Hpaec pad

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The HPAEC-PAD (High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection) is an analytical technique used for the separation, identification, and quantification of carbohydrates and other ionic compounds. It utilizes a combination of high-performance liquid chromatography and electrochemical detection to provide sensitive and precise analysis of these analytes.

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HPAEC (35 citations) HPAEC-PAD) system (10 citations) HPAEC-PAD ICS-5000 (10 citations) ICS-3000 HPAEC-PAD system (3 citations) DX 500 HPAEC-PAD system (2 citations) ICS-3000 HPAEC-PAD (2 citations) ED50 Detector (HPAEC-PAD) (2 citations) HPAEC-PAD (ICS 5000 system; (2 citations)

55 protocols using hpaec pad

Monosaccharide and Oligosaccharide Analysis

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Monosaccharide analysis. d-Glucose was measured enzymatically by the coupled GOD/POD assay, as described previously [20 (link)]. For the determination of d-galactose, the lactose/d-galactose test kit from Megazyme was used.
Oligosaccharide analysis. Capillary electrophoresis (CE) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex, Sunnyvale, CA, USA) were used for the qualitative and quantitative analysis of galacto-oligosaccharides. A capillary electrophoresis system with a UV-DAD detector (Agilent Technologies, Palo Alto, CA, USA) together with a fused silica capillary (internal diameter of 25 µm) equipped with a bubble cell detection window (bubble factor of five) was used for carbohydrate analysis. Carbohydrate samples were derivatized with 2-amino pyridine for CE analysis, as given in detail in [20 (link)]. HPAEC-PAD analysis was carried out on a Dionex DX-500 system consisting of a GP50 gradient pump (Dionex), an ED 40 electrochemical detector with a gold working electrode (Dionex), and an Ag/AgCl reference electrode (Dionex). Separations were performed at room temperature on a CarboPac PA-1 column (4 × 250 mm) connected to a CarboPac PA-1 guard column (Dionex).

Kittibunchakul S., Maischberger T., Domig K.J., Kneifel W., Nguyen H.M., Haltrich D, & Nguyen T.H. (2018). Fermentability of a Novel Galacto-Oligosaccharide Mixture by Lactobacillus spp. and Bifidobacterium spp.. Molecules, 23(12), 3352.

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Comprehensive Characterization of Apple Pomace

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Protein contents were analyzed using the Kjeldahl approach with ammonia being detected by an ammonia sensitive electrode (nitrogen conversion factor 6.25) [21] (link). Ash contents were determined after incineration for 4 h at 500 ℃. Starch was analyzed quantitatively following degradation into its glucose monomer units [12] (link). In brief, after enzymatic hydrolysis using a thermostable α-amylase and amyloglucosidase, liberated glucose was determined by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Thermo Fisher Scientific, Waltham, Massachusetts). Starch was calculated as the sum of anhydroglucose. Mono-and disaccharides were determined after aqueous extraction with ultrasonic treatment (four times) using HPAEC-PAD (Thermo Fisher Scientific, Waltham, Massachusetts) as described in detail in [12] (link). pH was measured with a pH electrode (500 mg of sample in 5 mL of water). To compare data, the pH of recently analyzed apple pomace and its extrudates [12, (link)13] (link) were also recorded (Table S2).

Trabert A., Schmid V., Keller J., Emin M.A, & Bunzel M. (2022). Chemical composition and technofunctional properties of carrot (Daucus carota L.) pomace and potato (Solanum tuberosum L.) pulp as affected by thermomechanical treatment. European food research and technology = Zeitschrift fur Lebensmittel-Untersuchung und -Forschung. A, 248(10).

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Enzymatic Hydrolysis of Galactomannans

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5.5 μmBoMan26B was incubated with 0.5% (w/v) LBG or guar gum and 1 mm CaCl₂ in 50 mm potassium phosphate buffer, pH 6.5, at 37 °C in duplicate for 24 h before boiling for 10 min. 50 μl of the reaction mixture was re-equilibrated to 37 °C before adding 0.5 units of guar α-galactosidase (Megazyme, Bray, Ireland) (1 unit is the amount of enzyme required to release 1 μmol of product/min), incubated for another 24 h at 37 °C, before boiling for 10 min. The samples containing hydrolysis products with or without treatment with guar α-galactosidase were analyzed by HPAEC-PAD with CarboPac PA-200 and PA-20 columns (Dionex, Sunnyvale).

Bågenholm V., Wiemann M., Reddy S.K., Bhattacharya A., Rosengren A., Logan D.T, & Stålbrand H. (2019). A surface-exposed GH26 β-mannanase from Bacteroides ovatus: Structure, role, and phylogenetic analysis of BoMan26B. The Journal of Biological Chemistry, 294(23), 9100-9117.

