The total polysaccharide content of EPS-160 was measured by the phenol-sulfuric acid (H2SO4) method (Yin et al., 2014 (link)). The presence of protein was determined using the Bradford method (Blum et al., 2008 (link)). For monosaccharide composition analysis, 20 mg EPS-160 was hydrolyzed with 2 mL of 2 M trifluoroacetic acid (TFA) at 120°C for 4 h. The hydrolyzate was evaluated by high-performance liquid chromatography (HPLC) (Agilent1200, United States) equipped with a ROA-Organic Acid H+(8%) column (Phenomenex, United States) (Zhang et al., 2017 (link)). Functional groups of EPS-160 were identified by measuring from 400 to 4000 cm–1 wavenumbers in a Nicolet 6700 infrared spectrometer (Thermo Fisher, America). Molecular weights (MWs) of EPS-160 were analyzed by a gel permeation chromatography (GPC) system (LC20, Shimadzu, Japan). The system was equipped with a refractive index (RI) detector (RID-20, Shimadzu, Japan) and a TSK G4000PWXL column (Tosoh, Japan). The MWs was calculated according to the weight-averaged molecular weight.
Roa organic acid h 8 column
Manufactured by Phenomenex
Sourced in United States
The ROA-Organic Acid H+ (8%) column is a type of liquid chromatography column designed for the separation and analysis of organic acids. The column features a stationary phase that is specifically tailored for the separation of these compounds. The column has a porous structure that helps in the efficient separation of organic acids, making it a useful tool for various analytical applications.
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Lab products found in correlation
Lc 20, by Shimadzu (1 mentions)
Rid 20, by Shimadzu (1 mentions)
Nicolet 6700 infrared spectrometer, by Thermo Fisher Scientific (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
C18 column, by Phenomenex (1 mentions)
Securityguard cartridge kit, by Phenomenex (1 mentions)
Lc 2030c plus, by Shimadzu (1 mentions)
Lcms 2020, by Shimadzu (1 mentions)
0.2 m cut off filter, by Merck Group (1 mentions)
Lc 20a hplc system, by Shimadzu (1 mentions)
Lc 20a hplc, by Shimadzu (1 mentions)
8 protocols using roa organic acid h 8 column
1
Comprehensive Characterization of EPS-160
2
Quantitative HPLC Analysis of Biomass Sugars
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High performance liquid chromatograph (HPLC) with a ROA-Organic Acid H+ (8%) column (Phenomenex, USA) was conducted to quantify d -glucose, D-xylose, xylitol, arabinose, and arabitol; 0.0025 M H2SO4 was served as the mobile phase at a column temperature of 75 °C and a flow rate of 0.3 mL/min. The concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) were analyzed using a C18 column (Phenomenex, USA) at a column temperature of 30 °C and a flow rate of 0.3 mL/min. The mobile phase was a mixture of water and methanol (80:20, v/v) with detection at a wavelength of 285 nm. The culture was centrifuged at 14,000×g for 10 min, and the supernatant was diluted 20-fold for HPLC analysis.
3
Comprehensive Metabolite Analysis Protocol
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Glucose and lactate concentrations were determined by High-Performance Liquid Chromatography (HPLC). Samples are analyzed on a Shimadzu Prominence LC-2030C Plus with an RI detector (RID-20A). An isocratic method of 15 min on a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex—part number 00F-0138-K0 + SecurityGuard Cartridge Kit (KJ0-4282) + SecurityGuard Cartridges Carbo-H 4 × 3.0 mm ID (AJ0-4490)) was used. The mobile phase was 5 mM sulfuric acid (Chem-lab CL00.2653.0050). The column temperature was 60°C and the temperature of samples was 4°C. The detection of glucose was done with an RI detector and the detection of lactate with a UV detector (210 nm).
Carboxylic acids (acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate, and caproate) were determined by gas chromatography (GC) with flame ionization detection (FID) as described by Candry et al. (2020) (link) after the extraction of acids into diethyl ether as described by Andersen et al. (2014) (link).
The gaseous head space composition was analyzed using a Compact Gas Chromatograph (Global Analyser Solutions, Breda, Netherlands), equipped with a Molsieve 5A pre-column and Porabond column (CH4, O2, H2, and N2), and a Rt-Q-bond pre-column and column (CO2). Concentrations of gases were determined using a thermal conductivity detector.
Carboxylic acids (acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate, and caproate) were determined by gas chromatography (GC) with flame ionization detection (FID) as described by Candry et al. (2020) (link) after the extraction of acids into diethyl ether as described by Andersen et al. (2014) (link).
