Luna 5 m nh2 column

The obtained products were also analyzed and purified by HPLC (Agilent Technologies 1260, Waldbronn, Germany) using a Luna^® 5 µm NH₂ column (4.6 × 250 mm, Phenomenex, Torrance, CA, USA) and detected with a RI detector. The maltitol product mixture was filtered through a nylon membrane, 0.45 μm disc filter (Merck Millipore, Darmstadt, Germany) before injection and elution with acetonitrile:water (75:25, v/v) using a flow rate of 1.0 mL/min at 55 °C [29 (link)]. Product yields were determined using Equation (2), where t₀ is the time the reaction commenced.

For quantification of soluble COS, measurement was done on a Hitachi LaChrom HPLC system (Merck, Darmstadt, Germany) equipped with a Luna 5 µm NH₂ column (100 Å, 250 × 4.6 mm, Phenomenex, Aschaffenburg, Germany) operated at 40 °C. Acetonitrile–water (70:30, volume ratio) was used as eluent at a flow rate of 1.5 mL/min. Refractive index detection was used. Calibration was done with authentic standards of G2 (2.5–50 mM), G3/G4 (2.5–15 mM), G5 (1.0–7.5 mM) and G6 (1.0–2.5 mM). The phosphate release was measured using colorimetric assay [39 (link)].
The measured concentration of substrate and products were assessed for internal consistency based on mass balance. Considering the reactions in which a substantial portion of products ended up insoluble, a so-called soluble mole ratio (mol.%) was defined. This is the ratio of glucosyl units in soluble products to the total glucosyl units transferred from αGlc1-P in the overall reaction. The released phosphate equals the αGlc1-P converted and the glucosyl residues not accounted for in the soluble products are considered to be present as insoluble products. The experimentally traceable soluble COS are limited to DP ≤ 6. Glucosyl residues not found in G3–G6 are thus taken as insoluble COS. The model-derived data were therefore treated in exactly the same way.