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Comprehensive Analysis of Rice Starch and Protein

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The total starch content of the rice was determined using an assay kit (Megazyme International Ireland, Ireland). Apparent amylose content was determined by iodine colorimetry (Juliano et al., 1981). The chain length distribution of amylopectin in starches isolated from the seven rice varieties tested was analyzed by high‐performance anion‐exchange chromatography on a CarboPac PA‐1 column equipped with a pulsed amperometric detector (HPAEC‐PAD, Dionex, USA). The sample solution was prepared using the method described by Nagamine and Komae (1996). After isoamylase debranching, the sample solution was eluted at 1 ml/min with a linear gradient of 50–1000 mM sodium acetate in 100 mM NaOH. Amylopectin branch chain types were classified as follows: DP6‐12 (A chain), DP13‐24 (B1 chain), and DP25‐36 (B2 chain) (Hanashiro, Abe, & Hizukuri, 1996). Protein content (N × 5.95) was determined using the Kjeldahl method.

Sasaki T., Matsuki J., Yoza K., Sugiyama J., Maeda H., Shigemune A, & Tokuyasu K. (2019). Comparison of textural properties and structure of gels prepared from cooked rice grain under different conditions. Food Science & Nutrition, 7(2), 721-729.

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Efficient Synthesis of Phosphorylated Heptasaccharide

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To a solution of the free heptasaccharide 14 (7.4 mg, 5.8 μmol) in H₂O (1 mL) were added Et₃N (45 μL, 0.32 mmol) and 2-chloro-1,3-dimethylimidazolinium chloride (DMC) (17.5 mg, 0.11 mmol) at 0 °C. The reaction mixture was monitored by DIONEX HPAEC-PAD. Within 1 h, the HPAEC analysis indicated that the free oligosaccharide was converted into a new oligosaccharide that eluted earlier than the reducing sugar under the HPAEC conditions. The product was purified by gel filtration on a Sephadex G-10 column that was eluted with 0.1% aq Et₃N to afford compound 15a (6.9 mg, 88%) after lyophilization together with NaOH (0.5 mg). HR-MS (ESI-TOF): [M – H]⁻ calcd for C₄₄H₇₄NO₃₈P, 1254.3554; found, 1254.3537. ¹H NMR (D₂O, 500 MHz): δ 6.10 (d, 1H, J = 7.2 Hz), 5.35 (s, 1H), 5.15 (s, 1H), 5.05 (s, 1H), 4,93 (s, 1H), 4.91 (s, 1H), 4.73 (s, 1H), 4.37 (1H, s), 4.21–3.67 (m, 39H), 3.58 (m, 1H), 3.43 (m, 1H), 2.08 (s, 3H). ¹³C NMR (D₂O, 125 MHz): δ 168.73, 102.69, 102.42, 101.41, 100.86, 100.17, 99.90, 99.50, 80.26, 79.02, 78.80, 78.57, 78.04, 74.46, 73.46, 72.87, 71.15, 71.05, 70.72, 70.54, 70.34, 70.26, 70.16, 70.03, 69.63, 69.44,67.14, 66.96, 66.71, 66.26, 66.09, 65.94, 65.49, 65.25, 63.26, 61.81, 61.17, 13.12 (selected peaks). ³¹P NMR (D₂O, 162 MHz): δ 4.51.

Yamaguchi T., Amin M.N., Toonstra C, & Wang L.X. (2016). Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition. Journal of the American Chemical Society, 138(38), 12472-12485.

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Chemoselective Tetrasaccharide Derivatization

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To a solution of the free tetrasaccharide (26) (20.0 mg, 0.023 mmol) in H₂O (2 mL) were added Et₃N (145 μL, 1.04 mmol) and 2-chloro-1,3-dimethylimidazolinium chloride (DMC) (58.5 mg, 0.35 mmol) at 0 °C. The reaction mixture was monitored by DIONEX HPAEC-PAD. Within 1 h, the HPAEC analysis indicated that the free oligosaccharide was converted into a new oligosaccharide that eluted earlier than the reducing sugar under the HPAEC conditions. The product was purified by gel filtration on a Sephadex G-10 column that was eluted with 0.1% aq Et₃N to afford compound 27 (21.7 mg, 97%) after lyophilization together with NaOH (2.8 mg). HR-MS (ESI-TOF): [M – H]⁻ calcd for C₂₆H₄₅NO₂₆P₂, 848.1632; found, 848.1664. ¹H NMR (D₂O, 500 MHz): δ 6.10 (d, 1H, J = 7.0 Hz), 5.10 (s, 1H), 4.95 (s, 1H), 4.90 (s, 1H), 4.79 (m, 1H), 4.39 (s, 1H), 4.21 (1H, s), 4.11–4.05 (m, 3H), 4.01–3.65 (m, 18H), 3.58 (m, 1H), 3.43 (1H, m), 2.08 (s, 3H). ¹³C NMR (D₂O, 125 MHz): δ 161.68, 102.81, 100.47, 100.07, 90.62, 80.81, 80.46, 80.13, 74.17, 72.89, 72.25, 70.16 69.15, 66.17, 65.91, 63.24, 62.89, 60.27, 53.80, 22.10 (selected peaks). ³¹P NMR (D₂O, 162 MHz): δ 4.00 (overlapped signals).

