At each OD measurement, subsamples of 5 mL were filtered through 0.22 μm polycarbonate filters into combusted glass vials and stored at −20°C. Alginate concentrations were quantified by High Performance Liquid Chromatography (HPLC) of its monomers mannuronate and guluronate after chemical hydrolysis (20 h, 100°C, 0.1 M HCl) in combusted and sealed glass ampoules. Samples were neutralized with 6 N NaOH, desalted using DionexOnGuard II Ag/H cartridges (Thermo Fisher Scientific, Waltham, MA), and eluted with 100 mM sodium acetate tri-hydrate in 100 mM NaOH. Concentrations were determined in three dilutions per sample (0.01, 0.002, 0.001%) using a Carbopac PA 1 column (Thermo Fisher Scientific) and pulsed amperometric detection according to Mopper et al. (1992) (link). A calibration curve was generated using hydrolyzed 1% alginate solution (R2 = 0.97). Glucose concentrations were measured using samples diluted to 0.001% with MilliQ followed by HPLC with NaOH (18 mM) as eluent and a Carbopac PA 1 column (Thermo Fisher Scientific). A calibration curve was generated using 24 concentrations from 0.025 to 10 μM glucose (R2 = 0.99).
Carbopac pa1 column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The CarboPac PA1 column is a high-performance anion-exchange chromatography column designed for the analysis of carbohydrates. It features a polymer-based packing material that provides excellent resolution and peak shape for a wide range of carbohydrate species.
Automatically generated - may contain errors
Lab products found in correlation
Carbopac pa 1 guard column, by Thermo Fisher Scientific (16 mentions)
Ics 3000, by Thermo Fisher Scientific (13 mentions)
Ics 5000 system, by Thermo Fisher Scientific (12 mentions)
Ics 3000 system, by Thermo Fisher Scientific (10 mentions)
Hpaec pad, by Thermo Fisher Scientific (9 mentions)
Carbopac pa guard column, by Thermo Fisher Scientific (9 mentions)
Dionex ics 5000 system, by Thermo Fisher Scientific (8 mentions)
Dionex ics 3000 system, by Thermo Fisher Scientific (7 mentions)
Ics 3000 workstation, by Thermo Fisher Scientific (7 mentions)
Chromeleon 7, by Thermo Fisher Scientific (7 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (6 mentions)
Ics 5000, by Thermo Fisher Scientific (6 mentions)
Hpaec pad system, by Thermo Fisher Scientific (5 mentions)
Pad detector, by Thermo Fisher Scientific (4 mentions)
Xylose, by Merck Group (3 mentions)
Mannose, by Merck Group (3 mentions)
Arabinose, by Merck Group (3 mentions)
Galactose, by Merck Group (3 mentions)
Dionex ics 3000, by Thermo Fisher Scientific (3 mentions)
D glucose, by Merck Group (3 mentions)
170 protocols using carbopac pa1 column
1
Quantifying Alginate and Glucose in Samples
2
High-Performance Anion Exchange Chromatography for Oligosaccharide Analysis
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Analytical scale HPAEC-PAD analyses
were performed on a Dionex ICS-3000 workstation (Dionex, Amsterdam,
The Netherlands) equipped with an ICS-3000 pulse amperometric detection
(PAD) system and a CarboPac PA-1 column (250 × 2 mm; Dionex).
The analytical separation was performed at a flow rate of 0.25 mL
min–1 using a complex gradient of eluents A (100
mM NaOH), B (600 mM NaOAc in 100 mM NaOH), C (Milli-Q water), and
D (50 mM NaOAc). The gradient started with 10% A, 85% C, and 5% D
in 25 min to 40% A, 10% C, and 50% D, followed by a 35 min gradient
to 75% A and 25% B, directly followed by 5 min washing with 100% B
and reconditioning for 7 min with 10% A, 85% B, and 5% D. External
standards of lactose, glucose, and fructose were used to calibrate
for the corresponding sugars. For determination of glucosylated lactose
compounds with a degree of polymerization (DP) of 3, maltotriose was
used as external standard.
Semipreparative HPAEC-PAD samples
were applied in a 4 mg mL–1 concentration in 250
μL injections on an ICS-5000 system, equipped with an ICS-5000
PAD detector, using a CarboPac PA-1 column (250 × 9 mm; Dionex).
