We prepared glycine (Sigma-Aldrich), creatine (Sigma-Aldrich), egg white protein (commercial), and D-glucose (Sigma-Aldrich) phantom solutions at different phosphate concentrations (ranging from 5 to 80 mM) and pH levels (ranging from 5.6 to 8.9). Detailed information on phantom compositions can be found in Table 1 . All sample solutions were pipetted from highly concentrated stock solutions to ensure the precision of concentrations. We used monobasic and dibasic potassium phosphate (KH2PO4 and K2HPO4, Sigma-Aldrich) as buffer agent and catalyst of the proton exchange process. KH2PO4 and K2HPO4 concentrations were calculated from phosphate pKa (6.82), desired pH, and total phosphate concentration for each sample. pH was measured at room temperature with Accumet pH meter (Fisher Scientific, Massachusetts, MN, USA), after mixing and pH adjusting using acid (HCl) and base (NaOH).
Accumet ph meter
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The Accumet pH meter is a precision instrument designed for accurate pH measurement. It features a digital display and allows for calibration and adjustment of the pH electrode.
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Lab products found in correlation
D glucose, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
K2hpo4, by Merck Group (1 mentions)
Creatine, by Merck Group (1 mentions)
Water pro ro, by Labconco (1 mentions)
Arl equinox 1000, by Thermo Fisher Scientific (1 mentions)
Mpeg oh, by Creative PEGWorks (1 mentions)
10 protocols using accumet ph meter
1
Phantom Solution Preparation for NMR Studies
2
Stability Analysis of Nanomedicine Formulations
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In addition to the −70
°C and 4 °C stability analyzes, short-term size stability
at 37 °C was monitored as a part of the cytotoxicity study, and
the results are presented elsewhere.17 (link) Also,
for selected representative vials, electrophoretic mobility and zeta
potential were measured on formulations 10-fold diluted in water by
mixed mode measurement phase analysis light scattering using a Zetasizer
Nano ZS.20 (link) Finally, the pH of the undiluted
formulations was monitored at room temperatures using an Accumet pH
meter and a micro probe (Fisher Scientific, Canada).
°C and 4 °C stability analyzes, short-term size stability
at 37 °C was monitored as a part of the cytotoxicity study, and
the results are presented elsewhere.17 (link) Also,
for selected representative vials, electrophoretic mobility and zeta
potential were measured on formulations 10-fold diluted in water by
mixed mode measurement phase analysis light scattering using a Zetasizer
Nano ZS.20 (link) Finally, the pH of the undiluted
formulations was monitored at room temperatures using an Accumet pH
meter and a micro probe (Fisher Scientific, Canada).
3
Comprehensive Characterization of MOF-5
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The common instruments used to perform the experiments were a Remi R-8C centrifuge, Remi orbital shaker (Model RS-36BL, Remi, Mumbai, India), Fisher Scientific Accumet pH meter (Model AB 250, Fisher Scientific, Loughborough LE11 5RG, UK), and Milli-Q Plant from Labconco Water Pro/Ro, Labconco, Kansas City, MO, USA. Skeletal analysis of MOF-5 was performed using Fourier transform infrared (FTIR) spectroscopy on a Benchtop Labtronics LT-4100, Labtronics, Welwyn Garden City AL7 1TW, UK, and crystallinity was investigated by powder X-ray diffraction (PXRD) on a Thermo Scientific ARL Equinox 1000, ThermoFisher Scientific, Waltham, MA, USA. Morphology, size, and elemental analyses were performed using field-emission scanning electron microscopy (FESEM) and energy-dispersive spectroscopy (EDS) (Zeiss EVO 18 from IIT Palakkad, Kerala, India). The experimental analysis was performed using a UV-vis spectrophotometer (Thermo Scientific Evolution 201, Fisher Scientific, Loughborough LE11 5RG, UK).
