4.4. Peptide Analysis by High-Performance Liquid Chromatography (HPLC)

The molecular mass distributions of extracts were performed in a modular Agilent Technologies liquid chromatograph (Agilent Technologies 1260 Infinity, USA, CA) with a UV detection at 280 nm. All samples were prepared twice and measured by duplicate. A TSK gel G 3000PWXL column, 7.8 mm I.D. × 30 cm (TOSOH Corporation, Tokyo, Japan) at a flow-rate of 0.3 mL/min and a temperature of 25 °C were used. The mobile phase consisted of 0.1 N NaCI 20 mM Tris-HCI buffer, pH 7.8. The approximate molecular weight (MW) was determined using standard protein samples (Sigma-Aldrich Co., USA, MO) as reference: bovine serum albumin (MW 66.3 kDa), egg albumin (MW 44.3 kDa), myoglobin (MW 18.0 kDa) cytochrome C (MW 12.4 kDa), aprotinin (MW 6.5 kDa), bacitracin (MW 1.4 kDa). All samples were filtered through 0.2 µm syringe filter (Whatman, PVDF) before injection. The MW of peptides was calculated by the elution time.