Fractions were manually collected and immediately neutralized with
20% acetic acid, followed by desalting over a CarboGraph SPE column
(Alltech, Breda, The Netherlands).
were performed on a Dionex ICS-3000 workstation (Dionex, Amsterdam,
The Netherlands) equipped with an ICS-3000 pulse amperometric detection
(PAD) system and a CarboPac PA-1 column (250 × 2 mm; Dionex).
The analytical separation was performed at a flow rate of 0.25 mL
min–1 using a complex gradient of eluents A (100
mM NaOH), B (600 mM NaOAc in 100 mM NaOH), C (Milli-Q water), and
D (50 mM NaOAc). The gradient started with 10% A, 85% C, and 5% D
in 25 min to 40% A, 10% C, and 50% D, followed by a 35 min gradient
to 75% A and 25% B, directly followed by 5 min washing with 100% B
and reconditioning for 7 min with 10% A, 85% B, and 5% D. External
standards of lactose, glucose, and fructose were used to calibrate
for the corresponding sugars. For determination of glucosylated lactose
compounds with a degree of polymerization (DP) of 3, maltotriose was
used as external standard.
Semipreparative HPAEC-PAD samples
were applied in a 4 mg mL–1 concentration in 250
μL injections on an ICS-5000 system, equipped with an ICS-5000
PAD detector, using a CarboPac PA-1 column (250 × 9 mm; Dionex).
Fractions were manually collected and immediately neutralized with
20% acetic acid, followed by desalting over a CarboGraph SPE column
(Alltech, Breda, The Netherlands).
3
Sugar Composition Analysis via HPAEC-PAD
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To analyze sugar composition, samples were hydrolyzed to be used for SO3− estimation, while the AIR and CL were treated with ice-cold 72% (w/w) H2SO4 at 4 °C for 1 h with sonication, followed by hydrolysis with 2 N H2SO4 at 121 °C for 1 h [29 ,30 (link)]. Monosaccharide in the hydrolysate was analyzed using high-performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD), using a Carbo Pac PA1 column (4 mm × 250 mm, Thermo Fisher Scientific, MA, USA). The column was eluted at a flow rate of 1 mL/min at 35 °C with 14 mM NaOH for neutral sugar, followed by a linear gradient program of 0–250 mM CH3COONa in 100 mM NaOH for UA.
4
Quantitative Analysis of Polyols in Samples
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The culture liquid samples were heated at 95 °C for 15 min and centrifuged for 5 min at 14,000 rpm. The supernatants were 10-fold diluted with MilliQ water (Merck, Amsterdam, The Netherlands) prior to analysis of xylitol and l -arabitol by HPLC (Dionex ICS-5000 + system; Thermo Scientific, Nieuwegein, The Netherlands) equipped with CarboPac PA1 column (2 × 250 mm with 2 × 50 mm guard column; Thermo Scientific), as described previously [30 (link)]. All selected monosaccharides and polyols as mentioned above with concentrations of 5–250 μM were used as standards for identification and quantitation.
5
Oligosaccharide Analysis by HPAEC
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Oligosaccharide analysis was carried out by high-performance anion exchange chromatography (HPAEC) on a Dionex ICS-3000 system (Thermo Scientific) equipped with a 4 × 250 mm CarboPac PA-1 column. A pulsed amperometric detector with a gold electrode and an Ag/AgCl pH reference electrode were used. The system was run with a gradient of 30–600 mM NaAc in 100 mM NaOH 1 mL/min. Chromatograms were analysed using Chromeleon 6.8 chromatography data system software (Thermo Scientific). A mixture of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose was used as reference. AmyC-modified product was dialyzed using dialysis tubing with a cutoff size of 100 Da to 500 Da in ultrapure water. Two milligrams of dry material was dissolved into 1 mL 5 mM sodium acetate buffer pH 5.0 with 5 mM CaCl2. Five hundred microliters of solution was mixed with 2.5 U isoamylase and 1.75 U pullulanase, and incubated at 40 °C for 16 h.
6
Quantifying Xyloglucan Oligosaccharides via HPAEC-PAD
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Degraded xyloglucan oligosaccharides were analyzed by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) on a ICS5000 (Thermo Scientific, Waltham, MA, USA) system equipped with a CarboPac PA-1 column (2 mm ID × 250 mm) in combination with a CarboPac PA guard column (2 mm ID × 50 mm). The mobile phases used were (A) 0.1 M NaOH, (B) 1 M NaOAc in 0.1 M NaOH and the column temperature was 20 °C. The elution program applied has previously been described [41 (link)]. Samples were diluted 5-fold prior to analysis. Standard cellodextrins [DP 2–6; Sigma-Aldrich (St. Louis, MO, USA)] were mixed, each in a concentration of 2.5 µg mL−1 and used for calibration.
