Extracellular metabolites (glucose, gluconate, pyruvate, acetate, lactate) were analyzed by HPLC (Shimadzu Scientific Instruments, Columbia, MD), equipped with differential refractive index and diode array detectors (Shimadzu Scientific Instruments, Columbia, MD), using a cation-exchange column (HPX-87H, BioRad Labs, Hercules, CA). The mobile phase was 5 mM H2SO4 at 0.5 mL min−1 flow rate and 45 °C.
Hpx 87h
Manufactured by Bio-Rad
Sourced in United States, Japan
The HPX-87H is a liquid chromatography column used for the separation and analysis of biomolecules. It is designed for high-performance size exclusion chromatography (SEC) applications. The HPX-87H column can be used to separate and analyze a variety of biomolecules, including proteins, peptides, and carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
Rid 10a, by Shimadzu (3 mentions)
High performance liquid chromatography (hplc), by Shimadzu (2 mentions)
Rid 10a, by Bio-Rad (2 mentions)
Model 1260, by Agilent Technologies (2 mentions)
Whatman, by Cytiva (2 mentions)
Optima 8000, by PerkinElmer (1 mentions)
Carbopac pa20 column, by Thermo Fisher Scientific (1 mentions)
High performance liquid chromatography (hplc), by Jasco (1 mentions)
Ultimate 3000, by Bio-Rad (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Chromeleon version 6, by Thermo Fisher Scientific (1 mentions)
2414 ri detector, by Bio-Rad (1 mentions)
Spd 10a, by Shimadzu (1 mentions)
Waters 2410, by Waters Corporation (1 mentions)
Du 800 uv vis spectrophotometer, by Beckman Coulter (1 mentions)
I series, by Shimadzu (1 mentions)
Shincarbon st 100 120 mesh, by Restek (1 mentions)
7890b gc system, by Agilent Technologies (1 mentions)
Eclipse xdb c18, by Agilent Technologies (1 mentions)
1100 series system, by Agilent Technologies (1 mentions)
62 protocols using hpx 87h
1
HPLC Analysis of Extracellular Metabolites
2
Cell Growth Monitoring and Metabolite Analysis
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Cell growth was monitored by measuring OD600 utilizing a spectrophotometer (Shimazu, Japan). Glucose and acetate were quantitatively analyzed by high-performance liquid chromatography (HPLC) (Shimazu, Japan) which equipped with a refractive index detector (RID-10A) and an Ion Exclusion column (Bio-Rad, HPX-87H). The samples were first centrifuged at 12,000 rpm for 10 min, and then the supernatant was filtrated with a 0.22 μm filter membrane. 5 mM sulfuric acid was utilized as the mobile phase of HPLC with the flow rate of 0.6 mL/min and the utilized column temperature was 65 °C.
3
Extracellular Metabolite Analysis by HPLC
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A spectrophotometer was used to measure the optical density at 600 nm (OD600) of the bacterial culture. For extracellular metabolite analysis, 1 mL of culture was centrifuged at 12,000g for 2 min. The supernatant was filtered through a 0.22-μm syringe filter for high-performance liquid chromatography analysis. Glucose, MVA, acetate, and pyruvate were measured on an ion exchange column (HPX-87H; Bio-Rad Labs) with a differential refractive index detector (Shimadzu RID-10A). A 0.5-mL/min mobile phase using a 5 mM H2SO4 solution was applied to the column. The column was operated at 65 °C.
4
Comprehensive Characterization of Starchy Hydrolysates
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Starchy hydrolysates in natura were characterized in triplicate for reducing sugars by DNS (2,5-dinitrosalicylic acid) method [68 (link)], total proteins [69 (link)], mineral content by inductively coupled plasma optical emission spectrometry (ICP-OES) (Perkin Elmer-Optima 8000), and pH. The presence of glucose, xylose, arabinose, and cellobiose in starchy hydrolysates and molasses were determined by high-performance liquid chromatography (HPLC) in Agilent Technology chromatograph equipped with an aminex HPX-87H (BIORAD) column (300 × 7.8 mm) at 45 °C, using 0.005 mol/L H2SO4 solution as eluent, with a flow rate of 0.6 mL/min, refractive index detection (RID6A), and injection of 20 μL of samples. Sugarcane molasses was also characterized regarding pH, reducing sugars (DNS method), and the concentration of sucrose and fructose by HPLC under the conditions of HPX-87H (BIORAD) column (300 × 7.8 mm) at 60 °C, deionized water as eluent, and 0.4 mL/min flow rate.
5
Quantification of Lactic Acid and Sugars
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The quantification of lactic acid as titer (g/L) was carried out by High Performance Liquid Chromatography (HPLC). The samples were prepared by centrifuging at 10,000 rpm for 10 min. The supernatant was 4-fold diluted and filtered through a 0.22 μm syringe filter (MERCK A/S, Søborg, Denmark). The samples were analyzed by HPLC directly after preparation. The HPLC was equipped with a refractive detector, a Bio-Rad HPX-87H (300 mm × 7.8 mm) column, and 12 mM H 2 SO 4 eluent operating at flow rate of 0.60 ml/min, with column oven temperature 63°C. For quantification of the compounds, a calibration curve was included with different dilutions (1, 2.5, 5, 7.5, 10 g/L) of a mix solution with glucose, succinic acid, lactic acid, formic acid and acetic acid. To determine the sugar concentration (mg/L) included in the substrate of interest, Ion Chromatography was used (IC; Dionex ICS-6000 HPIC System).
