HPLC analysis of reaction compounds

Quantification of main components in the reaction mixtures was performed by HPLC using a Merck Hitachi L-7100 system (Merck, Darmstadt, Germany) equipped with an autosampler (L-7250) and a refractive index detector (L-7490). Separation of sucrose, glucose, fructose and cellobiose was performed with an YMC-Pack Polyamine II/S-5 µm/12 nm column (250 mm × 4.6 mm; YMC Co., Ltd., Shimogyo-ku, Kyoto, Japan). Additionally, a guard column (20 mm × 4.0 mm; YMC Co., Ltd.) was installed. Elution was performed isocratically with an acetonitrile–water mixture (75:25, v:v) at a flow rate of 1 mL/min. Measurements were performed at room temperature, the injection volume per sample was set to 20 µL and the running time was 35 min. Soluble COS (DP 2–6) were quantified by a Luna 5 µm NH2 column (100 Å, 250 × 4.6 mm, Phenomenex, Aschaffenburg, Germany) operated at 40 °C. Acetonitrile–water (70:30, v:v) was used as eluent at a flow rate of 1.5 mL/min and a running time of 15 min.

Representative chromatograms of product solutions measured by the stated HPLC methods are shown in Additional file 1: Figure S6. Refractive index detected peaks were analyzed using the software Chromeleon Chromatography Data System (Thermo Fischer Scientific Inc.). Calibration was done with standards containing main components. Reagent-grade COS standards ranging from DP 2–6 were from CarboSynth (Compton).

Insoluble COS production was calculated based on the molar balance between the sum of glucose units used as primers for individual soluble COS species and the actually consumed glucose units by subtracting moles of soluble COS (DP 2–6) from moles converted glucose.