7
Carbohydrate and Lignin Quantification
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The carbohydrate
and lignin contents in the samples were determined after a two-stage
acid hydrolysis. Neutral monosaccharides in the acidic hydrolysate
were quantified by high-performance anion exchange chromatography
with pulse amperometric detection in a Dionex ICS-3000 (Thermo Fisher
Scientific, Waltham, MA, USA) instrument equipped with a CarboPac
PA1 column. The amount of Klason lignin was quantified gravimetrically,
and the amount of acid soluble lignin was determined by UV-spectrometry
at 215 and 280 nm, using the Goldschmid equation.37 The ash content was determined gravimetrically after combustion
of the lignin samples at 550 °C for 23 h (including heating time).
The elemental composition (C, H, N, O, and S) was determined using
a FLASH 2000 series organic elemental analyzer (Thermo Fisher Scientific,
Waltham, MA, USA). All samples were analyzed in duplicates.
and lignin contents in the samples were determined after a two-stage
acid hydrolysis. Neutral monosaccharides in the acidic hydrolysate
were quantified by high-performance anion exchange chromatography
with pulse amperometric detection in a Dionex ICS-3000 (Thermo Fisher
Scientific, Waltham, MA, USA) instrument equipped with a CarboPac
PA1 column. The amount of Klason lignin was quantified gravimetrically,
and the amount of acid soluble lignin was determined by UV-spectrometry
at 215 and 280 nm, using the Goldschmid equation.37 The ash content was determined gravimetrically after combustion
of the lignin samples at 550 °C for 23 h (including heating time).
The elemental composition (C, H, N, O, and S) was determined using
a FLASH 2000 series organic elemental analyzer (Thermo Fisher Scientific,
Waltham, MA, USA). All samples were analyzed in duplicates.
8
Monosaccharide and Amino Acid Analysis of PSM and EBN Diets
9
Quantification of Barley Glucans
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A 10mg aliquot of flour samples was used for analysis. Soluble sugars were removed by washing the powder in 80% (v/v) ethanol, in a boiling water bath for 30min and once for 10min, and 96% ethanol at room temperature for a final wash. The supernatant was removed at the end of every step after centrifugation at 16 000 g (Heraeus biofuge pico) for 5min and the pellet was dried at 40 °C. Complete hydrolysis of BGs was conducted using lichenase and β-glucosidase from the mixed-linkage BG kit (Megazyme International Ltd) following the method provided by the manufacturer (AOAC Method 995.16 AACC Method 32-23/ICC Standard Method 32-23 No. 168; the reaction volumes were scaled down according to the smaller sample mass). Samples treated in that way were centrifuged, filtered, and the quantification of the resulting glucose units was performed by a high performance anion exchange system (Thermo Fisher Scientific Inc.) with an AS50 autosampler, GS50 gradient pump, and ED50 PAD system equipped with a CarboPac® PA1 column using the run program described before (Lunde et al., 2008 (link)).
10
Carbohydrate Composition Analysis via HPLC
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The carbohydrate composition in the reaction mixture was analyzed by high-performance liquid chromatography (HPLC) equipped with a Dionex ICS-5000+ system (Thermo Fisher Scientific) consisting of an ICS-5000+ dual pump (DP) and an electrochemical detector (ED). Separations were performed at room temperature on CarboPac PA-1 column (4 × 250 mm) connected to a CarboPac PA-1 guard column (4 × 50 mm) (Thermo Fisher Scientific) with flow rate 1 mL/min. All eluents A (150 mM NaOH), B (150 mM NaOH and 500 mM sodium acetate) and C (deionized water) were degassed by flushing with helium for 30 min. Separation of D-glucose, D-galactose, lactose and allolactose was carried out with a run with the following gradient: 90% C with 10% A for 45 min at 1.0 mL/min, followed by 5 min with 100% B. The concentration of saccharides was calculated by interpolation from external standards. Total GOS concentration was calculated by subtraction of the quantified saccharides (lactose, glucose, galactose) from the initial lactose concentration. The GOS yield (%) was defined as the percentage of GOS produced in the samples compared to initial lactose.
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