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Quantifying Biomass Sugar and Lignin

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The total sugar in processed ball milled biomass was quantified in the hydrosylate after acid hydrolysis [38 ]. 30 mg of dried ball milled biomass was weighed and subjected to a two stage acid hydrolysis initially with 12 M sulphuric acid for 1 hour at 37°C followed by 1 M sulphuric acid for 2 hours at 100°C. The monosaccharide analysis was performed on fully acid hydrolysed residues and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex, UK) using a CarboPac PA20 column with 50 mM NaOH isocratic system at working flow rate of 0.5 ml/min at 30°C. Glucose, xylose, arabinose and galactose were used as standards with mannitol as an internal standard. The acetyl bromide method was performed to quantify lignin in ball milled biomass [39 (link)]. The details of the lignin analysis method are the same as described previously [5 (link)]. The sugar and lignin analyses were performed using three technical replicates.

Liaud N., Giniés C., Navarro D., Fabre N., Crapart S., Gimbert I.H., Levasseur A., Raouche S, & Sigoillot J.C. (2014). Exploring fungal biodiversity: organic acid production by 66 strains of filamentous fungi. Fungal Biology and Biotechnology, 1, 1.

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Gelatinization and Debranching of α-Glucan Samples

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Samples were gelatinized by boiling and enzymatically debranched by using 0.24U of isoamylase per 5 mg of sample at 40 °C. A 20 μL sample aliquot of such linearized α‐glucan (100 μg of linear α‐glucan) was analysed by high‐pressure anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD, Dionex, Sunnyvale, CA) as described (Blennow et al., 1998).

Blennow A., Skryhan K., Tanackovic V., Krunic S.L., Shaik S.S., Andersen M.S., Kirk H, & Nielsen K.L. (2020). Non‐GMO potato lines, synthesizing increased amylose and resistant starch, are mainly deficient in isoamylase debranching enzyme. Plant Biotechnology Journal, 18(10), 2096-2108.

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Xylan Hydrolysis by TlXyn11B Enzyme

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One milliliter of citric-Na₂HPO₄ buffered beechwood xylan (pH 3.5) was treated by 2 U of TlXyn11B at 50 °C overnight. The enzyme was then removed from the reaction system through the Nanosep centrifugal 3 K device (Pall, New York, NJ). Aliquots (100 μL) of 100-fold diluted hydrolysate samples were analyzed by high-performance anion-exchange chromatography (HPAEC-PAD) (Dionex, Sunnyvale, CA) equipped with a pulsed amperometric detector ICS-5000, a CarboPac PA100 guard column (4 × 50 mm) and an analytical column (4 × 250 mm). The xylo-oligosaccharides were eluted by 100 mM NaOH at the rate of 1 mL/min and at the temperature of 22 °C. Xylose, xylobiose, xylotriose, xylotetrose, xylopentose and xylohexose were used as the standards. Each experiment was repeated three times.

Wang X., Ma R., Xie X., Liu W., Tu T., Zheng F., You S., Ge J., Xie H., Yao B, & Luo H. (2017). Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering. Scientific Reports, 7, 15287.

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Quantification of Fruit Sugars by HPAEC-PAD

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For quantification of soluble sugars, 200 mg fresh weight of powdered fruit pericarp was extracted with 1 mL ethanol 80% (v/v) four times as described by [48 (link)]. Approximately 1 mL of the extract was vacuum dried in a SpeedVac system and resuspended in 1 mL of ultra-pure water. Glucose, fructose and sucrose were quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Dionex, Sunnyvale, CA, USA) using a Carbopac PA1 column (250 x 4 mm, 5 μm particle size, Dionex) in an isocratic run with 18 mM NaOH as mobile phase. Content of each sugar was calculated using standard curves made with pure glucose, fructose and sucrose. Total soluble solids were measured in ripe fruits with a refractometer DR201-95 (Kruss).

Lupi A.C., Lira B.S., Gramegna G., Trench B., Alves F.R., Demarco D., Peres L.E., Purgatto E., Freschi L, & Rossi M. (2019). Solanum lycopersicum GOLDEN 2-LIKE 2 transcription factor affects fruit quality in a light- and auxin-dependent manner. PLoS ONE, 14(2), e0212224.

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