High Performance Anion Exchange Chromatography (HPAEC) is coupled with a Pulsed Amperometric Detector (HPAEC-PAD) (Thermo Fisher Scientific, Waltham, MA, USA) for the separation and determination of monosaccharide constituents of polysaccharides (13 (link)). Each fucoidan sample (5 mg) was hydrolyzed in sulfuric acid (12 M, 0.5 mL) for half an hour. HPAEC-PAD analysis was performed using a Dionex ICS-2500 system (Dionex Corporation, Sunnyvale, California, USA) equipped with a CarboPac™ PA20 analytical column (250 mm x 4 mm ID, Dionex Corp., Sunnyvale, CA, USA) and CarboPac™ PA20 guard column (50 mm × 4 mm ID, Dionex). For the mobile phase, CH3COONa (1 M), H2O, and NaOH (250 mM) were used. The injection temperature was maintained at 30°C.
Carbopac pa20 guard column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CarboPac™ PA20 guard column is a laboratory equipment designed for use in high-performance liquid chromatography (HPLC) systems. The core function of this guard column is to protect the analytical column from contaminants and extend its lifespan, thereby improving the overall performance and reliability of the HPLC system.
Automatically generated - may contain errors
Lab products found in correlation
Carbopac pa20 column, by Thermo Fisher Scientific (3 mentions)
Carbopac pa20 analytical column, by Thermo Fisher Scientific (2 mentions)
Ics 6000 system, by Thermo Fisher Scientific (2 mentions)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Ics 2500 system, by Thermo Fisher Scientific (1 mentions)
Ics 3000 hpaec system, by Thermo Fisher Scientific (1 mentions)
Ed50 electrochemical detector, by Thermo Fisher Scientific (1 mentions)
Chromafil a 45 25, by Macherey-Nagel (1 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Chromeleon 7, by Thermo Fisher Scientific (1 mentions)
Gk80040, by Agilent Technologies (1 mentions)
5 protocols using carbopac pa20 guard column
1
Monosaccharide Profiling of Fucoidan
2
Compositional Analysis of Termite Dietary Materials
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The termite (and woodroach) dietary materials and their feces were taken for compositional analyses of total sugars, KL, and acid-soluble lignin using two-step acid hydrolysis procedure of National Renewable Energy Laboratory (Boulder, CO) (58 ). HPAEC was used to quantify the monosaccharides in the hydrolysate of each sample, using a Dionex ICS-3000 HPAEC system (Dionex, Sunnyvale, CA) equipped with an analytical CarboPacTM PA-20 column (4 mm by 250 mm, Dionex) in combination with the CarboPac PA-20 guard column (4 mm by 50 mm, Dionex) at the 30°C column temperature. The procedure was similar to that described by Sun et al. (59 (link)) but with modifications. Briefly, the monosaccharides and glucuronic acid were separated in 2 mM NaOH for 16 min at a flow rate of 0.4 ml/min, followed by a 100 mM NaOH and 50 mM NaOAc for 15 min. A 10-min elution with 2 mM NaOH was then used to reequilibrate the column before the next injection. The sugars including glucose, xylose, glucuronic acid, arabinose, galactose, and mannose were all purchased from Sigma-Aldrich (USA) and used as standards. Each sample analysis was biologically replicated (two distinctly pooled samples).
3
Quantitative Pectin Sugar Analysis
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High performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) was applied to quantify the content of the neutral sugars fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose in the pectin samples.
First, the pectin samples were hydrolyzed in 4 M trifluoroacetic acid at 110 °C for 1.5 h and immediately hereafter cooled in an ice bath. Subsequently, the remaining TFA was neutralized by consecutively drying the samples under N2 at 45 °C, washing the samples with 1 M ammonium hydroxide and finally drying the samples again under N2 at 45 °C (De Roeck, Sila, Duvetter, Van Loey, & Hendrickx, 2008; (link)Stolle-Smits, Beekhuizen, Recourt, Voragen, & Van Dijk, 1997) (link). Hydrolyzed samples were redissolved in ultrapure water and filtered (Chromafil® A-45/25, 0.45 μm pore size, Macherey-Nagel, Düren, Germany) before analysis. The hydrolysis was carried out in duplicate.
The HPAEC-PAD analysis was performed exactly as described in Neckebroeck et al. (2020) (link) using a Dionex ICS-6000 system equipped with a Dionex ICS-6000 Single Pump, a CarboPac™ PA20 column (150 x 3 mm) protected by a CarboPac™ PA20 guard column ( 30x 3 mm) and an ED50 electrochemical detector (Dionex, Sunnyvale, CA, USA).
First, the pectin samples were hydrolyzed in 4 M trifluoroacetic acid at 110 °C for 1.5 h and immediately hereafter cooled in an ice bath. Subsequently, the remaining TFA was neutralized by consecutively drying the samples under N2 at 45 °C, washing the samples with 1 M ammonium hydroxide and finally drying the samples again under N2 at 45 °C (De Roeck, Sila, Duvetter, Van Loey, & Hendrickx, 2008; (link)Stolle-Smits, Beekhuizen, Recourt, Voragen, & Van Dijk, 1997) (link). Hydrolyzed samples were redissolved in ultrapure water and filtered (Chromafil® A-45/25, 0.45 μm pore size, Macherey-Nagel, Düren, Germany) before analysis. The hydrolysis was carried out in duplicate.
The HPAEC-PAD analysis was performed exactly as described in Neckebroeck et al. (2020) (link) using a Dionex ICS-6000 system equipped with a Dionex ICS-6000 Single Pump, a CarboPac™ PA20 column (150 x 3 mm) protected by a CarboPac™ PA20 guard column ( 30x 3 mm) and an ED50 electrochemical detector (Dionex, Sunnyvale, CA, USA).
4
Polysaccharide Hydrolysis and Analysis
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Polysaccharides were firstly hydrolyzed with 12 M H2SO4 for 0.5 h and then with 2 M H2SO4 at 100°C for 3 h (22 (link)). The hydrolyzate was diluted to be 20 μg/ml and analyzed by a high-performance anion-exchange chromatography with pulsed amperometric detection (Dionex ICS-5000, Thermo Fisher, Waltham, MA, United States). A CarboPac™ PA20 Guard Column (Ø3 mm × 30 mm, Thermo Fisher, Waltham, MA, United States) and CarboPac™ PA20 Analytical Column (Ø3 mm × 150 mm, Thermo Fisher, Waltham, MA, United States) were equipped in series and eluted with the mobile phase at 0.5 ml/min (23 (link)).
5
Analysis of Protein Sialic Acid Content
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The dried protein sample (25 µg) was treated in triplicate with 5× Buffer B (10 µl) and Sialidase A (2 µL) (Agilent, GK80040) and adjusted to a final concentration of 0.48 µg/µL. The sample was kept at 37 °C for 18 h. The sample were then spun (14,000 rpm, 2 min), diluted 1:20 water, and 25 µL triplicate injections were eluted with a linear gradient of NaOH/sodium acetate buffer on a Dionex ICS-6000 system equipped with a CarboPac PA20 column (3 × 150mm, 060142), CarboPac PA20 guard column (3 × 30 mm, 060144) and a pulsed amperometric detector46 (link).
Both neutral sugar and sialic acid data collection and analysis have been performed with Chromeleon 7 (Thermofisher Scientific). The data was processed using GraphPad Prism 9.0.2.
Both neutral sugar and sialic acid data collection and analysis have been performed with Chromeleon 7 (Thermofisher Scientific). The data was processed using GraphPad Prism 9.0.2.
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