Filtrates of the reactions were analyzed by HPAEC–PAD on a Dionex ICS-5000 system (Sunnyvale, CA, USA) equipped with a CarboPac PA1 guard column (2 × 50 mm) and a CarboPac PA1 analytical column (2 × 250 mm). The flow was set at 0.25 mL/min, with an injection volume of 10 µL. For elution, two mobile phases were used: 0.1-M NaOH as eluent A, 1-M NaOAc in 0.1-M NaOH as eluent B. The following gradient was applied: linear increase from 0 to 10% B in 10 min, then to 30% B in the next 15 min, followed by an exponential gradient to 100% B in 5 min. The column was reconditioned before every injection by running 0% B for 9 min. Assignments of C1- and C4-oxidized peaks was based on comparison with products released from reference enzymes performing either C1- or C4-oxidation (NcLPMO9F and NcLPMO9C, respectively), which were produced as described elsewhere [45 (link)]. Gluconic acid and cellobionic acid were assigned and quantified using available standards (purchased from Megazyme and Synthose, respectively). To analyze the chromatograms, Chromeleon version 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA) was used.
Pa1 guard column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA1 guard column is designed to protect the analytical column from contaminants and particulates, helping to extend the life of the primary column. It is a short pre-column that is placed in front of the analytical column to prevent damage and ensure reliable and accurate chromatographic results.
Automatically generated - may contain errors
Lab products found in correlation
Ics 3000 system, by Thermo Fisher Scientific (2 mentions)
Pa1 column, by Thermo Fisher Scientific (2 mentions)
Pa1 analytical column, by Thermo Fisher Scientific (1 mentions)
Ics 5000 system, by Thermo Fisher Scientific (1 mentions)
Ed40 detector, by Thermo Fisher Scientific (1 mentions)
Q tof premier, by Waters Corporation (1 mentions)
Pulsed amperometric detector, by Thermo Fisher Scientific (1 mentions)
As3000 autosampler, by Thermo Fisher Scientific (1 mentions)
Carbopac pa1 column, by Thermo Fisher Scientific (1 mentions)
Chromeleon software, by Thermo Fisher Scientific (1 mentions)
Lc 6a hplc, by Shimadzu (1 mentions)
6 protocols using pa1 guard column
1
HPAEC-PAD Analysis of Oxidized Celluloses
2
Quantification of Galactinol, Raffinose, and Stachyose in Rice Leaves
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Galactinol, raffinose and stachyose contents in matured leaves and third leaves after cold treatment of transgenic rice plants were determined by high-performance anion exchange chromatography (HPAEC) analysis. Finely chopped leaves excised from rice plants were boiled in boiling deionized water for 1 h to extract total water-soluble sugars. The extract solution was filtered through 0.45 μm-pore-sized membrane filter. HPAEC analysis was performed by using a DX500 gradient chromatography system coupled with pulsed amperometry detection by using an ED40 detector (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac PA-1 (4 × 250 mm) analytical column and a CarboPac PA-1 guard column (4 × 50 mm). For the separation of galactinol, the sample solution was eluted at 1 ml min−1 in 100 mM NaOH for 2 min and then with a linear gradient of 0–100 mM sodium acetate in 100 mM NaOH for 18 min. For the separation of raffinose and stachyose, the sample solution was eluted at 1 ml min−1 in 150 mM NaOH for 2 min and then with a linear gradient of 0–40 mM sodium acetate in 150 mM NaOH for 18 min. Each sugar was identified and quantified by the external standard method using authentic galactinol, raffinose and stachyose.
3
Quantification and Characterization of Ah-MVPS
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About 100 mg of Ah-MVPS were precisely weighted and dissolved in 100 mL distilled water to prepare 1.0 mg/mL of stock solution. Ah-MVPS stock solution was diluted to anticipative concentrations to determine the content of total sugar and reducing sugar. The total sugar content was measured by the anthrone-sulfuric acid method (Laurentin and Edwards, 2003 (link); Piccolo et al., 2008 (link)), while the reducing sugar content was quantified by the 3,5-dinitrosalicylic acid (DNS) method in calibration with D-glucose (Zhao et al., 2008 (link)). The oligosaccharides of Ah-MVPS were initially analyzed by high-performance liquid chromatography (HPLC), adopting the methods of Chen (Chen et al., 2004 (link)) and using a Waters ™ Alliance 2695 HPLC Separation Module in combination with a Shodex Asahipak NH2P-4E column (4.6mm×250mm) and a Dionex PA-1 guard column (4.0mm×50mm). Further identification of the oligosaccharides of Ah-MVPS was executed on an HPLC-ESI-TOF-MS system. The system consisted of two successive coupled apparatuses: a Waters 2695 HPLC Separation Module equipped with a Shodex Asahipak NH2P-4E column (4.6mm×250mm) and a Time-of-Flight Mass Spectrometer (QTOF-MS, Waters QTOF Premier) equipped with an electrospray source.