4
Microbial Production of Polyhydroxyalkanoates
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Poly(3-hydroxyalkanoates) production in shake flasks was performed as previously described (Scheel et al., 2016 (link)). In brief, chemically competent E. coli LSBJ was transformed with MCL PHA biosynthesis plasmid pBBR-C1J4SII by heat-shock following standard procedures (Sambrook and Russell, 2001 ). Transformants were grown on LB-agar plates, and single colonies were used to inoculate three separate 2.5 mL LB seed cultures. Seed cultures were grown for 16 h at 37°C and 200 rpm rotary shaking, and 500 μL were used to inoculate 100 mL of growth media (LB, 4.0 g L–1 Brij-35, 10 mM decanoic acid, 8 mM sodium phosphate dodecahydrate, kanamycin, pH 7.0) in 500-mL baffled shake flasks. Cultures were grown for 12, 24, 36, or 48 h at 30°C and 220 rpm, then harvested by centrifugation as previously described (Tappel et al., 2012b (link)). Before harvesting, the pH of each culture was measured (Accumet® pH meter, Fisher Scientific) immediately after removal from the incubator.
5
Soil Characterization of Agricultural Land
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Soil sample was collected in an agricultural land located in St. Claude (Québec, Canada) on the south shore of the St. Lawrence River (45.6809°N, −71.9969°W). The field is managed with fallow, potatoes and maize crop rotation. Potato seedlings (approximately 10 cm height) were present on the site during sampling in July 2013. The top layer of the A-horizon (0–10 cm depth) was collected, stored in plastic bags at 4 °C and processed within a week. Soil was air-dried for 48 h in the laboratory and sieved (2 mm mesh size) through a vibratory sieve shaker AS 200® (Retsch GmbH, Haan, Germany) before preparation of soil microcosms. Soil was classified as sandy clay loam according to soil textural class parameters identified with the hydrometer method (Bouyoucos, 1936 (link)). Soil pH was determined with 1:2 soil-water suspensions with an Accumet pH-meter (Fisher Scientific, Hampton, NH). Total carbon (3.1 ± 0.3%) and total nitrogen (0.3 ± 0.0%) content were determined using an elemental combustion system using the protocol described in Khdhiri et al. (2015) (link).
6
Characterization of Dialdehyde Cellulose Beads
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The cellulose beads (100 g beads) were suspended in 400 or 200
mL of sodium periodate (NaIO4) at a concentration of 50
or 20 mM and reacted at 25 °C for 28 h under mild agitation using
an overhead stirrer to form DAC beads, and aliquots of beads were
withdrawn at 2, 4, 8, 24, and 28 h to calculate kinetics. Excess NaIO4 was removed by washing the beads (soaking and sieving with
a 1 mm mesh-size stainless-steel sieve) in abundant DI water
until the absorption of the supernatant at 290 nm was zero (periodate
adsorption peak). The dialdehyde beads (DAC beads) were stored in
DI water at room temperature. The DO was determined via acid–base
titration.20 (link) 2 g of oxidized beads were
homogenized with 3 mL of DI water using an Ika Ultra-Turrax T8 and
dispersed into 25 mL of a 0.25 M hydroxylamine hydrochloride solution
(adjusted with 0.1 M NaOH to pH 4). The HCl released in the reaction
between the aldehydes and hydroxylamine hydrochloride was titrated
against 0.1 M NaOH using an Accumet pH meter (Fisher Scientific),
and equivalent point peaks were obtained from the first order derivative
of pH changes against volume added (dpH/dV).
mL of sodium periodate (NaIO4) at a concentration of 50
or 20 mM and reacted at 25 °C for 28 h under mild agitation using
an overhead stirrer to form DAC beads, and aliquots of beads were
withdrawn at 2, 4, 8, 24, and 28 h to calculate kinetics. Excess NaIO4 was removed by washing the beads (soaking and sieving with
a 1 mm mesh-size stainless-steel sieve) in abundant DI water
until the absorption of the supernatant at 290 nm was zero (periodate
adsorption peak). The dialdehyde beads (DAC beads) were stored in
DI water at room temperature. The DO was determined via acid–base
titration.20 (link) 2 g of oxidized beads were
homogenized with 3 mL of DI water using an Ika Ultra-Turrax T8 and
dispersed into 25 mL of a 0.25 M hydroxylamine hydrochloride solution
(adjusted with 0.1 M NaOH to pH 4). The HCl released in the reaction
between the aldehydes and hydroxylamine hydrochloride was titrated
against 0.1 M NaOH using an Accumet pH meter (Fisher Scientific),
and equivalent point peaks were obtained from the first order derivative
of pH changes against volume added (dpH/dV).
