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7 protocols using waters 486

1

Mass Spectrometry and HPLC Analysis

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Reagents and solvents were purchased from Sigma Aldrich (Taufkirchen, Germany), Glen Research (Sterling, VA, USA), or Alfa Aesar (Thermo Fisher Scientific, Waltham, MA, USA) and were used without further purification. Phospholipids were purchased from Bachem AG (Bubendorf, Switzerland) or Avanti Polar Lipids (Alabaster, AL, USA). Fluorescent dyes were purchased from Sigma Aldrich (Germany). Cell culture media and additives were from Gibco® (Thermo Fisher Scientific, Waltham, MA, USA).
For the mass spectrometry analysis, an Ultraflex III MALDI-TOF-TOF instrument (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Smartbeam 2 laser with a repetition rate up to 200 Hz (500 laser shots) was used. The obtained mass spectrum was analyzed with Flex-analysis version 4.0 software (Bruker Daltonics GmbH).
The HPLC separation and analysis were performed with Waters® Alliance 2695 (Waters® Corporation, Milford, MA, USA) apparatus equipped with the ultraviolet (UV) detector Waters® 486 (Waters Corporation) and analyzed with Millennium 32 software (Waters Corporation). The reverse phase analytical C8 column (Macherey-Nagel, GmbH & Co, KG, Hoerdt, France) was used for all separations.
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2

Thiamine Speciation Analysis by HPLC

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1 mL of a 100 nM equimolar mixture of thiamine, TMP, and TDP in HPLC grade water was stored under static conditions in the dark for 1 h at 21 °C in HPLC autosampler vials (amber, silanized, non-silanized glass, and polypropylene). 874 µL of the solution was then transferred to a polypropylene 8 × 40 mm autosampler vial. 126 µL of an alkaline potassium ferricyanide solution was added and immediately mixed to convert forms of thiamine to their respective thiochromes. The composition of this solution, and the separation conditions below, were modifications of that reported by Brown et al.30 100 µL of this solution was injected using a Waters 717 autosampler, onto a Hamilton PRP-1, 5 μm 150 mm × 4.1 mm column, with a Waters 486 UV detector set at 372 nm, and Waters 474 fluorescence detector set at λex = 372 nm, λem = 433 nm. A Waters 600 pump and controller mediated the gradient between 25 mM ammonium bicarbonate, pH 8.4 (mobile phase A), and 65% (v/v) 25 mM ammonium bicarbonate, pH 8.4/35% (v/v) dimethylformamide (mobile phase B). The gradient was 3 min mobile phase A, changing to 70% mobile phase B over the next 9 min, then a hold at mobile phase B for 5 min before returning to the original condition over 5 min, followed by a 5-min hold in 100% mobile phase A. The flow rate was 1 mL/min. throughout.
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3

Comprehensive Nutrient Analysis of Diet Samples

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Diet samples were analyzed in triplicate for crude protein (Method 990.03), calcium, and phosphorus (method 985.01), and ether extract (method 920.39), according to Association of Official Analytical Chemists [16 ]. Gross energy of diets was measured by a bomb calorimeter (Model 1261, Parr Instrument Co., Moline, IL, USA). Amino acid composition of feed samples was analyzed by High Performance Liquid Chromatography (Waters 486, Waters Corp., Milford, MA, USA) after acid hydrolysis [17 (link)]. The methionine and cysteine were determined following oxidation with performic acid [18 ].
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4

HPLC Analysis of Sesame Seed Oil

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HPLC was performed to determine the content of β-sitosterol and δ-tocopherol in the S. mukorossi seed oil. The HPLC was equipped with a low-pressure mixing pump (L2130, Hitachi, Tokyo, Japan), controlled by a CBM-20A interface module (Shimadzu Technology, Kyoto, Japan), and had a UV detector (Waters 486, Waters Corporation, Milford, MA, US). The separation was achieved using a Mightysil RP-18 GP (250 mm × 4.6 mm i.d., Mightysil RP 18 GP Cica, Tokyo, Japan) at 30 °C. For the β-sitosterol analysis, the mobile phase consisted of 96% methanol, 3% tetrahydrofuran, and 1% deionized water. For δ-tocopherol detection, pure methanol was used as the mobile phase. For testing the two molecules, 20 μL of S. mukorossi seed oil were injected. The flow rates for the detection of β-sitosterol and δ-tocopherol were 0.5 mL/min and 0.5 mL/min, respectively. All the samples were monitored at a wavelength of 280 nm. The compounds were identified by comparing their retention times to those of authentic standards. The quantification was achieved using linear regression analysis.
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5

