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 analytical column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CarboPac™ PA20 analytical column is a high-performance ion exchange chromatography column designed for the separation and analysis of carbohydrates. The column features a stationary phase that enables the efficient separation of a wide range of carbohydrate compounds.
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Lab products found in correlation
Carbopac pa20 guard column, by Thermo Fisher Scientific (2 mentions)
Dionex ics 5000, by Thermo Fisher Scientific (2 mentions)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Ics 2500 system, by Thermo Fisher Scientific (1 mentions)
Ed50a, by Thermo Fisher Scientific (1 mentions)
Thermo ics5000 ion chromatography system, by Thermo Fisher Scientific (1 mentions)
Lc 10a system, by Shimadzu (1 mentions)
Hpaec pad ics 5000, by Thermo Fisher Scientific (1 mentions)
Total starch kit, by Megazyme (1 mentions)
9 protocols using carbopac pa20 analytical column
1
Monosaccharide Profiling of Fucoidan
2
Monosaccharide Composition Analysis of LPB8
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The monosaccharides of LPB8-0 and LPB8-1 were determined through high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The LPB8-0 and LPB8-1 samples (5.0 mg) were hydrolyzed with trifluoroacetic acid (2.0 M) at 121 °C for 2 h. Then, two hydrolysates were evaporated to dryness under an N2 stream blowing instrument and eluted with methanol. The released monomers and all standards were further measured using a Thermo ICS-5000 ion chromatography system (Thermo Scientific, Waltham, MA, USA) fitted with a Dionex CarboPac PA-20 analytical column and a Dionex ED50A electrochemical detector.
3
Molecular Weight and Monosaccharide Composition Analysis
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Gel permeation chromatography (GPC) was used to determine the molecular weights of Artp1 and Artp2 on a Shimadzu SPD-20A HPLC system equipped with a SUGAR KS-805 column (300 × 8.0 mm, Shodex, Japan) [7 (link)]. Briefly, 1.5 mg/mL of purified sample was dissolved in 0.02 M sodium phosphate buffer (pH 6.8) and percolated through 0.45 μm of millipore filter, and then applied to column (injection volume of 30 μL) with a flow rate of 1 mL/min at a column temperature of 55 °C. Preliminary calibration of the column was conducted using the Dextrans with different molecular weights (4320, 12,600, 73,800, 126,000, 289,000, and 1,000,000) The regression curve equation was as follows: LogMw = −1.1337Rt + 16.165 with a correlation coefficient of 0.9912.
The monosaccharide compositions of samples were determined by high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) (Dionex ICS-5000, Thermo Fisher, Waltham, MA, USA). Next, 5 mg of polysaccharide was dissolved in TFA and hydrolyzed at 121 °C for 2 h. Then, the hydrolyzed product was washed by evaporating at reduced pressure with methanol for 2–3 times. After this, the samples of hydrolyte were diluted and filtrated through 0.22 μm of hydrophilic membranes. The reaction products were analyzed with a Dionex™ CarboPac™ PA20 analytical column to elute with a mobile phase at 0.5 mL/min.
The monosaccharide compositions of samples were determined by high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) (Dionex ICS-5000, Thermo Fisher, Waltham, MA, USA). Next, 5 mg of polysaccharide was dissolved in TFA and hydrolyzed at 121 °C for 2 h. Then, the hydrolyzed product was washed by evaporating at reduced pressure with methanol for 2–3 times. After this, the samples of hydrolyte were diluted and filtrated through 0.22 μm of hydrophilic membranes. The reaction products were analyzed with a Dionex™ CarboPac™ PA20 analytical column to elute with a mobile phase at 0.5 mL/min.
4
Quantitative Analysis of Root Exudates
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Root exudates (10 ml) were lyophilized, redissolved in 40 μl of H2O, and filtered through a 0.2 μm PTFE spin filter (Thermo Scientific). Recovery control samples were prepared by spiking 0.06 μg of arabinose into 10 ml of ultra-pure water, lyophilized, and treated the same as the real samples.
Sugars in exudates were analyzed by anion exchange HPLC using a Dionex System equipped with a CarboPac PA20 Analytical Column (6.5 μm, 3.0 × 150 mm) and an AminoTrap guard-column (3 × 30 mm). Arabinose, galactose, glucose, fructose, sucrose and xylose were separated with a multi-step gradient of 10 mM NaOH, 200 mM NaOH and ultra-pure water. The flow rate was 0.3 ml/min, column temperature was 30°C and 20 μl of sample was injected. Sugars were detected with a Coulochem III detector (Dionex) configured with pulse-mode amperometric cell and gold electrode. Data analysis was performed using Chromeleon 6.80 SR10 software. Sugars were quantified with external standards using linear calibration curves (R2 = 0.99).
