Prominence auto sampler sil 20a hplc system

The phytochemical characteristics of NF50E were identified by HPLC using a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan) equipped with an SPD-M20A diode array detector (PDA) and LC solution 1.22 SP1 software. Protocatechuic acid, chlorogenic acid, catechin, epicatechin, and kaempferol were used as standard compounds. Reverse-phase chromatographic analysis was performed using a Phenomenex C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. A stepwise gradient of solvent A to B was used (A: 2% acetic acid and B: 50% acetonitrile (CAN) in 0.5% acetic acid). The flow rate was 0.8 mL/min, and the injection volume was 10 μL. A Q-Exactive™ Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific Inc., Rockford, IL, USA) was used to perform the mass experiments. The settings of the IT mass spectrometer were as follows: ESI voltage +4 kV, nebulization with N2 at 1.7 bar, dry gas flow 7 L/min, gas temperature 310°C, skimmer 1 voltage +12.4, collision energy set to 1 V, and ramped within 40%–200% of this value. The ion number accumulated within the trap was set to 10,000, and the maximum accumulation time was 200 ms. To determine the key chemical sdiagnostic product ions over the full range, the product ion spectrum was recorded in the targeted mode for the mass range m/z 50–1500.

The phytochemical characterization of LPE and the standard compounds gallic acid, p-coumaric acid, catechin, and epicatechin were identified by HPLC-diode array detection (HPLC-DAD) with a Shimadzu Prominence autosampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan). For reverse-phase chromatographic analysis, we used Phenomenex C18 column (4.6 × 250 mm) with 5 µm-diameter particle size. A stepwise gradient of solvent A (acetonitrile) to solvent B (1% formic acid solution) was applied by changing the ratio at every minute as follows: 10% A up to 10 min, at λ = 280 nm which was then shifted to find 30, 50, 60, 90, and 20 and 10% A in 15, 20, 25, 30, 35, and 40 min, respectively. The volume of injection was 20 µL, and the flow rate was sustained at 0.8 mL/min, as previously described [47 (link),48 ]. Based on the retention time, the phenolic components were identified by comparison with the standard compounds.

Only analytical grade chemicals were used in this study. Acetonitrile, ethyl alcohol, formic acid, catechin, Folin-Ciocalteu reagent and gallic acid were purchased from Merck (Darmstadt, Germany). Apigenin, rutin, orientin, luteolin, kaempferol, quercetin, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Trolox, 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), phenanthroline, amikacin, gentamicin, benzylpenicillin, cefotaxime, fluconazole and ketoconazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibiotics were dissolved in sterile water. High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT with reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.

The phytochemical characteristics of NNF and the standard compounds galic acid, catechin, epigallocatechin, epicatechin gallate, epicatechin, caffeic acid, quercetin, and apigenin were identified by high performance liquid chromatography-diode array detection (HPLC-DAD) with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with SPD-M20A diode array detector and LC solution 1.22 SP1 software. Reverse-phase chromatographic analysis was carried out using a Phenomenex C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. A stepwise gradient of solvent A (acetonitrile) to solvent B (1% formic acid solution) was used with the ration changing each minute as follows: 10% A up to 10 min, which was then changed to obtain 30%, 50%, 60%, 90%, 20%, and 10% A in 15, 20, 25, 30, 35, and 40 min, respectively, at λ = 280 nm. This is in accordance with a slightly varied protocol detailed by Brito et al. [37 (link)]. Polyphenolic compounds were identified by comparing retention times with those of available pure standards.

High‐performance liquid chromatography (HPLC‐DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL‐20A) HPLC system (Shimadzu, Kyoto, Japan) and equipped with Shimadzu LC‐20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD‐M20A diode array detector and LC solution 1.22 SP1 software. The quantification of phenolic compounds in the differently dried moringa leaf was carried out using the method described by Waczuk et al. (2015) with slight modifications. Reverse phase chromatographic analyses were carried out under gradient conditions using C₁₈ column (4.6 mm × 150 mm) packed with 5 μm diameter particles; the mobile phases A and B were Milli‐Q water, acidified to pH 2.0 with 1% of phosphoric acid and methanol respectively, solvent gradient was used as follows: 0–10 min, 5% B; 10–25 min, 15% B; 25–40 min, 30%; 40–55 min 50% B; 50–65 min 70% B; 65–80 min, 100% B, respectively. The flow rate was 0.6 ml/min and the injection volume was 50 μl. Quantifications were carried out by integration of the peaks using the external standard method, at 254 nm for gallic and ellagic acids; 280 nm for catechin and epicatechin; 327 nm for chlorogenic and caffeic acids; and 366 for quercetin, kaempferol, and rutin.

All chemical were of analytical grade. Acetonitrile, formic acid, gallic acid, chlorogenic acid, and caffeic acid were purchased from Merck (Darmstadt, Germany). Quercetin, theobromine, caffeine, rutin, catechin, and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Autosampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector, and LC solution 1.22 SP1 software.

The phytochemical characteristics of SSW and SSE were analyzed by high-performance liquid chromatography (HPLC) using standard compounds such as catechin, (−)-epigallocatechin gallate (EGCG, E4268, Sigma-Aldrich), epicatechin, gallic acid, syringic acid, and vanillic acid. The HPLC analysis was performed using a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with an SPD-M20A diode array detector (PDA) and LC solution 1.22 SP1 software. Before analysis, the samples were filtered through a 0.2 µm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA). The reverse-phase chromatographic analysis was carried out using a Phenomenex C18 column (4.6 mm × 250 mm) packed with 5 µm diameter particles and maintained at 25 °C. A stepwise gradient of solvent A to B was used (A: 2% acetic acid and B: 50% acetonitrile (ACN) in 0.5% acetic acid) according to a previous report [24 (link)] with a slight modification. The flow rate was 1 mL/min, and 10 µL was injected.