Hyperrez xp carbohydrate h column

Yeast concentration was determined by dry cell weight analysis. A 10 mL volume of cell suspension from the culture was washed twice with water (2 × 2.5 mL). After centrifuging (at 5000 rpm for 5 min), the biomass was separated using a 0.45 μm pore-sized membrane filter (Merck Millipore, Burlington, Massachusetts, USA) dried at 105 °C until a constant weight was reached, cooled in a desiccator, and weighed.
Mannitol, erythritol, glycerol, arabitol, and citric acid concentrations were determined using the HPLC method (Dionex—UltiMate 3000 LC Systems, Thermo Fisher Scientific, Waltham, MA, USA) on a HyperRez XP Carbohydrate H+ column (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a UV detector (λ = 210 nm) and an RI detector (Shodex, Resonac Europe GmbH, Munich, Germany). The column was eluted at 65 °C with 20 mM trifluoroacetic acid solution at a flow rate of 0.6 mL/min.

To observe cell growth during fermentations, the cell density of the collected samples (OD_600nm) was measured via spectrophotometer (Ultraspec 2100 pro spectrophotometer, GE Healthcare, USA). For metabolite analyses via HPLC, samples were centrifuged at 16,000×g for 5 min and 450 µL of supernatant was mixed and vortexed with 50 µL 5-sulfosalicylic acid for precipitation of proteins. After an additional centrifugation step (16,000×g for 5 min), the supernatant was analyzed via HPLC. The HPLC (Thermo Fisher, Germany) was equipped with a HyperREZ XP Carbohydrate H + column (300 × 700 mm, 8 micron; Thermo Fisher Scientific, Germany), coupled to a refractive index detector (Shodes RI-101, Shoko Scientific Co., Kanagawa, Japan). 5 mM of H₂SO₄ was used as mobile phase with a constant flow rate of 0.6 mL/min. The column temperature was kept constant at 65 °C [17 (link)].