Liquid chromatography of thin stillage

Anions and organic acids were analyzed by hydroxide-selective anion exchange chromatography. Samples were injected onto a 250 × 4 mm AS11-HC column (Thermo Scientific) equipped with a 50 × 4 mm AG11-HC IonPac guard column kept at 30 °C. The instrument used was a Thermo Scientific IC-5000+ with cooled autosampler, isocratic pump, eluent generator, thermostatted column compartment, and suppressed conductivity detector. Compounds were eluted at a flow rate of 2 mL/min and a hydroxide gradient of 0.2 mM isocratic for 6 min, then over 5 min to 5 mM, then over 16 min to 40 mM. Detection was performed by suppressed conductivity.

Monosaccharides were analyzed by anion exchange chromatography using a Thermo Scientific IC-5000+ instrument as described above except that a pulsed amperometric detector was used. The column used was a 150 × 3 mm CarboPac PA20 (Thermo Scientific) equipped with a 30 × 3 mm CarboPac PA20 guard column (Thermo Scientific). Compounds were eluted isocratically with 2 mM hydroxide at a flow rate of 0.4 mL/min and detected by pulsed amperometric detection.

Glycerol, ethanol, 5-HMF, and furfural were analyzed using a 300 × 7.8 mm HPX-87H column (BioRad, Richmond, CA, USA) equipped with a 30 × 4.6 mm Micro-Guard Cation H cartridge (BioRad). Compounds were eluted isocratically with 5 mM sulfuric acid at a flow rate of 0.6 mL/min and detected by refractive index detection (glycerol, ethanol) or UV (5-HMF, furfural).

All compounds were quantified by external calibration using mixtures of standards (purity ≥98 %) or using the combined seven anion standard (Thermo Scientific, San Jose, CA, USA).