Peptide Molecular Weight Analysis by HPLC

The molecular weight (MW) distribution of peptides was examined on a Waters e2695 HPLC system equipped with a TSKgel G2000 SW_XL column (7.8 mm × 300 mm, Tosoh, Tokyo, Japan) and a TSKgel SW_XL guard column (6.0 mm × 40 mm, Tosoh, Tokyo, Japan). The chromatographic conditions were as follows, according to Zhang et al. [17 (link)]: the eluent was 45% acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA), the flow rate was 0.5 mL/min, the injection volume was 10 μL and the absorbance was monitored at 220 nm. The standards used for the molecular weight calibration were as follows: HHL (429 Da), bacitracin (1422 Da), aprotinin (6.5 kDa) and cytochrome C (12.4 kDa).