Sidik Marsudi Muhammad Hanif Jin Yoshino Yoichi Atsuta Hiroyuki ...

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Sidik Marsudi Muhammad Hanif Jin Yoshino Yoichi Atsuta Hiroyuki ...

The Asian Conference on Sustainability, Energy & the EnvironmentOfficial Conference Proceedings 2012Osaka, JapanFigure 1 Analytical steps of the SFE with solid-phase trap and SFE without trap method.2.4 Supercritical Fluid ExtractionFigure 2 shows a schematic diagram of SFE-solid-phase method used in the present study.The system mainly consisted of an oven (GL Sciences GC-353B oven, Tokyo, Japan)equipped with an extraction vessel (1 mL internal volume, SUS 316.47 mm long, 10 mmI.D.), a backpressure regulator (Jasco-BPG 880-81, Tokyo Japan) used to control the pressureand a high-pressure pump: Jasco PU-1580 HPLC pump (Tokyo, Japan) used to deliver CO 2 .Besides, a solid-phase trap (octadecyl-silica cartridge, Zorbax-ODS 4.6 mm I.D. x 12.5 mm x5 µm, Agilent Technologies, Santa Clara,CA, USA) was directly mounted to the SFE systemto collect the extracted microbial quinones.In the SFE with solid-phase trap method, effects of static-dynamic extraction were studied.Approximately 100 mg of sample (freeze-dried digested sludge sample) was filled into theextraction vessel. After that, methanol – as a modifier – at various volumes (200 µL, 300 µL,400 µL, or 500 µL) was also added directly onto the sample inside to the vessel. The samplewas then extracted by supercritical CO 2 in static mode where pressure and temperature ofextraction vessel were 25 MPa and 45 !C, respectively. After 5 or 10 minutes of staticextraction time, the extracted microbial quinones were flushed from the vessel by thesupercritical CO 2 at a flow rate of 3 mL/min for 5 min, passed through to backpressureregulator (restrictor), and collected to the ODS solid-phase trap. The trap containingextracted microbial qinones then transferred manually to HPLC for quinones analysis.The effect of dynamic extraction time was performed by adding 400 µL of methanol as amodifier to the sample followed by extracting the sample for 10 min in static mode at acondition as describe above. After that, the extraction was continued by flowing supercritical440


The Asian Conference on Sustainability, Energy & the EnvironmentOfficial Conference Proceedings 2012Osaka, JapanFor UPLC analysis, the extracted microbial quinones were analyzed using UPLC (WaterAcquity system, Milford, MA, USA) equipped with a binary solvent delivery manager, asample manager and a photo-diode array detector (PDA-2996, Water), and an analyticalcolumn (Waters Acquity UPLC TM BEH C18 column, 1.7µm, 2.1mmx50mm). The mobilephase was 100% methanol and pumped to the column at a flow rate of 0.5 mL/min. Sampleinjection volume was 10 µL and the auto-sampler temperature was set at 40 ± 1 !C. Allanalyses were performed at a column temperature of 35±1 !C with a chromatographic runtime of 35 min. At this column temperature and the mobile flow rate, separation wasachieved in 10 min with a backpressure of 8,000 psi.Quinone species detected by HPLC or UPLC were identified base on the retention time onthe column and spectrum of each peak observed in the detector. The linear relationshipbetween the logarithm of the retention times of quinones and the equivalent number ofisoprenoid unit (ENIU) was also used to identify the quinone species (Hasanuddin et al.,2004; Katayama and Fujie, 2000). The amounts of quinone species were calculated based onpeak area and molar absorption coefficient of the quinones (ubiquinones 14,4 mM -1 cm -1 ,menaquinones 17.4 mM -1 cm -1 ) (Kroger 1978).3. RESULTS AND DISCUSSIONThe use of ODS solid-phase trap was evaluated as trapping media to collect extractedquinones after SFE. The SFE with solip-phase trap experiments were performed in staticdynamicmode. The effect of different operational parameters (e.g., static extraction time anddynamic extraction time) on extraction efficiency was studied. The extraction efficiency wascalculated based on the extracted quinones amount obtained by SFE with solid-phase trapmethod compared to quinones amount obtained by the SFE without trap method.1.1 The Effect of Static Extraction Time and Injection Volume of Methanol as a ModifierThe effect of static extraction time and methanol as a modifier was evaluated to the extractionefficiency of quinones in ODS solid-phase trap. Figure 3 shows the extraction efficiency ofSFE with solid-phase trap at 5 min or 10 min of static extraction time and at various injectionvolumes of methanol as a modifier. At 5 min static extraction time, the highest extractionefficiency was obtained when 400 µL of methanol was added as a modifier to the sample.There was no significant increase when 500 µL of methanol was added to the sample andthere was no effect at all when 200 µL and 300 µL of methanol were added. On the contrary,at 10 min of static extraction time, the extraction efficiency increased by increasing ofmethanol addition until reached a maximum efficiency of 91 % at an addition of 400 µL.However, the efficiency decreased when 500 µL was added to the sample.442


