Openlab chemstation software

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OpenLab ChemStation software is a data analysis and reporting solution used for laboratory instrument control and data acquisition. It provides a comprehensive platform for managing and analyzing chromatographic and spectroscopic data.

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24 protocols using openlab chemstation software

Caecal SCFA Analysis by GC-FID

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Caecal SCFA gas chromatography analysis was performed at the Gnotobiotics, Microbiology and Metagenomics Center (Boston, Massachusetts) as described before (Giri et al., 2019 (link)). Briefly, the chromatographic analysis was carried out using an Agilent 7890B system with a flame ionization detector (FID, Agilent Technologies, Santa Clara, CA). A high resolution gas chromatography capillary column 30 m × 0.25 mm coated with 0.25μm film thickness was used (DB-FFAP) for the volatile acids (Agilent Technologies). Nitrogen was used as the carrier gas. The oven temperature was 145 °C and the FID and injection port was set to 225 °C. The injected sample volume was 1 μl and the run time for each analysis was 12 min. Chromatograms and data integration was carried out using the OpenLabChemStation software (Agilent Technologies). A volatile acid mix containing 10 mM of acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic and heptanoic acids was used for standard solution (Supelco CRM46975, Bellefonte, PA). An internal standard control (stock solution containing 1% 2-methyl pentanoic acid, Sigma-Aldrich St. Louis, MO) was used for the volatile acid extractions.

LaGamma E.F., Hu F., Pena Cruz F., Bouchev P, & Nankova B.B. (2021). Bacteria - derived short chain fatty acids restore sympathoadrenal responsiveness to hypoglycemia after antibiotic-induced gut microbiota depletion. Neurobiology of Stress, 15, 100376.

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HPLC-RID Analysis of Organic Acids and Alcohols

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Organic acids and alcohols were analyzed by using HPLC-RID: High-performance liquid chromatography (HPLC-Agilent 1200 series, Santa Clara, CA, USA) coupled with a refractive index detector (Agilent Technologies, Santa Clara, CA, USA), as described by Chiș et al. [17 (link)]. Shortly afterward, 1 g of each sample was extracted with 4 mL ultrapure water, mixed using a Heidoph Reax top vortex (Merck, Darmstadt, Germany) for 1 min and sonicated for 30 min using an ultrasonic bath (Elma Schmidbauer, GmbH, Singen, Germany). An Eppendorf 5804 centrifuge (Eppendorf, Hamburg, Germany) was used to centrifugate samples at 2300× g, for 10 min. Afterward, the supernatant was filtered through Chromafil Xtra PA-45/13 nylon filter and injected into the HPLC-RID system. A Polaris Hi-Plex H, 300 × 7.7. column (Agilent Technologies, Santa Clara, CA, USA) was used to separate the compounds having H₂SO₄ (5 mM) as a mobile phase, with a flow rate of 0.6 mL/min. The column and RID temperatures were 70 °C and 35 °C, respectively; meanwhile, the compound elution time was 25 min.
OpenLab—ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was used for data acquisition and result interpretation. The compounds identification was realized through comparison with the standard retention times; meanwhile, the quantification of the compounds was realized using calibration curves.

Vasilica B.(., Chiș M.S., Alexa E., Pop C., Păucean A., Man S., Igual M., Haydee K.M., Dalma K.E., Stănilă S., Socaci S., Fărcaș A., Berbecea A., Popescu I, & Muste S. (2022). The Impact of Insect Flour on Sourdough Fermentation-Fatty Acids, Amino-Acids, Minerals and Volatile Profile. Insects, 13(7), 576.

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Quantification of Volatile SCFAs in Cecal Samples

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Volatile SCFAs were quantified as described in [51 ]. Acidified internal standards with 100uL of either ethyl anhydrous or boron trifluoride-methanol was added to 100 μL of supernatant from homogenized cecal contents. Chromatographic analyses were carried out on an Agilent 7890B system with a flame ionization detector (FID). Chromatogram and data integration were done using the OpenLab ChemStation software (Agilent Technologies, Santa Clara, CA). SCFAs were identified by comparing their specific retention times relative to the retention time in the standard mix. Concentrations were determined as mM of each SCFA per gram of sample for the raw cecal/fecal material. The Agilent platform cannot discriminate between isovalerate and 2-methylbutyrate, and so these are reported as a single peak value.

Dawkins J.J., Allegretti J.R., Gibson T.E., McClure E., Delaney M., Bry L, & Gerber G.K. (2022). Gut metabolites predict Clostridioides difficile recurrence. Microbiome, 10, 87.

