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.
Ics 2500 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ICS-2500 system is an ion chromatography system designed for the separation and analysis of ionic compounds. It provides high-performance liquid chromatography capabilities for the detection and quantification of ions in a variety of sample types.
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Lab products found in correlation
Carbopac pa20 analytical column, by Thermo Fisher Scientific (1 mentions)
Carbopac pa20 guard column, by Thermo Fisher Scientific (1 mentions)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Nano3, by Merck Group (1 mentions)
Na2hpo4 2h2o, by Merck Group (1 mentions)
Pa10 column, by Thermo Fisher Scientific (1 mentions)
1260 hplc system, by Agilent Technologies (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Delta 5 isotope ratio mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ionpac as18, by Thermo Fisher Scientific (1 mentions)
Am 500 mhz spectrometer, by Bruker (1 mentions)
11 protocols using ics 2500 system
1
Monosaccharide Profiling of Fucoidan
2
Monosaccharide Composition Analysis by HPAEC-PAD
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CMSPA90-1 (5 mg) was hydrolyzed with 10 mL of 2 M trifluoroacetic acid (TFA) at 120 °C in a sealed tube for 6 h. Excess TFA was removed by evaporation and co-distilled with methanol after the hydrolysis was completed. The monosaccharide composition was measured by HPAEC-PAD. The hydrolysate was re-dissolved in 0.5 mL of distilled water and then filtered with a 0.45 μm filter. The resulting solution (20 μL) was injected on the Dionex ICS-2500 system for the ionic chromatography analysis by HPAEC-PAD, and eluted with a solution of water and 200 mM NaOH with a volume ratio of 92 : 8.11 (link)
3
Quantification of Seed Nutrients
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To measure nitrate, phosphate, and phytate content, 5mg of seeds were boiled at 100°C for 15min in 0.5ml 0.5M HCl and 50mg l–1trans-aconitate (internal standard). After centrifuging for 2min at 13 000rpm, 200 µl of the supernatant was transferred to an HPLC-vial.
HPLC-analysis was performed on a Dionex ICS2500 system with an AS11-HC column and an AG11-HC guard column and eluted with NaOH. The elution procedure was: 0–15min linear gradient of 25–100mM NaOH, then 15–20min 500mM NaOH followed by 20–35min 5mM NaOH. Flow rates were 1ml min–1 throughout the run. Contaminating anions in the eluents were removed using an ion trap column (ATC), installed between the pump and the sample injection valve. Anions were determined by conductivity detection. Background conductivity was decreased using an ASRS suppressor, with water as a counterflow. Peaks were identified and quantified using known external standards. External standards of nitrate, phosphate, and phytate were NaNO3 (Merck), Na2HPO4.2H2O (Merck), and Na(12)-IP6 IP6 (Sigma-Aldrich), respectively.
HPLC-analysis was performed on a Dionex ICS2500 system with an AS11-HC column and an AG11-HC guard column and eluted with NaOH. The elution procedure was: 0–15min linear gradient of 25–100mM NaOH, then 15–20min 500mM NaOH followed by 20–35min 5mM NaOH. Flow rates were 1ml min–1 throughout the run. Contaminating anions in the eluents were removed using an ion trap column (ATC), installed between the pump and the sample injection valve. Anions were determined by conductivity detection. Background conductivity was decreased using an ASRS suppressor, with water as a counterflow. Peaks were identified and quantified using known external standards. External standards of nitrate, phosphate, and phytate were NaNO3 (Merck), Na2HPO4.2H2O (Merck), and Na(12)-IP6 IP6 (Sigma-Aldrich), respectively.