The gaseous head space composition was analyzed using a Compact Gas Chromatograph (Global Analyser Solutions, Breda, Netherlands), equipped with a Molsieve 5A pre-column and Porabond column (CH4, O2, H2, and N2), and a Rt-Q-bond pre-column and column (CO2). Concentrations of gases were determined using a thermal conductivity detector.
4
Cell Physiology Analysis by HPLC-MS
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Samples for the analysis of the cell physiology were taken every hour. One mL of culture was transferred to a 1.5 mL Eppendorf reaction tube on ice and centrifuged (5000 × g, 10 min). The supernatant was filtered with a 0.2 μm syringe filter and stored at -20°C until HPLC analysis (Nexera Shimadzu). The supernatant was analyzed with a refractive index (RI) detector and photodiode array (PDA) detector for quantification of the analytes and an electrospray ionization (ESI) ion source with a quadrupole mass analyzer for additional confirmation of the substances (LC–MS 2020 Shimadzu). Separation was performed with an ROA-Organic Acid H+ (8%) column (300 mm × 7.8 mm, Phenomenex) with an isocratic flow of 0.4 mL min-1 5 mM formic acid in water.
5
Fermentation Analysis Protocol for Sugars and Organic Acids
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Samples for offline analysis are drawn from fermentation at regular intervals. All analyses are done in triplicates. For each sample, 2 mL fermentation broth is centrifuged at 14,000 rpm for 10 min in a Sigma 1–15 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The supernatant is filtered with a 0.2 µm cut-off filter (Millipore-Sigma, Burlington, MA, USA) and analyzed for sugars and organic acid content by high performance liquid chromatography (HPLC). HPLC analysis is performed using a Thermo Fisher Ultimate 3000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an ERC RefractoMax 520 RID (Shodex, Munich, Germany). For separation, a ROA-Organic Acid H+ (8%) column (300 × 7.8 mm) (Phenomenex, Torrance, USA) column is heated to 30 °C and used with a mobile phase of 5 mM H SO running at 0.8 mL/min. Cell pellets are dried at 80 ° C for 48 h and then weighed to determine cell dry weight (CDW).
6
Hydrolytic Analysis of Xyl11 Enzyme
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To estimate the hydrolytic properties of Xyl11, the hydrolysis products from CXY and XOS standards (X1-X5) were analyzed. A total of 10 U/ml Xyl11 was incubated with 10 mg/ml CXY or 5 mg/ml XOS standards at 50°C for 2 h in glycine-HCl buffer (pH 3.0). Substrates containing inactive enzyme were used as blank controls. All the samples were boiled for 5 min after incubation and centrifuged at 12,000 rpm and 4°C for 15 min. The supernatants were further analyzed using an LC-20A HPLC system (Shimadzu, Japan) equipped with an ROA-Organic Acid H+ (8%) column (Phenomenex) and an RIDL10A refractive index detector, and a column temperature of 50°C, and 5 mM H2SO4 at a flow rate of 0.6 ml/min. X1-X5 standards were purchased from Megazyme.
7
Enzymatic Hydrolysis of Beechwood Xylan
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Hydrolytic activity of recombinant xylanase was estimated based on the hydrolysis of substrate BWX. For this purpose, reactions mixture containing 900 µL of 1% BWX (w/v) with 100 µL of appropriate diluted enzyme, in sodium phosphate buffer (pH 6.0) was prepared. The reaction mixture was then incubated at 50°C for 12 h and reducing sugars released from substrates were determined by DNS method (as above). The degradation rate (percent of total BWX) was calculated according to the following equation:
Hydrolysis products were analyzed by LC-20A HPLC (Shimadzu, Japan) with xylose and XOSs (X5, X4, X3, X2) as standards. HPLC system was equipped with a ROA-Organic Acid H+ (8%) column (Phenomenex) and a RIDL10A refractive index detector. The HPLC column was sustained at 50°C with H2SO4 (5 mM) as the mobile phase at a flow rate of 0.6 mL/min.
Hydrolysis products were analyzed by LC-20A HPLC (Shimadzu, Japan) with xylose and XOSs (X5, X4, X3, X2) as standards. HPLC system was equipped with a ROA-Organic Acid H+ (8%) column (Phenomenex) and a RIDL10A refractive index detector. The HPLC column was sustained at 50°C with H2SO4 (5 mM) as the mobile phase at a flow rate of 0.6 mL/min.
8
Yeast Growth Kinetics and Metabolites
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The growth of yeast was examined by measuring the OD600. Glucose and ethanol were quantified by high-performance liquid chromatography with a ROA-Organic Acid H+ (8%) column (Phenomenex, USA). The mobile phase was 0.0025 M H2SO4 at a column temperature of 75 °C and a flow-rate of 0.3 mL/min.
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