Dawn heleos 2 detector

Bio-oil samples were solubilized at 1 mg mL⁻¹ in tetrahydrofuran (THF). GPC analysis using RI and UV-DAD (or referred to as simply UV) was performed using an Agilent 1200 HPLC with 3 GPC columns (Agilent, 300 × 7.5 mm) packed with 10 μm polystyrene-divinyl benzene copolymer gel beads with nominal pore diameters of 10⁴, 10³ and 50 Å. The UV-DAD measured absorbance at 270 nm (80 nm bandwidth). An injection volume of 20 μL was used with an eluent (THF) flow rate of 1.0 mL min⁻¹ for a total run time of 45 min. Polystyrene standards (Agilent Technologies) were used to calibrate for relative M_w and M_n, with assumptions of Mark–Houwink parameters for relative comparison only. GPC analysis using MALS coupled with differential RI and viscometry analysis was performed using the same chromatographic method and columns described for UV detection. A Wyatt DAWN HELEOS II detector with a 785 nm MALS was used with a Wyatt ViscoStar III viscometer and Wyatt Optilab T-rEX differential refractometer (RI) with 785 nm LED. Data were collected and processed using ASTRA 7 for molecular weight analysis.

Size exclusion chromatography (SEC) followed by multi-angle laser light scattering (MALLS) and refractometry (RI) analysis allow the determination of the molecular mass of a protein or a complex in solution that is independent of its dimensions and shape [47 (link)]. SEC was performed with a column (Superdex^TM 200 increase 10/300 GL or Superdex 75 10/300 GL) equilibrated with 10 mM Hepes pH 7.5, 150 mM NaCl. Analytical runs were performed at 20 °C with a flow rate of 0.5 mL·min⁻¹. Multi-angle laser light scattering (MALLS) detection was performed with a DAWN-HELEOS II detector (Wyatt Technology, Toulouse, France) using a laser emitting at 690 nm and protein concentration was measured on-line with the use of differential refractive-index measurements, with an Optilab T-rEX detector (Wyatt Technology) and a refractive-index increment, dn/dc of 0.185 mL·g⁻¹. Data were analyzed and weight-averaged molar masses were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA, USA).

SEC combined with online detection by MALLS, refractometry, and UV-Vis was used to measure the absolute molecular mass in solution. The SEC runs were performed using a Superose^TM 6 increase 10/300 column (GE Healthcare/Cytiva) equilibrated in 0.025 M Tris–Base, 0.137 M NaCl, and 0.003 M KCl, pH 7.4. Protein sample of 50 μl, concentrated to about 1 mg/ml, was injected with a constant flow rate of 0.5 ml/min, and separation was performed at room temperature. Online MALLS and differential refractive index detection were performed using a DAWN-HELEOS II detector (Wyatt Technology Corp.) with a laser emitting at 690 nm and an Optilab T-rEX detector (Wyatt Technology Corp.), respectively. Weight-averaged molar mass determination was performed with ASTRA6, using the “protein conjugate” module. The following refractive index increments and UV-Vis absorbance values were used: dn/dc protein = 0.185 ml/g; dn/dc glycosylation = 0.15 ml/g; A₂₈₀ = 1.38 ml/mg.cm.

SEC-MALS was performed using a Superdex 200 Increase 10/300 Gl column (Cytiva) connected to an AKTA pure FPLC system (Cytiva). A DAWN HELEOS II detector (Wyatt) and an Optilab TrEX differential refractometer (Wyatt) were used to estimate the molecular weight of MRS2_58–333 under various experimental conditions. The entire in-line FPLC/MALS system was housed in cold cabinet maintained at ∼10°C. Data were obtained for four different protein concentrations: 0.45 mg/ml, 0.90 mg/ml, 2.5 mg/ml, and 5 mg/ml in 20 mM Tris (pH 8), 150 mM NaCl, and 1 mM DTT, using 100 µl injections of sample at each concentration. MALS molecular weights were determined in the accompanying ASTRA software (version 7.1.4; Wyatt) using Zimm plot analysis and a protein refractive index increment (dn/dc) = 0.185 L/g. Divalent cation containing experiments were performed by supplementing the running buffers and protein samples with 5 or 10 mM MgCl₂ and CaCl₂, as indicated.