The Asian Conference on Sustainability, Energy & the EnvironmentOfficial Conference Proceedings 2012Osaka, Japan100Extraction efficiency (%)7550255min10min0200µL 300µL 400µL 500µLInjection volume of methanol as modifierFigure 3 Extraction efficiency of SFE with solid-phase trap compared to SFE without trapmethod at various static extraction times and injection volumes of modifier.When static extraction time was increased from 5 min to 10 min, extraction efficiency wasincreased by 16% at a modifier volume of 400 µL. However, no significant effect could beobserved to other modifier volumes. It was reported that static extraction time influenced theextraction efficiency (Lehotaya ang Valverde-Garcia, 1997). The time was required to allowextraction fluid to fully penetrate the sample and dissolve the analyte. In the current study(Figure 3), a significant effect only could be observed at an addition volume of 400 µL.These results show the applicability of SFE with solid-phase trap using ODS. Based on theseresults, it could be decided that addition modifier of 400 µL at 10 min static extraction timegave the best result amount others. These operation parameters were used for further study.1.2 Effect of Dynamic Extraction Time and Supercritical Flow Rate of CO 2Instead of dynamic mode of SFE as performed in our previous works (Irvan et al., 2006a;Irvan 2006b; Hanif et al., 2012a; Hanif et al., 2012b), in this work, a static-dynamic modewas performed. The effect of dynamic extraction time (1-15 min) and supercritical flow rateof CO 2 (1-3 mL/min) were shown in Figure 4. At a flow rate of 2 mL/min and 3 mL/min,increasing dynamic extraction times were increased the extraction efficiency. When thedynamic extraction time was increased to 10 min, extraction efficiency reached maximumvalue of 66% and 109% for flow rate of 2mL/min and 3 mL/min, respectively. However, at aflow rate of 1 mL/min, the maximum value of 100% occurred faster, i.e. at a dynamicextraction of 5 min. In all experiments, after reaching a maximum value, increasing dynamicextraction times were decreased extraction efficiency. This decrease was probably caused bya decrease in trapping efficiency due to saturation of the ODS solid-phase trap with amodifier (methanol) (Nemoto et al., 1997; Mulchahey and Taylor, 1992; Howard and Taylor,1993). On ODS solid-phase trap with methanol, trapping efficiency increased by increasing443


The Asian Conference on Sustainability, Energy & the EnvironmentOfficial Conference Proceedings 2012Osaka, Japanthe methanol volume until a certain volume of methanol, then the efficiency decreased whenmethanol volume was increased due to saturation.It was demonstrated that at a dynamic extraction time of 10 min, the extraction efficiencyreached a maximum value that was higher than 100 %. It means that quinones could beextracted and trapped using ODS solid-phase trap and this trapping system showed a betterquinones recovery compared than that of without trapping system. Based on the results ofthose operating parameters, the optimum conditions were at a static extraction time of 10min, an addition of methanol as a modifier of 400 µL, a dynamic extraction time of 10 minand a CO 2 flow rate of 2 mL/min.Figure 4 Extraction efficiency of SFE with solid-phase trap method compared to theSFE without trap method at various dynamic extraction times andsupercritical flow rate of CO 2 .1.3 Comparison of SFE with solid-phase trap method to SFE without trap methodThe quinone content obtained by the SFE with solid-phase trap was compared to the SFEwithout trap method. The SFE with solid-phase trap method was carried out at an optimumcondition as previously determined (static extraction time, addition of methanol as a modifier,dynamic extraction time and CO 2 flow rate were 10 min, 400 µL, 10 min and 2 mL/min,respectively). The results are shown in Figure 5. Three types of ubiquinones (UQ-8, UQ-9and UQ-10) and eight types of menaquinones were identified based on the retention time andthe molar absorption coefficient. The same type and number of quinones were identifiedusing the two methods. The most dominant of bacterial quinones from the digested sludgesamples were MK-7, MK-8(H 2 ), UQ-9, MK-5(H 2 ), MK-9(H 2 ). The total amount of extractedquinones by SFE with solid-phase trap and SFE without trap method were 75.17 µmol/kgdry-sampleand 68.85 µmol/kg-dry-sample, respectively. It means the recovery of totalquinones obtained by SFE with solid-phase trap method was higher than that of SFE witouttrap method. In addition, the time required to extract and analyze the samples 2 times faster(1.5 h) than that of the SFE witout trap method (more than 3 h). The solvent required wasalso 3 times smaller (60 mL) than that of SFE without trap method (195 mL). Based on these444


The Asian Conference on Sustainability, Energy & the EnvironmentOfficial Conference Proceedings 2012Osaka, Japanresults, it can be concluded that the SFE with solid-phase trap can replace the SFE withouttrap method.Quinones content (µ mol/kg-dry sample)80706050403020100MK-9(H4)MK-9(H2)MK-8(H2)MK-5(H2)MK-9MK-8MK-7MK-6UQ-10UQ-9UQ-8SFE without trapSFE with solid-phase trapFigure 5 Comparison of SFE-solid-phase trap method to SFE+UPLC method.4. CONCLUSIONSIt was demonstrated that ODS solid-phase trap (Zorbax-ODS) could be used as trappingmedia to collect microbial quinones after SFE. This method performed an online system ofSFE extraction to HPLC analysis for determination of microbial quinones from digestedsludge sample. Recovery of quinones obtained by SFE with solid-phase trap method washigher than that of SFE without trap method. In SFE with solid-phase trap method, the timeand solvent amount required to determine microbial quines were shorter and smallercompared to SFE without trap method, respectively. The SFE with solid-phase trap methodwas simpler and stepped ahead compared to our previous works in term of extended topossibilities of automation in application for analyzing of microbial quinones inenvironmental samples.ACKNOWLEDGMENTThe first author would like to express his gratitude to Greater Nagoya Initiative Program forthe financial support of all his activities in Japan including attending this conference. Theauthors appreciate Mr. Beppu on his support of our laboratory work and Maezawa Industries445


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