HPLC Analysis of Hydrolysate Composition

Using a Shimadzu LC-20A HPLC (Shimadzu, Tokyo, Japan) with a refractive index detector, the amount of monosaccharides, 2-furaldehyde, 5-HMF, and organic acids in the resulting hydrolysates were quantified. Cellobiose, glucose, xylose, arabinose, galactose, mannose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid were used as reference standards. We employed a Shodex Sugar SH1821 column at 60 °C, with an eluent of 0.008 M H₂SO₄ at a rate of 0.6 mL/min^-1, to separate the cellobiose, glucose, 2-furaldehyde, acetic acid, 5-HMF, levulinic acid, and formic acid. For the carbohydrate analysis, we used a Shodex Sugar SP0810 column at 80 °C, with deionized water as the mobile phase under a flow rate of 0.6 mL·min⁻¹. Samples were neutralized to pH 5–7 with BaCO₃ and filtered through a 0.2 μm membrane filter before injection. All samples were tested three times. For each analyzed standard, the equations for the calibration curves are given in our previous publication [15 (link)].