Agilent 1100 series sec system

The structure of extracted whole starch and leached starch molecules was characterized by SEC using an Agilent 1100 Series SEC system (Agilent Technologies, Waldbronn, Germany) equipped with GRAM 30 and 3000 analytical columns (PSS) and a refractive index (RI) detector (RID-10A, Shimadzu Corp., Kyoto, Japan), following a method described elsewhere46 (link)47 . The molecular size distribution of branched starch was plotted as the SEC weight distribution, w_br(logR_h). For branched starch molecules, as for any branched polymer, there is no unique relation between size and the molecular weight. For the debranched samples, which are linear, the relation between R_h and molecular weight M was obtained as follows. The assumption of universal calibration for SEC is that the elution time of the analyte depends only on its R_h and not on its structure, whence one has for two linear polymers, a sample and a standard, the relation:

where K and α are the Mark-Houwink parameters for the polymer, solvent and temperature being used. Pullulan standards with known peak molecular weights were used for calibration to obtain a relationship between SEC elution volume and R_h of starch molecules following the Mark-Houwink equation:

Here N_A is Avogadro’s constant. The Mark-Houwink parameters K and α of pullulan in DMSO/LiBr solution at 80 °C are 2.424 × 10⁻⁴ dL g⁻¹ and 0.68, respectively46 (link).

According to Zhou et al. and Zhang et al., the chain length distributions of amylopectin were quantitatively analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Dionex-ICS 3000; Dionex Corporation, Sunnyvale, CA, USA) for DP6-90, and the Agilent 1100 Series SEC system was used with GRAM precolumn, GRAM 100 and GRAM 1000 columns (PSS, Mainz, Germany) for measurement of longer chain distributions [46 (link),47 (link)]. To obtain the chain length distributions (CLDs) of debranched starch molecules, the starch samples (5 mg) were debranched using isoamylase (I5284; Sigma, Louis, USA) before analysis.

The CLD of debranched starch was analyzed by SEC following the method of Li et al. (2011 (link)) Debranched starch (2 mg) was dissolved in DMSO with 0.5 % (w/w) LiBr for 2 h at 80 °C. This solution was analyzed with an Agilent 1100 Series SEC system (Agilent Technologies, Waldbronn, Germany) equipped with GRAM 100 and GRAM 1000 analytical columns (Polymer Standards Service (PSS), Mainz, Germany) set at 80 °C and a differential refractive index (DRI) detector (Wyatt, Santa Barbara, CA, USA). DMSO containing 0.5 % w/w LiBr was used as the eluent and the flow rate was 0.6 mL min⁻¹. Calibration was with two pullulan standards of molecular weight 2.35 × 10⁶ and 1.22 × 10⁶; while the small range offered by these standards means that the conversion of elution volume to molecular weight (or DP), implemented by methods given elsewhere (Vilaplana and Gilbert 2010 (link)), will be inaccurate, use of even this limited calibration does mean that the data are reproducible. The results were collected and analyzed with Astra software (Wyatt) using a dn/dc value of 0.0717 mL g⁻¹ (Li et al. 2011 (link); Syahariza et al. 2013 (link)).