4
Monosaccharide Quantification by HPAEC-PAD
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ASPA80-1 (5 mg) was hydrolyzed in 2 mL of 2 M TFA for 5 h at 110 °C. Thereafter, the excess TFA was removed by evaporation on a water bath at 40 °C and co-distilled with MeOH. The monosaccharide content was measured using high performance anion exchange chromatography (HPAEC-PAD). The hydrolysate (1 mg) was dissolved in distilled water (1 mL) and then filtered using a 0.45 μm filter. The solution (20 μL) was injected into HPAEC-PAD on the Dionex ICS-2500 system and eluted with a mixture of water and 200 mM NaOH in a volume ratio of 92 : 8 [36 ].
5
Molecular Weight and Monosaccharide Composition Analysis
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The molecular weight of SCP_A and SCP_N was determined by high-performance gel permeation chromatography (HPGPC), which was performed in an Agilent 1260 HPLC system equipped with an KS-802 column (8 mm × 300 mm) detected by a refractive index detector. The monosaccharide composition was determined by HPAEC pulsed amperometric detection (PAD), a CarboPacTM PA10 column (2.0 mm × 250 mm), and a Dionex ICS-2500 system. Standard monosaccharides (Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru, Gala, and Glca) were used as references.
6
Quantifying Nitrate in Plant Seeds
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Nitrate measurements were performed as described in He et al. (2014) (link). In brief, 5 mg of seeds were boiled at 100°C for 15 min in 0.5 ml of 0.5 M HCl and 50 mg l–1trans-aconitate (internal standard). After centrifuging for 2 min at 13,000 rpm, 200 μl of the supernatant was transferred to an HPLC vial.
HPLC analysis was performed on a Dionex ICS2500 system with an AS11-HC column and an AG11-HC guard column, and eluted with NaOH. The elution procedure was: 0–15 min linear gradient of 25–100 mM NaOH, then 15–20 min of 500 mM NaOH followed by 20–35 min of 5 mM NaOH. Flow rates were 1 ml min–1 throughout the run. Contaminating anions in the eluents were removed using an anion trap column (ATC), installed between the pump and the sample injection valve. Anions were determined by conductivity detection. Background conductivity was decreased using an ASRS suppressor, with water as a counterflow. Peaks were identified and quantified using known external standards. The external standard of nitrate was NaNO3.
HPLC analysis was performed on a Dionex ICS2500 system with an AS11-HC column and an AG11-HC guard column, and eluted with NaOH. The elution procedure was: 0–15 min linear gradient of 25–100 mM NaOH, then 15–20 min of 500 mM NaOH followed by 20–35 min of 5 mM NaOH. Flow rates were 1 ml min–1 throughout the run. Contaminating anions in the eluents were removed using an anion trap column (ATC), installed between the pump and the sample injection valve. Anions were determined by conductivity detection. Background conductivity was decreased using an ASRS suppressor, with water as a counterflow. Peaks were identified and quantified using known external standards. The external standard of nitrate was NaNO3.
7
Geochemical Porewater Analysis
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Sulfate and chloride concentrations were measured by suppressed ion chromatography with an ICS2500 system (Dionex) equipped with an eluent generator (EG50) and KOH eluent generator cartridge (EGC III KOH). The column was a Dionex IonPac™ AS18 operated at 30°C. KOH concentration started at 20 mmol L−1 and was raised to 32 mmol L−1 at the end of the analysis run at 15 min. Hydrogen sulfide (sum of H2S, HS− and S2−) was determined spectrophotometrically at 670 nm (FLUOstar Omega, BMG Labtech GmbH, Orthenberg, Germany) on zinc-preserved porewater samples by the methylene blue method (Cline, 1969 ; Reese et al., 2011 (link)). DIC was measured immediately after the cruise on headspace-free porewater samples stored at 4°C. Samples were transferred to sealed exetainers and acidified with 85% (v:v) phosphoric acid. After 24 h of equilibration time, the produced CO2 was measured from the headspace of the exetainer by a Delta V™ isotope ratio mass spectrometer (IRMS, Thermo Scientific).
