717 plus

Peptide purification was performed by semi-preparative RP-HPLC. Separation of peptides was performed on an HPLC system (Waters Corp., Mildford, MA, USA) equipped with two pumps (module Delta 600), a pump controller (module 600), an autosampler (module 717 plus), and a diode array detector (module 996). The data-processing software was Empower 2 (Waters Corp.). A 250 × 21.5 mm Hi-Pore 318 reverse phase column (BioRad, Hercules, CA, USA) was used. Peptide fractions were dissolved in solvent A (water: trifluoroacetic acid (TFA), 1000:1, v/v) at a concentration of 30 mg/mL. Fractions (400 μL) were injected and eluted at a flow rate of 10 mL/min, with a linear gradient of solvent B (acetonitrile:TFA, 1000:0.8, v/v) in A, going from 5% to 45% B in 50 min. Each chromatographic run was repeated 15–20 times and the subfractions were collected automatically with a Fraction Collector (Model II, Waters Corp.). The collection times were from min 6 to 12 for F1, min 12 to 16 for F2, min 16 to 22 min for F3, and min 22 to 35 for F4. The collected fractions were pooled, freeze-dried, and stored at −20 °C until further analysis. Quantification of peptides in each subfraction was performed by the Quantitative Colorimetric Peptide Assay, according to the manufacturer’s protocol.

For measuring the photosynthetic pigment Chl-a one liter of seawater samples were taken from Niskin bottles, immediately filtered on GF/F filters, frozen in liquid nitrogen, and stored at -80°C until further analyses by high-performance liquid chromatography (HPLC) at the home laboratory of the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research after the cruise. The samples were measured using a Waters 600 controller equipped with an auto sampler (717 plus), a photodiode array detector (2996) and the EMPOWER software. Chl-a was analyzed by reverse-phase HPLC using a VARIAN Microsorb-MV3 C8 column (4.6 3 100 mm) and HPLC-grade solvents (Merck). The solvents gradient and routine of analysis are fully described in Taylor et al. [54 ]. Chl-a concentrations were quantified based on peak area of the external standard, which was spectrophotometrically calibrated using extinction coefficients published in Jeffrey et al. [55 ].

Conventional HP-SEC analyses were performed with a Waters 515 pump (Water Inc., Milford, MA, USA), equipped with an auto-injector (Waters717plus), ultraviolet (UV) detector (Waters 2487) and differential refractometer (RI) detector (Waters 2410). Both polymeric gel based columns and silica based columns were used. In the first case, TSK G3000 PWXL (7.8 × 300 mm) and G2500 PWXL (7.8 × 300 mm) columns (Tosoh Corp.), connected in series, were eluted with 0.1 M NaNO₃ at a flow rate of 0.6 mL/min, according to HP-SEC/TDA procedure. In the second case, TSK SW guardcolumn, TSK G3000 SWXL (7.5 × 300 mm) and TSK G2000 SWXL (7.5 × 300 mm) (Supelco), connected in series, were eluted with 0.2 M Na₂SO₄, at a flow of 0.5 mL/min, according to the EP indications. In both cases, processes were aided by Chromatography Manager Software Millennium–32 (Waters), with its GPC Empower option.

Drug content (assay), entrapment efficiency (EE) and in vitro permeability samples were analyzed using high performance liquid chromatography (HPLC) method. The HPLC system comprising of Waters 717 plus auto sampler, Waters 2487 dual absorbance detector, 600 Waters controller pump, and an Agilent 3395 Integrator. Luna^® C₁₈ (4.6 mm × 250 mm) column, with a mobile phase of 1:1 isocratic solution of water and acetonitrile, at a flow rate of 1 mL/min and detection wavelength (λ_max) of 254 nm, and AUFS of 1, was used for analysis of TA [26 ].

The HPLC system used was from Breeze with a dual λ absorbance detector (Waters 2487, Massachusetts, USA) isocratic HPLC Pump (Waters 1515 Massachusetts, USA), autosampler (Waters 717 plus Massachusetts, USA). The analysis of phyllanthin (1), hypophyllanthin (2), niranthin (3), and corilagin (4) in P. amarus extract was obtained from Wu, et al. [31 (link)] and Murugaiyah and Chan [32 (link)] with modification.
Isocratic mode with a flow rate of 1.0 mL/min and an injection volume of 20 µL using Column Waters X Bridge C18 (4.6 mm × 250 mm, 5 um particle size) was used for the quantification of P. amarus. Acetonitrile:water in the ratio of 55:45 was used as a solvent system to quantify phyllanthin (1), hypophyllanthin (2), and niranthin (3) using UV wavelength of 344 nm, whereas, 1% acetic acid in acetonitrile was used as a solvent in quantifying corilagin (4) with a UV wavelength of 274 nm.

Qualitative and quantitative changes in PAH mixtures have been achieved using high-performance liquid chromatography (Waters HPLC 600E) for aromatic fractions. PAH standards purchased from Supelco CRMs for chromatography solutions (https://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=120241095) have been also subjected successively to be analyzed by high-performance liquid chromatography (HPLC). The apparatus consists of an autosampler, Waters 717 plus, attached to a computerized system with Millennium 3.2 software and a dual UV absorbance detector, Waters 2487. The separation has been performed according to the conditions mentioned in [19 (link)].

A HPLC system (Waters, Milford, MA) was applied to quantify the concentration and size distribution of the FITC‐Ficoll samples. Size separation of the plasma and urine samples was accomplished using an Ultrahydrogel 500 column (Waters) connected to a guard column (Waters). A phosphate buffer (0.15 M NaCl, pH 7.4) was used as the mobile phase, driven by a pump (Waters 1525). Fluorescence in the samples was detected at an excitation wavelength 492 nm and emission wavelength of 518 nm. Samples were loaded onto the system using an autosampler (Waters 717 plus). As is described at some length elsewhere (Asgeirsson et al., 2006), the system was calibrated using protein standards and narrow Ficoll standards. The urine FITC‐Ficoll concentration for each size (a_e) was multiplied by the urine‐to‐plasma FITC‐Inulin concentration ratio to approximate the concentration of Ficoll in primary urine (C_u). The glomerular sieving coefficient was calculated by dividing C_u by the plasma concentration of Ficoll.