Methanol in samples producing lactic acid were quantified via UPLC (UPLC Ultimate 3000) equipped with a Rezex ROA UPLC column (Rezex ROA-Organic Acid H+ (8%): 300 × 7.8 mm; Phenomenex) coupled to a refractive index detector (RefractoMax 521, Thermo Fisher Scientific). As the solvent, 2.5 mM sulfuric acid in ultrapure water was used. To separate the metabolites, a flow rate of 600 μl min−1 was used. Peaks were identified and the peak areas were quantified using Chromeleon 7 software (Thermo Fisher Scientific) on the basis of an external standard curve of pure methanol (Sigma-Aldrich).
Refractomax 521
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RefractoMax 521 is a laboratory refractometer designed for precise measurement of refractive index. It utilizes a compact optical system to determine the refractive index of liquid samples with high accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Chromeleon 7, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
System gold hplc, by Beckman Coulter (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Ri 2300, by Knauer (1 mentions)
Hpx 87p column, by Aminex (1 mentions)
Dionex ultimate 3000 rapid separation lc system, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Dad 3000, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 hplc system, by Phenomenex (1 mentions)
6 protocols using refractomax 521
1
UPLC-Based Methanol Quantification in Lactic Acid Samples
2
Saccharide Analysis via HPLC-RI
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Saccharide analysis was performed using a Thermo Scientific Ultimate 3000 HPLC system coupled to a RefractoMax 521 refractive index detector (Thermo Fisher Scientific, Waltham, MA, USA). Saccharide components were separated using two sugar columns in series, SUGAR KS-802 and KS-801 (8.0 mmID × 300 mm each), with ultrapure water as a mobile phase. The columns were operated at 80 °C with an isocratic flow rate of 0.5 mL/min. Samples were run for 60 min, and the injection volume was 10 µL. Chromatograms were recorded and processed using Chromeleon 7 software (Thermo Fisher Scientific, Waltham, MA, USA).
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.
4
Glucose Quantification via HPLC
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Glucose concentration was determined using High Performance Liquid Chromatography with an Aminex HPX-87P column. Supernatant from 1 mL of sample culture as filtered with a 0.2 μm Nylon filter before analysis using a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC system with RefractoMax 521 detector. Filtered and degassed water was used as the mobile phase with a flow rate of 0.6 mL/min. Column temperature was maintained at 85 °C. As necessary, supernatant samples were diluted with deionized water before filtration to stay within the linear range of the detector.
5
Analyzing Gut Microbiota and VFAs in Piglets
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On Day 16 and two weeks post-weaning (Day 39 ± 1), feces were collected from two female piglets per litter to further determine microbiota and quantify the level of VFAs. The VFA profile in the feces was determined by high-performance liquid chromatography (HPLC) with a method adapted from Htoo et al. [18 (link)]. Briefly, feces samples that had been previously weighed and frozen at −20 °C with 1 mL of phosphoric acid (25%, w/v) were thawed. Following defrosting, 1 mL of internal standard (pivalic acid at 1%, w/v) and 18 mL of distilled water were added into the tube. This preparation was stirred for 3 h at room temperature before being centrifuged for 5 min at 4000 g. The supernatants were filtered and analyzed for VFA level using HPLC (Ultimate 3000, Thermo Fisher Scientific, Reinach, Switzerland) with an exchange ion column (Nucleogel ION 300 OA 300 × 7.8 mm, Marcherey-Nagel AG, Oensingen, Switzerland) and equipped with a refractive index detector (RefractoMax 521, Thermo Fisher Scientific, Reinach, Switzerland). During the experiment, growth performance of the selected female piglets was recorded by weighing them on Days 0 (at birth), 2, 5, 16, 25 ± 1 (weaning), 32 ± 1, and 39 ± 1 after birth. From weaning to Day 39 (±1), piglets were not mixed and stayed in the farrowing pens.
6
HPLC-based Mannitol Quantification
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Samples for HPLC analysis were prepared by combining cell-free supernatants with an equal volume of mobile phase spiked with 50 mM valerate as internal standard. The mobile phase was composed of 5 mM H 2 SO 4 . Subsequently, the mixture was passed through a cellulose acetate syringe filter with a pore size of 0.22 μm. Samples were analysed using a Thermo Scientific UltiMate 3000 HPLC system equipped with a Phenomenex Rezex ROA-organic acid H+ (8%) 150 mm × 7.8 mm × 8 μm column, a diode array detector DAD-3000 with the wavelengths set at 210 and 280 nm and a refractive index detector RefractoMax 521 (Thermo Fisher Scientific). The flow rate of the mobile phase was set to an isocratic 0.5 mL/min with a column temperature of 35 • C. Samples were run for 30 min and the injection volume was 20 μL. Data analysis was performed using Chromeleon 7 (Thermo Fisher Scientific). Mannitol concentrations were quantified using calibration curves generated from running standards of known concentrations, which were prepared the same as the samples.
DCW was quantified by pelleting 1 mL of cell culture in a pre-dried and pre-weighed 1.5 mL Eppendorf tube. The supernatant was removed, and the cell pellet was dried for 48 h at 100 • C and weighed using a fine balance. DCW was calculated as grams per litre.
DCW was quantified by pelleting 1 mL of cell culture in a pre-dried and pre-weighed 1.5 mL Eppendorf tube. The supernatant was removed, and the cell pellet was dried for 48 h at 100 • C and weighed using a fine balance. DCW was calculated as grams per litre.
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