Ohpak sb 804 hq

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).

HPMCs were dissolved in N,N-dimethylformamide (DMF) (0.3 g/30 mL) and mixed with 1.5 mg of fluorescein-5-carbonyl azide diacetate. The mixtures were purged with N₂ gas and agitated at 90°C for 3 h. Excess fluorescein reagent was removed by repeated precipitation and solubilization steps; briefly, labeled HPMCs were recovered by the addition of hexane to the reaction mixture, and residual solids were redissolved in DMF. The resultant labeled HPMCs were dissolved in aqueous NaHCO₃ solution (100 mM, pH 8.0) at room temperature for 8 h for the deprotection of acetyl groups according to the manufacturer’s instructions. The resulting solution was dialyzed against deionized water on an acetyl cellulose membrane (MW cut-off: 3,500 Da) for 2 days. Fluorescein-labeled HPMC solutions were filtered through a 0.45-μm-pore filter and lyophilized. No fluorescent signal was detected in low molecular weight fractions by gel permeation chromatography (GPC) analysis using a fluorescence detector (column, OHPak SB-804 HQ [Showa Denko, Tokyo, Japan]; detection, excitation at 494 nm and recording at 520 nm).

The purified RTP1S_C2-strep and RTP1S_C3-Strep were analyzed using an HPLC system with OH-Pak SB-804HQ (Showa Denko, Tokyo, Japan). An aliquot (100 μl) of the RTP1S solution (0.3 mg/ml) was injected onto the column with buffer (20 mm phosphate buffer, pH 8.0, 100 mm NaCl). Measurement of molecular mass was carried out under the same conditions by SEC-MALS using a multiangle light-scattering detector (mini-DAWN DSP, Wyatt Technology) and a differential refractive index detector (Shodex RI-71, Showa Denko).