Structural Characterization of Polymers

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1H NMR spectra were recorded on a Bruker Avance DRX 600 at 20 °C by using DMSO-d6 or CDCl3 (99.9%) as solvents. Chemical shifts referenced to the solvent value δ = 2.5 ppm for DMSO-d6 and δ = 7.26 ppm for CDCl3. SEC-MALS measurements were carried out on a combined system comprising the following elements: refractive-index detector Optilabrex (Wyatt Technologies, laser wavelength 658 nm), three-angle light-scattering detector miniDawn TREOS (Wyatt Technologies, laser wavelength 658 nm, detector angles at 43.5°, 90.0° and 136.5°), UV detector Waters 486 (Waters), column set of HEMAbio 300 and HEMAbio 100 (MZ-Analysentechnik), pump, degasser and autosampler (Agilent 1200, Agilent technologies). The eluent was ultrapure water at a flow rate of 1 mL/min. The molecular weight was calculated with Astra5 software from static-light-scattering data by using the Zimm model. Dynamic light scattering (DLS) experiments were carried out with a Malvern Zetasizer Nano ZS ZEN 3600 at a temperature of 25 °C. The particle size distribution is derived from a deconvolution of the measured intensity autocorrelation function of the sample by a general purpose method, i.e., the non-negative least squares algorithm, included in the DTS software. The TEM images have been obtained with a TEM Zeiss 902.
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6

Phytate Quantification by HPIC

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Phytate content, as myo‐inositol hexakisphosphate, was analyzed using high‐performance ion chromatography (HPIC) following the method described by Carlsson et al.25 and modified by Lazarte et al.5 Duplicate samples of 0.5 g were extracted with 20 mL of 0.5 mol L–1 HCl for 2 h at room temperature under constant stirring. Supernatant was recovered after centrifugation (Model Allegra® X‐15r, Beckman Coulter, Brea, CA, USA) at 2851 g for 10 min at 20 °C. Supernatant was frozen overnight, thawed, and centrifuged (Model Optima™ LE‐80 k, Beckman Coulter Brea, CA, USA) at 12348 g for 10 min at 20 °C. Supernatant (2 mL) was filtered through a 0.2 μm syringe filter disk and 50 μL of supernatant were injected and analyzed by HPIC with an OmniPac PA‐100 (4 × 250 mm) analytical column and a PA‐100 (4 × 50 mm) guard column (Dionex Corp., Sunnyvale, CA, USA). Detection and quantification of phytate were made after a post‐column reaction with 0.1 g L−1 Fe(NO3)3.9H2O (99.99% trace metal basis) (Sigma‐Aldrich) in 20 g L−1 HClO4; the absorbance was monitored at 290 nm in a UV detector (Waters 486, tunable absorbance detector, Waters Corporation, Milford, MA, USA). Phytate dodecasodium salt hydrate (Sigma‐Aldrich, Staad, Switzerland) was used as standard. The limit of detection of the method was 0.13 g kg−1.
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7

SCFA and BCFA Analysis in Fermentation

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Fermentation supernatants and inoculum of the twelve donors used in the fermentation study were analysed for SCFA and BCFA contents, by using a Waters 2690 HPLC system (Waters) fitted with an Aminex HPX-87 H column (300 mm × 78 mm; Bio-Rad Laboratories) combined with a Waters 486 tuneable absorbance detector set at 210 nm.
In vitro fermentation data were corrected for the content of the blanks, as well as the inocula. The sterile bottle containing 15 ml of fermentation supernatants was vortexed for 1 min, and 2 ml aliquot was centrifuged at 13 000 rpm for 15 min. Aliquot (1•5 ml) of the supernatants was transferred to a vial, and pH was adjusted to between one and three, using 0•1M HCl.
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