Lc 20at quaternary pump

Chromatographic quantification of the pomegranate compounds was performed by high-performance liquid chromatography using a Shimadzu system (Shimadzu Corporation, Kyoto, Japan) consisting of a pump (LC-20AT), diode array detector (SPD-M20A), system controller (CBM-20A), autoinjector (SIL-20A), LC-20AT quaternary pump, and Shimadzu LC solution software. The chromatographic separation and the ellagic acid and punicalagin determination were performed using a reverse-phase Shimadzu Shim-Pack GIST analytical column C18 (100 mm × 4.6 mm × 3 μm) at 30 °C, as described by Santiago et. al, with some modifications [80 ]. The mobile phase consisted of acetonitrile (phase B) and water containing 5% formic acid (v/v) (phase A) using the following gradient program: 0–5 min, 97–95% A; 5–10 min, 95–85% A; 10-16 min, 85-70% A; 16–18 min, 70–97% A; 18–25 min, 97% A. The flow rate was 0.8 mL min⁻¹. Peaks were determined by comparison with an authenticated ellagic acid (Sigma-Aldrich, St. Louis, MO, USA) and punicalagin α/β (Sigma-Aldrich) standard. PPE samples were diluted in methanol, homogenized in an ultrasonic bath for 30 min, and then filtered (0.45 µL). The injection volume was 5 μL, and we used a wavelength of 260 nm. All samples were prepared in triplicate.

Dried powder samples (0.02 g) were accurately weighed and extracted with hydrochloric acid-methanol solution (10 mL, 1:100, v/v) for 30 min in an ultrasonic bath at room temperature. Then, the decrease in weight was replenished with the extraction solvent. The sample solution was filtered through a 0.45 μm membrane filter prior to the HPLC-UV analysis. A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with a LC-20AT quaternary pump, a SIL-20A XR autosampler, a CTO-20AC column oven and a SPD-20A UV/Vis detector was used to determine the objective compounds. The chromatographic column Xtimate C18 (250 × 4.6 mm, 5 μm, Welch, Shanghai, China) was applied to perform chromatographic separation and the column temperature was constantly kept at 25 °C The binary mobile phase consisted of acetonitrile (A) and 30 mmol/L ammonium bicarbonate solution containing 0.7% ammonia and 0.25% triethylamine (B) at a continuous flow rate of 1 mL/min in the following gradient program: 0–15 min, 10%–25% A; 15–25 min, 25%–30% A; 25–50 min, 30%–45% A. The injection volume was 5 μL and the detection wavelength was acquired at 270 nm. The contents of eight alkaloids (berberine, palmatine, coptisine, epiberberine, columbamine, jatrorrhizine, magnoflorine and groenlandicine) in each sample were calculated from standard curves.

On silica gel (70–230 mesh) dried plant extract was adsorbed and loaded on silica column (600 mm height × 55 mm diameter). Elution were obtained with an increased polarity gradient of hexane–dichloromethane i.e., 9.5:0.5, 9:1, 8.5:0.5, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10. After that elutions were collected with 100% dichloromethane, and 100% ethyl acetate, ethyl acetate and methanol (9.5:0.5, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9) and 100% methanol.
The entire process was supervised at 280 nm and 254 by a Dual λ absorbance detector (Waters Milford, MA 01757, USA). 5 µL Aliquots of 225 fractions were marked on large TLC glass plates (20 cm × 20 cm) and placed in glass jars (20 cm × 10 cm × 20 cm), with mobile phase at room temperature. The plates were monitored using ultra-violet (UV) light at 254 and 360 nm.
Using Shimadzu, LC-20AT system equipped with LC-20AT quaternary pump, a on-line degasser, a photodiode array detector HPLC was done. The mobile phase of 30:70 H₂O and acetonitrile was used.

