Each 10 mL fraction was collected by a fraction collector, and 180 µL of each fraction was neutralized with 10 µL of 1 M HCl and stained with 10 µL of 1 % KI / 0.1 % I2. Starch in each fraction (#1-6 in Fig. 1 ) was precipitated with three volumes of ethanol at －30 °C overnight. Precipitates were washed with 70 % ethanol and dried. α-glucans of each fraction were debranched at 37 °C for 24 h by 354 U of isoamylase derived from Pseudomonas amyloderamosa (Hayashibara Co., Ltd., Okayama, Japan) according to the previous report.17) (link) GPC analysis of debranched starch was performed using a Toyopearl HW55S column and three columns of HW50S connected in series (2.2 cm diameter × 30 cm each; Tosoh Corp.) following the method reported by Toyosawa et al.18) (link) Chain length distribution analysis of each fraction was performed by fluorophore-assisted capillary electrophoresis (FACE) with a P/ACE MDQ Carbohydrate System (Sciex LLC, Framingham, MA, USA) according to the methods of the previous reports.17) (link)19) The difference in chain length distribution was expressed as molar change (%), equivalent to the rate of molar change of each chain (%) in Fujita et al.,20) (link) and was calculated for each DP as the differences in molar (%) at each DP value vs. that of Fraction #1. Differences are expressed by the percentage of the molar % value of a given DP for Fraction #1.
Hw50s
Manufactured by Tosoh
Sourced in Japan, United States
The HW50S is a high-performance liquid chromatography (HPLC) system designed for analytical applications. It features a compact and modular design, providing efficient and reliable performance for various separation and detection techniques. The HW50S is capable of handling a wide range of sample types and is suitable for a variety of laboratory environments.
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Lab products found in correlation
2 protocols using hw50s
1
Analyzing Starch Fractions by GPC and FACE
2
Starch Characterization in Rice Endosperm
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Starch was extracted from mature rice endosperm to assess the amylopectin chain-length distribution according to the method of Fujita et al. (2001) (link). The chain-length distribution of endosperm starch was analysed by capillary electrophoresis (O’Shea and Morell, 1996 (link)) using the P/ACE MDQ Carbohydrate System (Beckman Coulters, CA, USA).
Gel filtration chromatography of starches (from mature and developing endosperm) and amylopectin (from mature endosperm) was performed as described previously (Fujita et al., 2007 (link), 2009 (link)) using a Toyopearl HW55S gel filtration column (300×20mm) connected in series to three Toyopearl HW50S columns (300×20mm) equipped with a refractive index (RI) detector (Tosoh RI-8020).
Estimation of starch content in rice seeds and determination of the amylopectin molecular weight were performed by HPSEC-MALLS-RI according to the method of Fujita et al. (2003) (link). Purified starch granules were coated with gold using a fine coater (JEOL JFC-1200) for 120 s. Starch granule morphology was examined by scanning electron microscopy (SEM; JEOL-500, Tokyo, Japan). SEM was performed in a secondary electron mode at 15kV according to the method of Fujita et al. (2003) (link). Observation of iodine-stained endosperm thin sections was performed according to the method of Matsushima et al. (2010) (link).
Gel filtration chromatography of starches (from mature and developing endosperm) and amylopectin (from mature endosperm) was performed as described previously (Fujita et al., 2007 (link), 2009 (link)) using a Toyopearl HW55S gel filtration column (300×20mm) connected in series to three Toyopearl HW50S columns (300×20mm) equipped with a refractive index (RI) detector (Tosoh RI-8020).
Estimation of starch content in rice seeds and determination of the amylopectin molecular weight were performed by HPSEC-MALLS-RI according to the method of Fujita et al. (2003) (link). Purified starch granules were coated with gold using a fine coater (JEOL JFC-1200) for 120 s. Starch granule morphology was examined by scanning electron microscopy (SEM; JEOL-500, Tokyo, Japan). SEM was performed in a secondary electron mode at 15kV according to the method of Fujita et al. (2003) (link). Observation of iodine-stained endosperm thin sections was performed according to the method of Matsushima et al. (2010) (link).
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