Chemstation for lc systems software

For optimization of chromatographic conditions, the effects of various method parameters such as mobile phase, column, flow rate and solvent ratio, and detection system were evaluated, and the chromatographic parameters such as asymmetric factor, resolution, and column efficiency were calculated. The best results were obtained with a mixture of 10 mM phosphate buffer, pH 6.8, and acetonitrile, in a gradient phase mobile composition obtained using a gradient program (Table 1) at 0.7 mL/min. Chromatograms were recorded at 273 nm with a run time of 15 min.
At the end of each work day, the column was washed with acetonitrile : water (90 : 10 v/v) during 30 min. Chromatographic data were processed using Chemstation for LC systems software (Agilent Technologies).

HPSEC chromatograms of tested alginates and SWE were performed in a 1260 series Hewlett-Packard chromatograph (Agilent, Waldbornn, Germany) with a refractive index detector. The sample was injected with autosampler and the volume was 5 µL. Two columns 300 × 7.8 mm (TSKGel G2500PW_XL and TSKGel G3000PW_XL, Tosoh Bioscience, Zürich, Germany) in series and one 40 × 6 mm PWX-guard column were used as measuring systems. Measurements were run using Milli Q water (0.4 mL/min) as mobile phase and dextrans (1000–80,000 g/mol) (Fluka, St. Louis, MO, USA) as calibration standards. The corresponding molar mass distribution chromatograms were recorded using the ChemStation for LC systems software (Agilent Technology, Germany).

Purity of collected peptides was checked by RP-HPLC on an analytical column Luna C₁₈ 100 × 2.0, 3 µm, 100Å (Phenomenex, Torrans, CA, USA). Mobile phase was the same as the previous experiments. Analysis was carried out at a flow rate of 0.27 mL/min in linear gradient of Solvent B (5–50% for 10 min; 50–75% for 3 min; 75–90% for 1 min; 90% for 3 min; 90–5% for 3 min, after that the column was re-equilibrated with 5%B for 4 min). Purity was estimated using the ChemStation for LC systems software (Agilent Technologies, Santa Clara, CA, USA).