The analysis was performed as described by Alarcón et al. [20 (link)] using a high-performance liquid chromatography–diode array detection (HPLC-DAD) system (Shimadzu, Tokyo, Japan) equipped with an LC-20AT quaternary pump, a DGU-20A5R degassing unit, a CTO-20A oven and a diode array detector (SPD-M20A). Identity assignments were performed using an HPLC-DAD system coupled to an MDS Sciex system QTrap3200 liquid chromatography–tandem mass spectrometry instrument (Applied Biosystems, Foster City, CA, USA) and confirmed by comparison with the retention time when utilising commercial standards. A C₁₈ column (250 × 4.6 mm, 5 μm) (Kromasil, Supelco, Bellefonte, PA, USA), a C₁₈ precolumn (22 × 3.9 mm, 4 μm) (Novapak, Waters, Milford, MA, USA) and an oven temperature of 40 °C were used. Two mobile phases were used: mobile phase A was composed of water:acetonitrile:formic acid (87/3/10 v/v/v), while mobile phase B was composed of water:acetonitrile:formic acid (40/50/10 v/v/v). Hydroxycinnamic acid (HCAD) detection was performed at 320 nm by external calibration using chlorogenic acid as a standard.
Total phenols were determined using the method described by Parada et al. [48 (link)] in 96-well microplates using a microplate reader SYNERGY HTX (BioTek Instruments, Winooski, VT, USA) to obtain the absorbance at 750 nm. Gallic acid was used as a standard.

The sample preparation and components analysis for the FEPE samples were carried out according to the Chinese Pharmacopeia [20 ]. The dried fruits were crushed in a small medicine crusher and screened through an 80-mesh pharmacopeia sieve to obtain some brown powder. The powder of 0.100 g was weighed and placed in a dry conical flask, added to methanol, and extracted ultrasonically for 1 h. The methanol was added again to compensate for the lost mass and filtered through a 0.45 μm microporous membrane to obtain the test solution. The main chemical components of gallic acid, corilagin, chebulinic acid, and ellagic acid in FEPE were monitored by using our previously established method [21 (link)]. A Shimadzu system (Shimadzu, Kyoto City, Japan) equipped with an LC-20AT quaternary pump, a SIL-20A XR autosampler, a CTO-20AC column oven, and an SPD-20A UV/Vis detector was utilized to determine bioactive compounds of FEPE. An Agilent ZOR-BAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 mm) column was applied to separate objective compounds. Other HPLC conditions are listed below: column temperature: 30 °C; mobile phase: methanol (A) and 0.1% phosphoric acid (B); flow rate: 1 mL/min⁻¹; elution gradient: (0~15 min, 5% A; 15–35 min, 5~37% A; 35~39 min, 37~47% A; 39~60 min, 47~60% A); injection volume: 5 μL; detection wavelength: 273 nm.

Twenty liters of the LTPNA 08 strain were grown. After one month of cultivation, the material was centrifuged (4000× g for 6 min) and the cell pellet was lyophilized, yielding 3 g of dry biomass. Since the strain of Microcystis LTPNA 08 produces both microginins and microcystins, the classical method reported by Lawton et al. [21 (link)] was adapted to reliably separate these peptides. A pre-purification was performed on a solid phase extraction column from the crude extract. Dried material was extracted three times with 75% methanol. The extract was eluted with a gradient of 20%, 40%, 60%, 80% and 100% methanol to obtain fractions of different polarities, and these fractions were analysed in HPLC-DAD, according to the methodology described by Lawton et al. [21 (link)]. In order to isolate the microginins on a semi-preparative scale (Luna C18(2); 250 mm × 10 mm, 5 μm, Phenomenex^®), the method described by Lawton et al. was optimized. The best optimized gradient reduced the running time from 55 min to 24 min, using 100% methanol as mobile phase B. 0.1% formic acid was acidifying the water and the methanol. These conditions were used for isolation of microginins in the semiprep column. Analyses were carried out in a Shimadzu Prominence system equipped with a LC-20AT quaternary pump and a SPDM20A photodiode array detector.