India, the largest consumer of vegetable oil, generates massive amounts of wastewater during its production, adversely affecting the aquatic ecosystem. Various methods are used to degrade vegetable oils. Conventionally, oil degradation was quantified by measuring a sample’s end products, such as free fatty acid (FFA) or glycerol. None of these techniques measures intermediate compounds (monoglycerides or diglycerides), which leads to erroneous oil degradation calculations. We propose a high-performance thin-layer chromatography (HPTLC) based method to quantify the percentage degradation of soybean oil. The mobile phase consists of n-hexane, ethyl acetate, acetic acid (9:1:0.1, v/v/v) and spots scanned at 196 nm. Soybean oil in water (1%, v/v) degraded using immobilized lipase at 37 °C temperature, pH 7, and 180 rpm for 6 h. The change of bonds in FTIR qualitatively confirms the degradation of oil. The proposed method was validated by comparing the quantity of FFA obtained from the HPTLC chromatogram (22.62 ± 0.07%, w/w) with the data calculated from acid value (21.98 ± 0.56%, w/w). The percentage degradation was calculated as 45.67 ± 0.67 (w/w). The present study shows that the developed method is a consistent, precise, reproducible, accurate, and rapid analytical technique for quantifying degraded oil in water.
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