2.6 Analytical methods
The reaction was stopped by placing it in a boiling bath, and the
reaction solution was centrifuged at 16,200 × g for 2 min, and
the supernatant was filtered with a 0.22-µm organic phase membrane. The
concentrations of galactose, galactitol, ethanol, and tagatose were
determined using high-performance liquid chromatography equipped with a
refractive index detector (RI detector L2490, HITACHI, Japan) using a
Shodex SP-0810 column (300 × 8 mm, Showa Denko K.K., Tokyo, Japan). The
mobile phase was double distilled water, and the samples were separated
at 70 °C at a flow rate of 1 mL min−1. The retention
times of galactose, galactitol, and tagatose were 9.91, 21.83, and 14.29
min, respectively. The formation of scaffolds was confirmed by negative
stain transmission electron microscopy, as previously
described.[15]