8
Spectrophotometric Soil Solution Analysis
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Stock solutions of bromocresol purple (BCP) and phenol red (PR) at a total concentration of 3 × 10−3 mol L−1 were used. The absorbance maxima (Abs) of acid and base forms of PR were read at 433 nm, 558 nm (λ1 and λ2) and BCP at 432 nm, 589 nm (λ1 and λ2), respectively, using the spectrophotometer Cintral™ software and used for R (= λ2Abs./λ1Abs) calculation (see [32 (link)]). The value for molar absorbance ratios (e1-e3) and pK2 of indicators used in this study (PR and BCP) are those of [36 (link)].
The ionic strength (µ) of each soil extract was determined via electrical conductivity (EC, mS cm−1) measurement using a calibrated conductivity electrode (TPS Glass K = 1.0 Cond Sensor) using the equation µ = EC × 0.0127 [32 (link), 41 (link), 42 (link)].
The concentration of dissolved organic carbon (DOC) in filtered soil solutions was also estimated using a spectrophotometer at an absorbance of 250 nm [43 (link)] using the regression equation [DOC] = 33.99 A250 + 8.16 [43 (link)–45 (link)].
Concentrations of major cations (Ca2+, Mg2+, Na+, K+) were measured by inductively coupled plasma optical emission spectroscopy (ICPOES) [46 ] and concentrations of anions (NO3−, SO42−, Cl−) were determined by ion chromatography using a Dionex ICS-2500 system [46 ] (Table1 ).
The ionic strength (µ) of each soil extract was determined via electrical conductivity (EC, mS cm−1) measurement using a calibrated conductivity electrode (TPS Glass K = 1.0 Cond Sensor) using the equation µ = EC × 0.0127 [32 (link), 41 (link), 42 (link)].
The concentration of dissolved organic carbon (DOC) in filtered soil solutions was also estimated using a spectrophotometer at an absorbance of 250 nm [43 (link)] using the regression equation [DOC] = 33.99 A250 + 8.16 [43 (link)–45 (link)].
Concentrations of major cations (Ca2+, Mg2+, Na+, K+) were measured by inductively coupled plasma optical emission spectroscopy (ICPOES) [46 ] and concentrations of anions (NO3−, SO42−, Cl−) were determined by ion chromatography using a Dionex ICS-2500 system [46 ] (Table
9
HPAEC-PAD Sugar Composition Analysis
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The sugar composition was determined by HPAEC-PAD using a Dionex ICS-2500 system.
Sugar analysis was by methanolysis followed by TFA hydrolysis using myo-inositol as internal standard as reported previously [7] .
Sugar analysis was by methanolysis followed by TFA hydrolysis using myo-inositol as internal standard as reported previously [7] .
10
Comprehensive Carbohydrate Characterization
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Total sugar content was determined by the phenolsulfuric acid colorimetric method using glucose as the standard (12) . Sulfate content was measured according to the literature (13) . Uronic acid content was evaluated by the carbazole-sulfuric acid method using glucuronic acid as the standard (14) . The homogeneity and molecular weight of LCP70S-1 were determined by gel permeation chromatography (GPC) on a Sephacryl S-300HR column (1.6 × 70 cm) with standard dextrans (T-4, T-7, T-10, T-70, T-200, and blue dextran) and glucose. The elution volumes were plotted against the logarithm of their respective molecular weights. The elution volume of LCP70S-1 was plotted in the same graph, and the molecular weight was measured (15) . The monosaccharide composition was analyzed by high-performance anion exchange chromatography (HPAEC) after hydrolization and UV detection, coupled with pulsed amperometric detection (PAD), equipped with a Carbo PAC TM PA10 (2.0 × 250 mm) column. The hydrolysate (1 mg) was dissolved in pure water (1 mL). The solution (25 mL) was used for the ionicchromatographic analysis by HPAEC-PAD of Dionex ICS-2500 system, eluted with a mixture of water and 200 mM NaOH in the volume ratio of 92:8 (16).
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