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Chromatographic separation was carried out on a Shimadzu scientific instrument (Shimadzu Corporation; Kyoto, Japan) with a Luna C18 (2) 100A column (100 × 4.6 mm, 5 μm) (Phenomenex).
The absence of co-eluting peaks as well as interference from matrix ions was minimal with a Luna C18 (2) column in combination with mobile-phase ammonium acetate buffer and acetonitrile (90 10, v/v).
The resulting solution was injected (5 μl) onto a 1200 series LC system (Agilent Technologies), consisting of 1200 series binary pump, autosampler, vacuum degasser, column compartment and control module, with a Luna 3 μm C18(2) 100 Å, 100 mm × 2.00 mm column and a SecurityGuard cartridge kit (Phenomenex, Macclesfield, UK) as a guard column.
LC separation was achieved with a Luna reverse-phase C5 column (Phenomenex).
The chromatographic separation was performed with a Luna Phenomenex C-18 column (250 mm × 4.6 mm × 5 μm).
HPLC-UV analysis was performed on a Shimadzu Prominence HPLC system with a Luna 5μm C18 (2) 100Å (250 × 4.60 mm I.D).
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For separation of polar metabolites, normal-phase chromatography was performed with a Luna-5 mm NH2 column (50 mm × 4.60 mm, Phenomenex).
Briefly, a Phenomenex C18(2) Luna column (250 mm×4.6 mm×5 µm) in combination with a C18 Luna guard column were used.
The concentration of HMF and furfural in the pretreated liquor were analyzed on a Phenomenex Luna C18(2) reversed phase column equipped with a Phenomenex Luna C18(2) precolumn (Separations, Johannesburg, South Africa) with column temperature set to 25°C and a flow rate of 0.7 ml/min.
The intermediate was purified by a semi-prep HPLC system equipped with a Phenomenex Luna C18 column and mobile phase of 55 % MeCN in water at a flow rate of 5 mL/min.
The LC system was equipped with a Phenomenex Luna HILIC column (100 × 4.6 mm, 3 m) and a Phenomenex SecurityGuard 4.0 mm × 2.0 mm pre-column.
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