Thapanee Pruksatrakul ^a, Pattamaporn Phoopraintra ^a, Prapin Wilairat ^b, Pimchai Chaiyen ^c, Rattikan Chantiwas ^a,⁎

^a Department of Chemistry and Center of Excellence for Innovation in Chemistry and Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand

^b National Doping Control Centre, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
^c Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology,
Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
*Corresponding author: rattikan.cha@mahidol.ac.th, rattikan.cha@mahidol.edu (R. Chantiwas)

Short-chain fatty acids, such as acetic, propionic, butyric, iso-valeric and valeric acids, play an important role in methanogenesis activity for biogas production processes. Thus, simple and rapid procedures for monitoring the levels of short-chain fatty acids are requisite for sustaining biogas production. This work presents the development of a sequential injection-liquid microextraction (SI-LME) procedure with GC-FID analysis for determination of short-chain fatty acids. GC-FID was employed for detection of the short-chain fatty acids. Calibration curves were linear with good coefficients of determination (r2 > 0.999), using methacrylic acid as the internal standard. Limits of quantification (LOQ) were in the range of 0.03–0.19 mM. The SI-LME procedure employed tert-butyl methyl ether (TBME) as the extracting solvent. Various SI-LME conditions were investigated and optimized to obtain the highest recovery of extraction. With these optimized conditions, an extraction recovery of the five key short-chain fatty acids of 67–90% was obtained, with less than 2% RSD (n=3). The final SI-LME procedure employed two fluidic zones of TBME with a single aqueous fluidic zone of sample sandwiched between the TBME zones, with 5 cycles of flow reversal at a flow rate of 5 µL/s for the extraction process. Intra- and interday precision values were 0.5–4.0% RSD and 3.3–4.8% RSD, respectively. Accuracy based on percentage of sample recovery were in the range of 69–96, 102–107, and 82–101% (n=4) for acetic, propionic and butyric acids, respectively. The proposed method was applied for the measurement of short-chainfatty acids in palm oil mill effluents used in biogas production in a factory performing palm oil extraction process. The SI-LME method provides improved extraction performance with high precision, and is both simple and rapid with its economical extraction technique. The SI-LME procedure with GC-FID has strong potential for use as a quality control process for monitoring short-chain fatty acid levels in biogas production.