Hydrothermal liquefaction of pine biomass: 5-hydroxymethylfurfural formation as a function of temperature and residence time quantified by high-performance thin-layer chromatography

5-Hydroxymethylfurfural (5-HMF) is a key lignocellulosic-derived platform molecule formed during biomass conversion processes such as hydrothermal liquefaction (HTL). Its determination in this kind of matrices relies mainly on chromatographic techniques requiring extensive sample preparation and high solvent consumption. In this work, a method based on high-performance thin-layer chromatography (HPTLC) combined with densitometric detection and mass spectrometry (HPTLC-densitometry-MS) was developed for the separation, on-plate UV and MS identification and quantification of 5-HMF in bio-oils obtained from the HTL of pine biomass ( Pinus radiata ). Samples produced under different temperatures (150–350 °C) and reaction times (0–90 min) were analysed, allowing the influence of process severity on 5-HMF formation during HTL to be evaluated. The method enables the simultaneous analysis of multiple samples and standards on a single plate and provides both qualitative chromatographic fingerprinting of complex HTL-derived bio-oils and quantitative determination of 5-HMF, with good linearity, precision, and sensitivity. Maximum 5-HMF contents reached 15.2 wt % in the aqueous phase and 7.1 wt % in the oily phase. Carbon yields ranged from 3 to 16 % C in the aqueous phase and from 5 to 31 % C in the oily phase. Overall, the results showed that intermediate temperatures and short residence times favoured 5-HMF formation, whereas higher temperatures or prolonged reaction times promoted its degradation through secondary reactions. The proposed method represents a simple, high-throughput and environmentally friendly analytical approach for the determination of 5-HMF in HTL-derived bio-oils, as indicated by the calculations of National Environmental Methods (NEMI) and the Blue Applicability Grade (BAGI) indices.