Agilent 1200 series chromatographic system

The HPLC experiments were performed on: Agilent 1200 series chromatographic system encompassed with a quaternary pump, a micro vacuum degasser, a thermostatted column compartment and a variable wavelength UV–VIS detector. In addition, Agilent 1200 series autosampler was used for sample injection. Agilent ChemStation software, version A.10.01 was utilized to collect and process data. Separation was performed on Agilent Zorbax SB-C₁₈ (150 × 4.6 mm, 5 μm) column which is manufactured by Agilent Technologies (Polo Alto, CA, USA). To adjust the pH, a “Jenway 3505” pH-meter (Jenway, UK) was used.

Ten milligrams of the sample were dissolved in 1 mL of distilled water. The solution was 25 times diluted, and 50 μL of the solution were dried up in an ampoule. Then, 100 μL of 6 M HCl were added, and the ampule was sealed under vacuum. Acidic hydrolysis was performed at 110 °C for 24 h. After that, the ampoule was opened, and the solution was dried up in the Eppendorf 5301 vacuum concentrator (Eppendorf, Hamburg, Germany). Finally, 50 μL of 0.1 M HCl were added to the dried sediment.
The amino acid analysis was performed using Agilent 1200 series chromatographic system equipped with a fluorescent detector and ZORBAX Eclipse AAA (5 μm; 4.6 mm × 150 mm) column (Agilent Technologies, Santa Clara, CA, USA). The mobile phases were a 40 mM phosphate buffer solution with pH 7.8 (solution A) and 80% water solution of acetonitrile (solution B). Borate buffer with pH 10.2 and o-phtalaldehyde were used for amino acid derivatization. Each measurement was made in triplicate. See [23 (link)] for details.