Hpx 87p column

The crude enzymatic extracts produced by C. cubensis and com- mercial cellulase (Multifect® CL) were applied in a biomass saccharification experiment. The C. cubensis enzymatic extract were concentrated 5-fold before the experiment using an Amicon Ultra- filtration system (Millipore Co. – Billerica, MA, USA) and an YM-10 (Cut-off Mr 10,000 Da) membrane filter. Enzymatic saccharification of alkali-treated sugarcane bagasse was performed in 2 mL sample tubes at an initial solid concentration of 2% dry matter (w/v) in 1.5 mL of 50 mM sodium acetate buffer at pH 4.5. Enzyme loading was specified as 10 FPase units per gram of biomass with the addition of sodium azide (10 mM) and tetracycline (40 μg mL⁻¹) to the reaction mixture to inhibit microbial contamination. The reaction was carried out in an orbital shaker at 250 rpm and 50 °C for different time intervals up to 72 h. These samples were immediately heated to 100 °C to denature the enzymes, cooled and then centrifuged for 5 min at 15,000 g. Products of the saccharification assays were analyzed by high performance liquid chromatography (HPLC) with a Shimadzu series 10 A chromatograph. The HPLC was equipped with an Aminex HPX-87P column (300 × 7.8 mm) and refractive index detectors. The column was eluted with water at a flow rate of 0.6 mL min⁻¹ and 80 °C.

The sugar contents were quantified by the HPLC (Hitachi, Japan) equipped with an Aminex HPX-87P column. Nano-pure water was used as a mobile phase running at 0.6 mL/min. The column temperature was 80 ℃, and the elution time was 40 min. Acetic acid, HMF, and furfural were quantified by the HPLC system equipped with an HPX-87H column. The mobile phase was composed of 5 mM of sulfuric acid running at 0.6 mL/min. The column temperature was kept at 55 °C, and the elution time was 50 min.

Twenty microliters of S. mutans culture (OD600 = 0.01), 20 µL of L. pentosus MJM60383 supernatant, and 160 μL of BHI medium containing 1% sucrose were added to the wells of a 96-well plate and incubated at 37°C for 24 h. The experiment treated with MRS broth instead of L. pentosus MJM60383 supernatant served as a control. The sucrose content in the supernatant was assessed by HPLC with a refractive index detector (Agilent Technologies, Boeblingen, Germany) [26 (link)]. All samples were conducted on the Aminex HPX-87P column and eluted with distilled water as the mobile phase at a flow rate of 1 mL/min for 30 min. The injection volume was 30 μL. This experiment was performed in triplicate.

The quantitative analysis of monosaccharides (xylose, arabinose, galactose, and mannose) in the Klason and saccharification hydrolysate was determined by a high-performance liquid chromatography system (HPLC- YL9100 model) equipped with an Aminex HPX-87P column at 85 °C, which was preceded with the appropriate pre-column and eluted with HPLC grade water at a flow rate of 0.6 mL min⁻¹. The components were detected by a refractive index detector and quantified by external calibration.

Previous analysis, the hydrolysates were centrifuged at 1368 × g for 6 min and filtered using 0.20-μm filters. Monosaccharides were quantitatively determined on an Aminex HPX-87P column at 85°C with 0.6 mL/min water as eluent. The HPLC system used as a Waters 2695e (Milford, MA, USA), comprising a Waters performancePLUS vacuum degasser, a Waters four channels solvent delivery system, a Waters high temperature column oven, a Waters 2414 refractive index (RI) detector and a Waters plus auto-injector. All experiments were performed in duplicates, and the analysis was carried out at least three times for each sample.