7
Rumen Fluid Sampling Protocol
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Forty eight hours before starting sampling, special probes screwed to a plastic tube and covered with thin socks for collection rumen fluid were placed in the rumen. Four hours after morning feeding on day 18 of each sampling period, rumen fluid was sampled using 50-mL plastic syringe to determine the concentration of total volatile fatty acids (VFA) (0.375 mL of orthophosphoric acid and the solution was added to 1.5 mL rumen fluid). On d 19 of each sampling period at 0, 2, 4, and 6 h after morning feeding for determining ruminal ammonia N concentration, a sample of rumen fluid was taken (5 mL of 0.2 N HCL was added to 5 mL rumen fluid). Also, 40 mL of rumen fluid at 2 and 6 h after morning feeding was taken for determining tangestic acid soluble N (TAN) and trichloroacetic acid soluble N (TCAN). Samples frozen at −20°C for later determination of VFA and ammonia nitrogen (N), large peptide (LPepN) and small peptide plus amino acid N (Spep+AAN) concentrations. On d 20, ruminal pH was measured every 15 minutes to 8 h after morning feeding (Accumet pH meter, Fisher Scientific, Montreal, Quebec, Canada).
8
Carbonyl Quantification via Hydroxylamine Oximation
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Carbonyl groups were
quantified by titrating HCl that was liberated as a consequence of
the oximation reaction of hydroxylamine hydrochloride with carbonyls
as described previously.27 (link) The oxidized
beads (2 g of wet weight) were homogenized with 3 mL of DI water using
an Ultraturrax homogenizer and dispersed into 25 mL of a 0.25 M hydroxylamine
hydrochloride solution in an acetate buffer (adjusted with 0.1 M NaOH
to pH 4). The HCl released in the reaction between the aldehydes and
hydroxylamine hydrochloride was titrated against 0.1 M NaOH using
an Accumet pH meter (Fisher Scientific), and equivalent points peaks
were obtained from the first order derivative of pH changes against
volume added (dpH/dV).
quantified by titrating HCl that was liberated as a consequence of
the oximation reaction of hydroxylamine hydrochloride with carbonyls
as described previously.27 (link) The oxidized
beads (2 g of wet weight) were homogenized with 3 mL of DI water using
an Ultraturrax homogenizer and dispersed into 25 mL of a 0.25 M hydroxylamine
hydrochloride solution in an acetate buffer (adjusted with 0.1 M NaOH
to pH 4). The HCl released in the reaction between the aldehydes and
hydroxylamine hydrochloride was titrated against 0.1 M NaOH using
an Accumet pH meter (Fisher Scientific), and equivalent points peaks
were obtained from the first order derivative of pH changes against
volume added (dpH/dV).
9
Polymer Dissolution and pH Measurement
10
Rumen Fluid Characterization Protocol
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Ruminal pH, VFA, and ammonia were measured in ruminal fluid collected before (0 h) and at 1, 2, 4, 6, and 8 h after the a.m. feeding. For this purpose, ruminal fluid (~250 mL) was collected from the anterior dorsal, anterior ventral, medium ventral, posterior dorsal, and posterior ventral locations within the rumen using a 50-mL syringe screwed to a stainless tube ending with a probe covered by a fine metal mesh (RT Rumen Fluid Collection Tube, Bar Diamond Inc.). Ruminal pH was measured immediately after sampling (Accumet pH meter; Fisher Scientific, Montreal, QC, Canada). Samples (15 mL) were stored at -20°C for the determination of VFA. Ammonia was measured in samples conserved with 50% sulfuric acid and stored at -20°C until analysis.
Protozoa enumeration was performed as described in Benchaar et al. (2013) (link). Ruminal content (~1 L) was collected 4 h after the a.m. feeding and squeezed through 4 layers of cheesecloth. Samples (5 mL) of the filtered ruminal fluid were preserved with methyl green formalin-saline solution (5 mL) for later enumeration of protozoa (Ogimoto and Imai, 1981) using a counting chamber (Neubauer Improved Bright-Line counting cell, 0.1-mm depth; Hausser Scientific, Horsham, PA).
Protozoa enumeration was performed as described in Benchaar et al. (2013) (link). Ruminal content (~1 L) was collected 4 h after the a.m. feeding and squeezed through 4 layers of cheesecloth. Samples (5 mL) of the filtered ruminal fluid were preserved with methyl green formalin-saline solution (5 mL) for later enumeration of protozoa (Ogimoto and Imai, 1981) using a counting chamber (Neubauer Improved Bright-Line counting cell, 0.1-mm depth; Hausser Scientific, Horsham, PA).
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