4
Quantification and Identification of Xylooligosaccharides
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The DP of XOs was determined by a Dionex ICS 3000 system equipped with an AS3000 auto sampler using high-performance anion exchange chromatography with a pulse amperometric detection (HPAED-PAD) system (Thermo Scientific). The types of XOs were identified by comparing the peak areas of standard xylobiose (X2), xylotriose (X3) (Wako, Osaka, Japan) xylotetraose (X4) (Biocon, Nagoya, Japan). A Carbopac PA1 column (4 × 250 mm, Dionex, Thermo Scientific) with PA1 guard column (4 × 50 mm, Dionex) was used at a flow rate of 1.0 mL/min and the column temperature was set at 35 °C. A pulsed amperometric detector with an Au electrode operating in the integrated amperometric mode (Dionex) was used for the detection of XOs which was separated with a gradient of 10–100 mM NaOH for 15 min, followed by 0–20 mM sodium acetate gradient in 100 mM NaOH for 25 min.
5
Carbohydrate Profiling of S. flexneri
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The sugar composition of the S. flexneri PS was determined by HPAEC-PAD using a Carbo Pac PA1 column (50 mm × 250 mm) coupled to a CarboPac PA1 guard column and connected to Dionex KS5000 system. Samples diluted at 10 µg/ml of saccharide were treated with NaOH at a final concentration of 2 M, heated at 110 °C for 6 h in a closed screwcap test tube and filtered with 0.45 µm filter before the analysis. The separation was performed with a flow rate of 1 ml/min using gradient elution of 100 mM NaOH for 32 min with increased concentration of NaNO 3 ranging from 8 to 50%, followed by a washing step for 22 min. The chromatography was monitored using the pulsed amperometric mode with a gold working electrode and an Ag/AgCl reference electrode. A quadruple-potential waveform for carbohydrates was used. The chromatographic data were processed using Dionex Chromeleon TM software. The calibration curve was set up with glucose (Fluka) in the range of 1.0-25.0 µg/ml which was treated the same way as the samples mentioned above [20] . All the above experiments have been performed in triplicates and the results are the mean of those three readings.
6
Lignin, Carbohydrate, and Pulp Analysis
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The amount of acid-insoluble lignin (Klason lignin) in the materials was determined according to a modification of the TAPPI test method, T222 om-01 [22] . Extractive and ash content was determined following TAPPI test methods T204 om-88 and T211 om-93, respectively. Carbohydrate compositions were determined by ion chromatography of the filtrates of the acid hydrolysis, which had been diluted 1000 times using a Dionex ICS 3000 system (Dionex, Sunnyvale, CA, USA) equipped with a single pump (SP-1), an electrochemical detector, a CarboPac PA 1 column ( φ 4 mm × 250 mm), a CarboPac PA 1 guard column ( φ 4 mm × 50 mm), and an auto sampler [23] . The amount of acid-soluble lignin was determined according to TAPPI test method T222 om-88 from the ultraviolet absorbance of the filtrate at 205 nm using a spectrophotometer. Kappa number, viscosity, and α-cellulose content of the pulps were determined according to TAPPI test methods T236 om-13, T230 om-94, and T203 cm-09, respectively. The ISO brightness was measured based on TAPPI test method T452 om-92 using a digital color meter (TC-1500 SX, Tokyo-Denshoku, Japan). The hexenuronic acid (HexA) content of the pulps was determined using a high-performance liquid chromatograph (Shimadzu LC-6A HPLC) with a Zorbax ODS column: φ 4.6 × 250 mm (Shimadzu, Kyoto, Japan).
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