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Characterization of Capillary Monolithic Columns

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Measurements on the ZIC-pHILIC column were done on an Agilent 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a quaternary pump, an autosampler, and a diode array detector with a flow cell of 1 μL. Measurements on the capillary monolithic columns were executed on an Ultimate 3000 RSLC nano system (Dionex, Amsterdam, the Netherlands), with a Binary Rapid Separation Nano Flow pump with nano flow selector, an autosampler, a four-port injection valve with a 20 nL internal loop (VICI, Houston, TX, USA) and a variable wavelength detector (VWD) with a 3 nL flow cell. Experiments were executed at room temperature (21.5 ± 0.5 °C), using an injection volume of 20 nL for the monolithic columns, and 1 μL for the ZIC-pHILIC column. The detection wavelength was set to 254 nm, and the data acquisition rate was 40 Hz for all experiments. Data acquisition and processing were done with Chromeleon software (version 6.8, Dionex) or OpenLab Chemstation software (edition C.01.07, Agilent Technologies).
pH values were measured using a Metrohm 691 pH meter (Antwerp, Belgium). Scanning electron microscopy (SEM) experiments were performed using a TESCAN MIRA4 system (Brno, Czech Republic), using an energy between 5 and 15 keV. Magnifications were between 700× and 50.000×.

Li H., Jiang Z., Desmet G, & Cabooter D. (2023). In-Depth Performance Analysis and Comparison of Monolithic and Particulate Zwitterionic Hydrophilic Interaction Liquid Chromatography Polymer Columns. Molecules, 28(7), 2902.

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Molecular Weight Determination of EPSwk

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The molecular weight (Mw) of EPSwk was determined using chromatographic analyzes carried out in an Agilent™ 1260 Infinity II LC System chromatograph, equipped with Agilent™ 1290 Infinity II Evaporative Light Scattering Detector (ELSD), with N2 flow of 1.2 slm, an evaporator temperature of 70 °C and a nebulizer temperature of 50 °C. PL Aquagel-OH MIXED-M columns (part number: PL1149-6801, 4.6 × 250 mm, 8 µm) and Aquagel-OH 20 (part number: PL1120-6520, 300 × 7.5 mm, 5 µm) were used as the stationary phase, with a pre-column of PL Aquagel-OH (part number: PL1149⁻¹240, 7.5 × 50 mm, 15 µm). Ammonium acetate solution (10 mmol L⁻¹) was used as a mobile phase at a flow rate of 0.6 mL.min⁻¹. The sample was prepared in ammonium acetate solution (10 mmol L⁻¹) and filtered with a 0.22 μm syringe filter to ensure homogeneity. The system was calibrated with the standard pullulan kit of varying molecular weights (736 kDa P-800; 348 kDa P-400; 200 kDa P-200; 113 kDa P-100; 49 kDa P-50; 23 kDa P-20; 9.9 P-10 kDa; and 6.6 kDa P-5). All chromatograms were analyzed using OpenLab ChemStation software (Agilent™, Santa Clara, CA, USA).

Lucena M.D., Ramos I.F., Geronço M.S., de Araújo R., da Silva Filho F.L., da Silva L.M., de Sousa R.W., Ferreira P.M., Osajima J.A., Silva-Filho E.C., Rizzo M.D., Ribeiro A.B, & da Costa M.P. (2022). Biopolymer from Water Kefir as a Potential Clean-Label Ingredient for Health Applications: Evaluation of New Properties. Molecules, 27(12), 3895.

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Quantification of Cecal Short Chain Fatty Acids

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Volatile short chain fatty acids from specifically-associated mice (n=6 mice/group across two experimental replicates) were quantified as described (Moore, 1993 ). In brief, acidified internal standards with 100 μL of ethyl ether anhydrous or boron trifluoride-methanol was added to 100μl of supernatant from homogenized cecal contents. Chromatographic analyses were carried out on an Agilent 7890B system with flame ionization detector (FID). Chromatogram and data integration were carried out using the OpenLab ChemStation software (Agilent Technologies, Santa Clara, CA). SCFA in samples were identified by comparing their specific retention times relative to the retention time in the standard mix. Concentrations were determined and expressed as mM of each SCFA per gram of sample for the raw cecal/fecal material. The Agilent platform cannot discriminate the isomers isovalerate and 2-methylbutyrate and thus reports these compounds out as a single peak and interpolated value.

Girinathan B.P., DiBenedetto N., Worley J.N., Peltier J., Arrieta-Ortiz M.L., Immanuel S.R., Lavin R., Delaney M.L., Cummins C.K., Hoffman M., Luo Y., Gonzalez-Escalona N., Allard M., Onderdonk A.B., Gerber G.K., Sonenshein A.L., Baliga N.S., Dupuy B, & Bry L. (2021). In vivo commensal control of Clostridioides difficile virulence. Cell host & microbe, 29(11), 1693-1708.e7.

