Xylose and xylonate concentrations in the supernatant were analyzed in a Beckmann Coulter System Gold High Performance Liquid Chromatography (HPLC) (Beckmann Coulter, Brea, CA, USA) with a Metab-AAC 300 × 7.8 mm separation column (particle size: 10 μm, ISERA GmbH, Düren, Germany), a UV detector 166 (Beckmann Coulter, Brea, CA, USA) at 210 nm and a refractory index detector (RI 2300, Knauer GmbH, Berlin, Germany). Elution was performed with 5 mM H2SO4 at a flow rate of 0.5 ml min−1 at 40°C.
Ri 2300
Manufactured by Knauer
Sourced in Germany
The RI 2300 is a refractive index detector designed for high-performance liquid chromatography (HPLC) applications. It provides reliable and accurate measurements of the refractive index of sample components as they elute from the HPLC column. The RI 2300 is a sensitive and stable detector that can be used with a variety of HPLC systems and mobile phases.
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3 protocols using ri 2300
1
HPLC Analysis of Xylose and Xylonate
2
Pentose Sugar and Furfural Analysis by HPLC
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The pentose sugar components (xylose, lyxose, and xylulose) were analyzed by the HPLC-RI technique (RI 2300, Knauer). ACE excel 5 NH2 (250 mm × 4.6 mm × 5 μm) was used to separate and to quantify these sugars at a temperature of 40 °C. 85 : 15 (v/v) using acetonitrile and DI water as mobile phase at a volumetric flow rate of 0.7 mL min−1. The injection volume for each analysis was 10 μL. Analytical standards were applied as reference peaks for sample identification. The concentration of sugar sample (g L−1) was calculated based on the calibration curve of each analytical standard which was prepared for the range of 0.5 to 30 g L−1.
The furfural content was analyzed by the HPLC-UV technique (UV 2500, Knauer). The quantitative analysis was carried out on ACE excel C18 (250 mm × 4.6 mm × 5 μm). The mobile phase of acetonitrile and water (10 : 90 v/v) at a flow rate of 0.7 mL min−1 was used under the separation temperature of 40 °C and the wavelength of 320 nm. The injection volume for each analysis was 10 μL. The analytical standard of furfural was used to identify and quantify this compound. The calibration curve of furfural standard was in the range of 0.6 to 19.2 g L−1.
The furfural content was analyzed by the HPLC-UV technique (UV 2500, Knauer). The quantitative analysis was carried out on ACE excel C18 (250 mm × 4.6 mm × 5 μm). The mobile phase of acetonitrile and water (10 : 90 v/v) at a flow rate of 0.7 mL min−1 was used under the separation temperature of 40 °C and the wavelength of 320 nm. The injection volume for each analysis was 10 μL. The analytical standard of furfural was used to identify and quantify this compound. The calibration curve of furfural standard was in the range of 0.6 to 19.2 g L−1.
3
HPLC-based Glucose Quantification Protocol
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The glucose concentration in the experiments to determine the optimal induction point was determined by HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, United States) using a refractive index detector (RefractoMax 521, Thermo Fisher Scientific, Waltham, MA, United States). An organic acid resin column (250 mm × 8 mm, CS-Chromatographie Service, Langerwehe, Germany) was used for separation at a temperature of 40°C. 1 mM sulfuric acid was used as a mobile phase. A flow rate of 0.8 mL/min was used.
For the experiments regarding minimal medium and chassis optimization, glucose, gluconate, and ketogluconate were quantified by using a Beckmann Coulter System Gold HPLC with a UV detector 166 (Beckmann Coulter, Brea, CA, United States) at 210 nm and a refractory index detector RI2300 (Knauer GmbH, Berlin, Germany). For separation, a Metab-AAC 300 mm × 7.8 mm column (particle size: 10 μm, ISERA GmbH, Düren, Germany) was used. Elution was performed with 5 mM H2SO4 at a flow rate of 0.5 mL/min at 40°C.
For the experiments regarding minimal medium and chassis optimization, glucose, gluconate, and ketogluconate were quantified by using a Beckmann Coulter System Gold HPLC with a UV detector 166 (Beckmann Coulter, Brea, CA, United States) at 210 nm and a refractory index detector RI2300 (Knauer GmbH, Berlin, Germany). For separation, a Metab-AAC 300 mm × 7.8 mm column (particle size: 10 μm, ISERA GmbH, Düren, Germany) was used. Elution was performed with 5 mM H2SO4 at a flow rate of 0.5 mL/min at 40°C.
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