1260 series liquid chromatograph

Glucose decanoate was determined by HPLC using a Kinetex EVO C18 (2.6 μm, 250 mm × 4.6 mm) column from Phenomenex (Aschaffenburg, Germany) with an accompanying guard column (4 mm × 3.0 mm ID) of the same phase, using an Agilent 1260 series liquid chromatograph (Waldbronn, Germany) equipped with a quaternary pump, an autosampler and a column oven. An evaporative light scattering detector (ELSD) from BÜCHI Labortechnik (Essen, Germany) was used for detection. The mobile phase, solvent A, was water and solvent B was acetonitrile. The flow rate was 1 mL/min and a gradient was used for separation of products and substrates: starting from 40% A-60% B, then 0–10 min a linear gradient up to 35% A-65% B, followed by another linear gradient from 10 to 15 min up to 25% A-75% B. This gradient was held for 5 min, followed by a reconditioning step of the column with 40% A-60% B for 5 min. The injection volume was set to 10 μL. The column was operated at 50°C. The detector was operated at 38°C with a gas flow (air) of 1.5 mL/min. The gain was set to 1.

The phenolic profile of Alhagi graecorum ethanolic extract was determined using High-Performance Liquid Chromatography (HPLC). HPLC analysis was carried out according to Kim et al. (2006 (link)) using Agilent Technologies 1260 series liquid chromatograph equipped with an auto-sampler and a diode-array detector. The separation was carried out using Eclipse XDB-C18 (4.6 mm × 250 mm i.d., 5 μm) with a C18 guard column (Phenomenex, Torrance, CA). The mobile phase consisted of water (A) and 0.05% tri-fluoro-acetic acid in acetonitrile (B) at a flow rate 1 mL/min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (82% A); 0–5 min (80% A); 5–8 min (60% A); 8–12 min (60% A); 12–15 min (82% A) and 15–16 min (82% A). The multi-wavelength detector was monitored simultaneously at 280, 320, and 360 nm. The injection volume was 10 μL for each of the sample solutions and peaks were monitored simultaneously at 280, 320, and 360 nm. The column temperature was set during the separation process to 35 °C.

HPLC analysis was performed according to Hollenbach et al., using a Kinetex EVO C18 (2.6 µm, 250 × 4.6 mm) from Phenomenex (Aschaffenburg, Germany) with an accompanying guard column (4 x 3.0 mm ID) of the same phase using an Agilent (Waldbronn, Germany) 1260 series liquid chromatograph equipped with a quaternary pump, an autosampler and a column oven [49 (link)]. For detection, an evaporative light scattering detector from BÜCHI Labortechnik (Essen, Germany) was used. Mobile phase was a gradient of acetonitrile (A) and water (B) with a total flow rate of 1mL/min. This method reliably separates monoesters from substrates and by-products such as diesters [49 (link)]. Only products with a purity of at least 95% determined by the area % of the HPLC chromatograms were used for further investigations.

Modified and unmodified MS2 CP variants
were analyzed with an Agilent 1260 series liquid chromatograph connected
in-line with an Agilent 6530 LC/QTOF mass spectrometer with an electrospray
ionization source. The expected mass of each MS2 CP variant was confirmed
via QTOF-ESI-MS (Figure S1).