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HPLC Analysis of Ionic Liquids

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HPLC analyses were performed with an Agilent 1260 Infinity HPLC–DAD system (Agilent Technologies, Santa Clara, CA, USA). For the separation, an HPLC LiChrospher^® RP-18 Column, 5 μm, 4.6 × 250 mm, (Merck, Darmstadt, Germany) was used.
Experimental designs were run with acetonitrile–water phosphate buffer using the ionic liquids BMIM[BF4] and BMIM[PF6] as shown in Table 2.
For the mobile phase H₂O:ACN 60:40 containing 1.0 mM BMIM[BF₄] and 1.0 mM BMIM[PF₆], respectively, pH variation was conducted at 3.5, 5.2 and 9.5 with 25%phosphoric acid or ammonia. The flow rate was set at 1.0 mL/min. The total run time of the method was 30 min. The detection wavelengths were 225 and 245 nm, and the full spectra were recorded over a range of 195–380 nm with a step of 1 nm. Data were evaluated by the OpenLAb ChemStation software from Agilent.

Axente R.E., Stan M., Chitescu C.L., Nitescu V.G., Vlasceanu A.M, & Baconi D.L. (2023). Application of Ionic Liquids as Mobile Phase Additives for Simultaneous Analysis of Nicotine and Its Metabolite Cotinine in Human Plasma by HPLC–DAD. Molecules, 28(4), 1563.

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Quantification of Cecal Short-Chain Fatty Acids

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Approximately 35 mg of cecal content was weighed, snap-frozen and stored at −80°C until analysis. The samples were extracted with water and proteins precipitated with phosphotungstic acid. 2-Ethylbutyrate was added to supernatants at a ratio of 1: 4 as an internal standard. The SCFA content was determined from a 0.3 µL volume of supernatant by gas chromatography (Agilent 7890B gas chromatograph, Agilent Technologies, Les Ulis, France) equipped with a split-splitless injector, a flame-ionization detector and a fused silica capillary column (15 m × 0.53 mm × 0.5 µm; Supelco, Saint-Quentin-Fallavier, France). The carrier gas (H₂) flow rate was 10 ml/min. The oven temperature was initially set at 100°C for 10 min, then increased from 100 to 180°C at a rate of 20 C/min and held for 2 min. The detector temperature was 240°C. Samples were analyzed in duplicate. The peaks obtained were integrated using OpenLAB Chemstation software (Agilent Technologies, Les Ulis, France).

Fréville M., Estienne A., Ramé C., Lefort G., Chahnamian M., Staub C., Venturi E., Lemarchand J., Maximin E., Hondelatte A., Zemb O., Canlet C., Guabiraba R., Froment P, & Dupont J. (2022). Chronic dietary exposure to a glyphosate-based herbicide results in total or partial reversibility of plasma oxidative stress, cecal microbiota abundance and short-chain fatty acid composition in broiler hens. Frontiers in Physiology, 13, 974688.

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Quantifying SCFAs in Bacterial Supernatant

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Acetate, butyrate, and propionate were quantified in the bacterial supernatant using gas chromatography (Agilent Technologies, Les Ulis, France) [28 (link)]. 2-Ethylbutyrate (Sigma, Angers, France, 2-ethylbutyric acid, 99%) at a concentration of 20 mM was used as internal standard in a 1:4 ratio to the bacterial supernatant for normalization of the data per run. An external standard (Sigma, Angers, France, volatile free acid mix) was used for identification and quantification of acetate, butyrate, and propionate in the samples in each run. Analyses were made using the OpenLab Chemstation software (Agilent, Les Ulis, France). Concentrations are given in mM.

Verstraeten S., Sencio V., Raise A., Huillet E., Layec S., Deruyter L., Heumel S., Auger S., Robert V., Langella P., Beney L., Trottein F, & Thomas M. (2022). Description of a Newly Isolated Blautia faecis Strain and Its Benefit in Mouse Models of Post-Influenza Secondary Enteric and Pulmonary Infections. Nutrients, 14(7), 1478.

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Cecal Short-Chain Fatty Acid Analysis

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Ceca were isolated, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C until further use. Cecal samples were sent for SCFA analysis to the Gnotobiotics, Microbiology, and Metagenomics Center (Boston, MA, USA). Chromatographic analysis was performed using an Agilent 7890 B system with a ﬂame ionization detector and OpenLab ChemStation software (Agilent Technologies, Santa Clara, CA, USA). A volatile acid mix (10 mM acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic, and heptanoic acids) was used as the standard solution (Supelco CRM46975, Bellefonte, PA, USA). An internal standard control (1% 2-methyl pentanoic acid, Sigma-Aldrich, St Louis, MO, USA) was used for volatile acid extraction.

Tanelian A., Nankova B., Cheriyan A., Arens C., Hu F, & Sabban E.L. (2023). Differences in gut microbiota associated with stress resilience and susceptibility to single prolonged stress in female rodents. Neurobiology of Stress, 24, 100533.

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