High performance liquid chromatography system

The phosphorus content of cells was determined as the total phosphorus concentration in the fresh medium divided by the biovolume concentration of the culture at sampling, assuming that, under P-limitation and axenic conditions, all phosphorus was incorporated into the cells. Biovolume was measured with a cell counter (CASY, Modell TTC, Schärfe System, Germany). Carbon content was measured with a C/N analyser (Vario EL, Elementar Analysensysteme, Hanau, Germany) after collecting cells on pre-rinsed, pre-fired and pre-weighed filters (Munktell 25 mm). Chlorophyll a was measured for each culture at each dilution rate and experimental treatment with a Waters Alliance (Milford, USA) high performance liquid chromatography (HPLC) system with photodiode array detector, according to the method of Shatwell et al. [38] . In addition, each time a culture was sampled, chlorophyll content was measured using the chlorophyll fluorescence, F_o, calibrated against the HPLC measurements. Chlorophyll content for each culture was then taken as the mean of the HPLC and fluorescence measurements.

The encapsulation efficiency of HCPT was determined by HPLC system after dissolving 1 mg/mL HANP/HCPT in distilled water (DW) and diluting with 100x the volume of the mobile phase. The drug was assayed using a Waters high-performance liquid chromatography (HPLC) system combined with a separation module, a fluorescence detector, and a reverse-phase C-18 column (5 μm, 120 Å, 250 mm × 4.6 mm) using 5 to 65% acetonitrile containing 0.1% TFA versus distilled water containing 0.1% TFA over 30 min at a flow rate of 1 mL/min. Wavelength for the detection of HCPT was 254 nm. The loading efficiency of SWCNTs was calculated using the following equation: loading efficiency = W_{loaded drug}/W_{loaded drug} + W_HANP × 100%

A Waters high-performance liquid chromatography (HPLC) system (Milford, CT, USA) and a Waters 2414 refractive index detector (RID) were employed for chromatographic analysis of fructose, glucose, and sucrose. The separation of sugar compounds was performed on a Luna^® NH2 100 Å column (250 × 4.6 mm; 5 µm particle size) purchased from Phenomenex (Torrance, CA, USA). For the chromatographic analysis of sugars, the following conditions were used. The mobile phase consisted of acetonitrile and water (80:20, v/v), which were degassed before use; the injection volume was 10.0 µL; a flow rate of 1.3 mL/min was used; and a run time of 15 min per sample was maintained. The retention times of glucose, fructose, and sucrose were 5.368 min, 6.255 min, and 8.057 min, respectively. The method used for the HPLC-RID analysis was validated according to international guidelines. The preparation procedure was carried out by measuring 5 g of sample and adding water and methanol (3:1, v/v). After filtering, the sample was introduced into the HPLC-RID autosampler vial. Each sample was assessed in triplicate.

The catechins, theanine, and caffeine were analyzed on a Waters High Performance Liquid Chromatography (HPLC) system supported with a Waters 600 controller and Waters 2489 UV/Visible Detector (280 nm). Chromatographic separation was performed on a Phenomenex Gemini C18 column. The column temperature was set at 25 °C. The injection volume of sample was 5 μL, the elution rate was 1 mL/min, and the detection wavelength was set at 278 nm. The mobile phase consisted of mobile phase A (deionized water with 0.17% acetic acid) and mobile phase B (100% acetonitrile). The linear gradient at a flow rate of 1.0 mL/min was set as follows: mobile phase B from 8–28.4% (v/v) in 30 min was initiated, from 28.4–100% (v/v) for 8 min, and from 100–8% (v/v) for another 10 min.

The concentration of AIF in samples obtained from in vitro and in vivo assays in this study was determined using a Waters high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) equipped with Waters 2695 separations module and Waters 2996 photodiode array detector. The Fortis C18 chromatography column (5 μm, 4.6 × 250 mm) was used and maintained at 30 °C during the analysis. AIF and genistein (internal standard) were eluted using the isocratic mode with a mobile phase composed of ACN/water (70:30, v/v), which was freshly prepared for each run and degassed before use. The sample injection volume was 10 μL and flow rate was 1.0 mL/min for the mobile phase. The retention times of genistein and AIF were 3.4 and 12.7 min, respectively and their concentrations were calculated by comparing the peak areas with the standard curve (Figure 1).

Tea cultivar Yunkang 10 (Camellia sinensis var. assamica) was cultivated in Menghai County, Yunnan Province, China. Yunkang green tea (YKGT) was provided by Tea Research Institute of the Yunnan Province. Tea cultivar Longjing 43 (Camellia sinensis var. sinensis) was from tea garden of Anhui Agricultural University, Anhui Province, China. Longjing green tea (LJGT) was prepared by a professional tea producer following a standard protocol. The main catechins and caffeine were analyzed following our previous protocol [14 (link)] on a Waters High Performance Liquid Chromatography (HPLC) system supported with a Waters 600 controller and Waters 2489 UV/Visible Detector (280 nm).

We used the Waters High-Performance Liquid Chromatography (HPLC) system to analyze the chemical composition of GJXLT. The system comprised a 626 pump, a 600 s controller, and a 996 photodiode array detector. A C18 column (250 mm × 4.0 mm, 5 μm, ACE, UK) was used as solid phase while acetonitrile (DUKSAN)-H₂O containing 0.05% KH₂PO₄ (pH = 2.5) was utilized as mobile phase. The flow rate was 1 mL/min, and the detection wavelength was 254 nm.