Refractive index detector

As described by Zhang et al. (14 (link)), the molecular structure was analyzed by determining the molecular weight distribution. Completely dissolved solution (starch samples/dimethyl sulfoxide was 2 mg/mL, 90°C, 24 h) was evaluated with an absolute molecular weight analysis system including multi-angle laser light-scattering detector (Wyatt Technology Corporation, Santa Barbara, CA, USA), refractive index detector (Wyatt Technologies), and high-performance size-exclusion chromatography (Wyatt Technology Corporation). The guard column, Shodex OHpak SB-804 HQ and Shodex OHpak SB-806 HQ (Showa Denko K.K., Tokyo, Japan) Phenogel columns were used. The column temperature was 60°C, and flow rate of the dimethyl sulfoxide mobile phase was 0.3 mL/min. The sample injection consisted of 100 μL. Data obtained using this system were analyzed with Astra software (version 5.3.4, Wyatt Technology).

Molecular weights of RP, RPS, RPG, RPI, RPI-6 h, RPI-12 h, RPI-24 h, and RPI-48 h were measured by size exclusion chromatography collected with a multi-angle laser light scattering detector and a refractive index detector (Wyatt Technology Co., Santa Barbara, CA, USA), as previously reported [18 (link)]. The Shodex OHpak SB-806 M HQ (300 mm × 8.0 mm, i.d.) column was used, and the mobile phase was 0.9% of NaCl solution. Additionally, infrared spectra of RP, RPS, RPG, RPI, RPI-6 h, RPI-12 h, RPI-24 h, and RPI-48 h were measured by a Nicolet iS 10 FT-IR (Thermo Fisher Scientific, Waltham, MA, USA) as previously reported [21 (link)]. Besides, the esterification degree (DE) values of RPs were also calculated based on the absorption bands around 1741 cm⁻¹ and 1629 cm⁻¹.

SEC-multi-angle light scattering experiments were conducted using an AKTA™ pure HPLC system (GE Healthcare, USA) connected in-line with a DAWN HELEOS II (Wyatt Technology, Santa Barbara, CA, USA) eight-angle light-scattering detector, followed by a refractive-index detector (Wyatt Technology, Santa Barbara, CA, USA). SEC-MALS system was equilibrated with the corresponding running buffer at 0.5 mL/min for 12 h prior to the sample loading. A series of PrpA, PrpA^2–54, and PrpTA samples were prepared in several kinds of buffers (including 50 mmol/L tris–HCl and 100 mmol/L NaCl pH 8.0, 50 mmol/L tris–HCl and a300 mmol/L NaCl pH 8.0, 50 mmol/L tris–HCl and 500 mmol/L NaCl pH 8.0, 50 mmol/L MES and 500 mmol/L NaCl pH 5.5, and 100 Mm (NaH²PO⁴/Na²HPO⁴) pH 8.0), and 0.1 ml was injected into the loop for each dilution. Accordingly, the absolute molar mass of each protein sample could be determined based on the data processed by the ASTRA (v7.0.1) offered by Wyatt company.

The method referred to the literature adopted high performance size exclusion chromatography coupled with multi angle laser light scattering and refractive index detector (Wyatt Technology Co., Santa Barbara, CA, United States ) to detect the molecular weight of CLRP-1 and CLSP-1 (Wu et al., 2016 (link)). ShodexOHpak SB-806 M HQ (300 mm × 8.0 mm, id) column was used at 30°C to separated sample. The mobile phase was 0.9% NaCl aqueous solution at a flow rate of 0.5 ml/min. The injection volume was 100 μL. Data acquisition and analysis adopted Astra software (version 7.1.3, Wyatt Technology Co., Santa Barbara, CA, United States).