The sample preparation process was identical to that for HPLC. After the preparation the samples were analyzed by IC equipped with a Dionex™ CarboPac™ PA20 column (3 × 150 mm). The quantification was by applying a calibration curve (1; 2.5; 5; 7.5; 10; 15; 20 mg/L), able to identify mannitol, fucose, rhamnose-arabinose, galactose, glucose, xylose, and mannose.
The salinity, expressed as NaCl content was measured with as CDM230 conductivity meter (MeterLab, Hach, Brønshøj, Denmark).
The sample preparation process was identical to that for HPLC. After the preparation the samples were analyzed by IC equipped with a Dionex™ CarboPac™ PA20 column (3 × 150 mm). The quantification was by applying a calibration curve (1; 2.5; 5; 7.5; 10; 15; 20 mg/L), able to identify mannitol, fucose, rhamnose-arabinose, galactose, glucose, xylose, and mannose.
The salinity, expressed as NaCl content was measured with as CDM230 conductivity meter (MeterLab, Hach, Brønshøj, Denmark).
6
HPLC Analysis of Fermentation Byproducts
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A high performance liquid chromatography (HPLC, Jasco International Co., Tokyo, Japan) with an ion-exchange Aminex column (HPX-87H, Bio-Rad, Richmond, CA, USA) was used to analyze ethanol, acetic acid, and 2,5-anhydromannose at 60 °C with 0.6 mL/min eluent of 5 mM sulfuric acid. Ethanol and 2,5-anhydromannose were detected with RI detector while acetic acid was detected using a UV-Vis detector.
Sucrose, glucose, and fructose were measured by the assay kit Megazyme K-SUFRG (Megazyme, Bray, Ireland). The samples were diluted 50 times and filtered through 0.2 μm syringe filter before analyses.
Sucrose, glucose, and fructose were measured by the assay kit Megazyme K-SUFRG (Megazyme, Bray, Ireland). The samples were diluted 50 times and filtered through 0.2 μm syringe filter before analyses.
7
HPLC Analysis of Sugars and Aldehydes
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After clarifying the culture by centrifugation and filtration, the supernatant was subjected to high performance liquid chromatography (HPLC). Quantification was performed using an Aminex HPX-87H cationic exchange column connected to an Aminex 85H Micro-Guard Column (Bio-Rad Labs, Richmond, CA, USA). The chromatographic conditions for sugars and aldehydes were as follows: mobile phase, 4.5 mM H2SO4 or 8 mM H2SO4; flow rate, 0.6 mL · min−1; and the column oven temperature, 65°C or 35°C. Sugars and aldehydes were detected using a Jasco RI-2031 Plus Intelligent Refractive Index Detector (Jasco, Tokyo, Japan) or a Jasco UV-2070 Plus Intelligent UV/VIS Detector at 278 nm (Jasco).
8
Volatile Fatty Acids and Organic Acid Analysis
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Total volatile fatty acids (VFA), acetate, propionate, butyrate, valerate, isobutyrate and isovalerate were analyzed as described by Brandao et al. (under review). Lactate, formate and succinate were analyzed following the method of Weimer et al. (1991) (link) and analyzed by high performance liquid chromatograph (HPLC). The column used for analysis was a Bio-Rad HPX-87H, 300 mm length with 4.6 mm i.d (Bio-Rad, Bio-Rad Laboratories, Inn, CA, United States) with a flow rate of 0.7 mL/min. The VFA components were identified by comparison of retention time with a mixture of fermentation product standards (10 mM each); previous studies have shown that the refractive index detection used for this assay provides a linear response with VFA concentration up to at least 300 mM (Dill-McFarland et al., 2014 (link)).
9
HPLC Analysis of Metabolites
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The metabolites were detected by HPLC system (Dionex Ultimate3000) equipped with Bio-Rad HPX-87H ion exclusion column. The mobile phase was 0.005 mol/L H2SO4 at the rate of 0.6 mL/min using IR and UV detection.
10
Quantifying Microbial Growth and Metabolites
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Cell growth was estimated from the optical density at 600 nm (0.35 g dry cell weigh per OD600). Fermentation samples were centrifuged at 8,000 rpm for 10 min. The supernatant was then treated with H2SO4 and centrifuged again to remove the precipitate (CaSO4). The supernatant was then filtered through a 0.22 μm membrane and analyzed for sugar and organic acid concentrations by an Agilent HPLC (column, BioRad HPX 87H; temperature, 35 °C; flow rate, 0.5 ml min−1; mobile phase, 4 mM H2SO4). Optical purity was determined by HPLC using a chiral column (EC 250/4 NUCLEOSIL Chiral-1, Germany) (HPLC condition: 35 °C, 0.5 ml min−1 of 0.2 mM CuSO4 as the mobile phase).
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