agricultural soil and composts is of interest to researchers. It has been reported that the technique of quinone profiles has an advantage in terms of quantification, simplicity, and reproducibility [1]. The quinone profile analysis is also considered to be a useful tool for the analysis of microbial population dy- namics in mixed cultures, since it gives the information on the microbial biomass, the diversity and the structure of microbial community [2,3]. Current method for extrac- tion of quinone is performed by organic solvent extrac- tion with chloroform–methanol mixture. Identification and analysis was then performed using high performance liquid chromatography (HPLC). However, this method suffers many drawbacks, such as the use of a large amount of organic solvents, labour intensive and time consuming. Our previous work on quinones analysis from activated sludge sample has shown that supercritical car- bon dioxide (scCO 2 ) and HPLC is a promising technol- ogy to overcome the above mentioned problems [4]. The scCO 2 has been utilized in many applications since the solvent has favourable properties such as low critical temperature and pressure (Tc=31.1 o C, Pc=7.39 MPa). Moreover, scCO 2 is non toxic, environmentally benign solvent, and abundantly available at low cost. Recently, an improvement in chromatographic performance has been achieved by the introduction of UPLC. The UPLC is a relatively new technique in liquid chromatography giv- ing new possibilities to identify bacterial quinones. This technique provides higher peak capacity, greater resolu- tion, increased sensitivity and high speed of analysis over conventional HPLC [5]. In this study, the SFE and UPLC were applied to mi- crobial quinones analysis of sludge sample obtained from methane fermentation process. 2. EXPERIMENTAL The experiments were conducted using an SFE system. The system is mainly equipped with two high-pressure pumps, a back pressure regulator, a 1-ml extraction vessel and an oven which controls the temperature of the extrac- tion vessel. The details of the experimental apparatus have been described in our previous paper [4]. In a typi- cal extraction, 0.1 g of the dried sample of sludge ob- tained from methane fermentation process was placed into the extraction vessel. The system is then sealed. At the temperature and pressure reached the desired value, the CO 2 and modifier were continuously supplied by the high pressure pump to the extraction vessel. This moment was defined as the beginning of extraction. During the extraction, the extracted microbial quinones were col- lected in collection flask and then evaporated using rotary vacuum evaporator after completed extractions. These dried extracted quinones, containing menaquinones and ubiquinones, were then loaded into two Sep-Pak ® Plus Silica cartridges using 15 ml hexane solution at a fixed flow rate. The sample was then analyzed by HPLC and UPLC analysis. 3. RESULTS AND DISCUSSION Previous study on microbial quinones extraction with scCO 2 has shown that significant amount of quinones could be extracted from activated sludge sample at a temperature of 55 o C, a pressure of 25MPa, and 10% (v/v) methanol for 15 min. At these conditions, the quinone contents obtained by scCO 2 extraction with methanol as modifier were compared to those obtained by organic solvent extraction (Irvan, 2006). The results were com- parable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Based on these results, the same extraction conditions were con- ducted in current experiments. Figure 1 shows the analysis results of quinones by us- ing HPLC and and UPLC. Each experiment was repeated three times under the same conditions to verify the re- Corresponding author: M. Faisal, faisal@fujielab.eco.tut.ac.jp 1196