Sugars in exudates were analyzed by anion exchange HPLC using a Dionex System equipped with a CarboPac PA20 Analytical Column (6.5 μm, 3.0 × 150 mm) and an AminoTrap guard-column (3 × 30 mm). Arabinose, galactose, glucose, fructose, sucrose and xylose were separated with a multi-step gradient of 10 mM NaOH, 200 mM NaOH and ultra-pure water. The flow rate was 0.3 ml/min, column temperature was 30°C and 20 μl of sample was injected. Sugars were detected with a Coulochem III detector (Dionex) configured with pulse-mode amperometric cell and gold electrode. Data analysis was performed using Chromeleon 6.80 SR10 software. Sugars were quantified with external standards using linear calibration curves (R2 = 0.99).
5
Molecular Weight Analysis of Polysaccharides
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The molecular weight was analyzed by a high-performance gel permeation chromatography (HPGPC) method on a Shimadzu(LC-10A)system with a BRT105-104-102™Linear column (8.0 mm×300 mm) (Tang.et al., 2017). The Dextrans with molecular weights of 5, 11.6, 23.8, 48.6, 80.9, 148, 273, 409.8 and 667.8 kDa were served as the control for a standard curve.
The monosaccharide compositions of WMCP & DMCP were determined by Xie et al. (2013) (link) with minor appropriate modifications. Briefly, five mg sample was hydrolyzed with 2 mL of TFA (2 M) under 120°Cfor 3 h. Then the hydrolyzation was continued at 105°Cfor 4 h. The hydrolysate was finally made up to 50 mL with deionized water. The compositions of WMCP and DMCP were determined by the DionexICS-2500 ion chromatography system (Dionex, USA) coupled with a Dionex Carbopac™ PA20 analytical column (3.0×150 mm).
The monosaccharide compositions of WMCP & DMCP were determined by Xie et al. (2013) (link) with minor appropriate modifications. Briefly, five mg sample was hydrolyzed with 2 mL of TFA (2 M) under 120°Cfor 3 h. Then the hydrolyzation was continued at 105°Cfor 4 h. The hydrolysate was finally made up to 50 mL with deionized water. The compositions of WMCP and DMCP were determined by the DionexICS-2500 ion chromatography system (Dionex, USA) coupled with a Dionex Carbopac™ PA20 analytical column (3.0×150 mm).
6
Monosaccharide Composition Analysis of Fermented Black Tea
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Monosaccharide composition analysis of fermented black tea beverage was performed following the method (Shi et al., 2017 ) described previously with slight modification. Briefly, the aliquots obtained at different time intervals were centrifugated at 15000 g for 10 min, and the supernatants were collected after being filtered with 0.22 μm microporous filter membrane; and next, the supernatants were transferred to a 50 mL volumetric flask and diluted 5 or 10 times using ultrapure water. Finally, the composition of monosaccharide in the fermented black tea beverage was determined using a high-performance anion exchange chromatographic (HPAEC) system coupled with a pulse amperometric detector (PAD) (Dionex-5500, Dionex Corporation, Sunnyvale, California, USA). NaOH solution (100 mM) was used as isocratic eluent and a Dionex Carbo-Pac PA20 analytical column (3 mm × 150 mm) was used and maintained at 30 °C. Meanwhile, the injection volume was 10 μL, and the flowrate of mobile phase was 0.5 mL/min.
7
Comprehensive Lignocellulose Analysis Protocol
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Determination and characterization of lignin, cellulose and hemicellulose content was performed according to the NREL Laboratory Analytical Procedure (NREL/TP‐510‐42618, 2012). Extracted monosaccharides and uronic acids were identified and quantified using an HPAEC‐PAD (ICS‐5000, Thermo Scientific, Sunnyvale, CA, USA) equipped with a CarboPac PA20 analytical column (150 mm × 3 mm; 6 µm) as well as a guard column (30 mm × 3 mm), as previously described in Falck et al. (2014 ). For the separation of arabinose and rhamnose, the mobile phase concentration was increased to 10 mM sodium hydroxide. Uronic acids were analysed according to the same method, with a mobile phase consisting of 90 mM sodium hydroxide and 150 mM sodium acetate.
8
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)).
9
Lignocellulose and Starch Analysis of Biomass
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The lignin, cellulose, hemicellulose and starch content of the 200 g scale unbleached and bleached samples was analysed. Before analysis, the whole hulls were milled in a coffee grinder for one cycle at grind size 8 (Model: BCG820BSSUK; Sage). The lignocellulose fraction was characterized according to the NREL Laboratory Analytical Procedure (NREL/TP-510-42618, 2012). The extracted monosaccharides were identified and quantified utilizing HPAEC-PAD (ICS-5000, Dionex, Thermo Scientific) equipped with a CarboPac PA20 analytical column (150 mm × 3 mm, 6 μm) as well as a respective guard column (30 mm × 3 mm) as previously described by Falck and colleagues [18 (link)]. The starch content was determined utilizing the “Total Starch” kit from Megazyme (https://www.megazyme.com/total-starch-assay-kit ); method “a” of protocol K-TSTA 09/14 was followed.
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