A semi-preparative HPLC instrument (1290 infinity series II, Agilent Tech., Santa Clara, CA, USA) consisting of a degasser, quaternary pump, UV–Vis detector, thermostated autosampler, and oven, equipped with an automatic fraction collector, was used. The separation was performed on a Develosil diol column (250 mm × 10 mm, 5 μm) at 50 °C. The flow rate was set at 5 mL/min, and the injection volume was equivalent to 80 mg/load. The mobile phase was based on a previous work by Kelm et al. [48 (link)], using acetonitrile/acetic acid (98/2 v/v, A) and methanol/water/acetic acid (95/3/2 v/v, B). The optimal linear gradient applied was: 0 min, 10% B; 0.5 min, 12% B; 1.5 min, 12% B; 6.0 min, 18% B; 12.5 min, 35% B; 12.6 min, 100% B; 13.6 min, 100% B. Then, it was returned to the initial condition (10% B) and re-equilibrated for 10 min. The sample was dissolved in acetone/water/acetonitrile/acetic acid (60/29.5/10/0.5 v/v/v/v) and filtered through a 0.45-µm (Durapore PVDF, Millipore, USA) membrane. The thawed fractions were pooled and stored at −80 °C for less than 5 days. After that, collected samples were concentrated and stored as explained in Section 4.3.1 . The relative composition of every fraction was determined by UHPLC-QTOF-MS method described in Section 4.4 .
1290 infinity 2 series
Manufactured by Agilent Technologies
Sourced in Germany
The Agilent 1290 Infinity II series is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It offers advanced features and capabilities to support a wide range of laboratory workflows.
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Lab products found in correlation
Durapore pvdf, by Merck Group (1 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
Uhplc system, by Agilent Technologies (1 mentions)
Openlab cds 3, by Agilent Technologies (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
Openlab cds, by Agilent Technologies (1 mentions)
Hss t3, by Waters Corporation (1 mentions)
Kinetica 4, by Thermo Fisher Scientific (1 mentions)
7 protocols using 1290 infinity 2 series
1
Semi-preparative HPLC Purification Protocol
2
UPLC-PDA-MS Protocol for Reaction Yield Analysis
3
Quantification of Stilbenoids by UHPLC-UV
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For quantification of stilbenoids, a UHPLC system from Agilent Technologies (Waldbronn, Germany), with a high-speed pump (1290 Infinity II series), multicolumn thermostat (1290 Infinity II series), vialsampler (1290 Infinity II series), a diode array detector (1290 Infinity II series), and Open Lab CDS 3.4 (Agilent Technologies, Waldbronn, Germany) was used. The separation was achieved on a Zorbax Eclipse Plus C18 column (1.8 µm, 50 × 2.1 mm, Agilent Technologies, Waldbronn, Germany) according to Kiene and co-workers [35 (link)]. For this, the mobile phase consisted of water/acetonitrile/formic acid (100/10/0.1; v/v/v) (A) and acetonitrile (B). The separation was carried out at 60 °C with a flow rate of 0.4 mL/min, under the following conditions: 0 min (0% B), 1.73 min (0% B), 2.73 min (16% B), 6.2 min (45% B), 6.4 min (0% B), and 7.2 min (0% B). Trans-resveratrol was quantified at λ 306 nm and trans-ε-viniferin, r-2-viniferin, and r-viniferin were quantified at 324 nm, respectively. The quantification parameters of the UHPLC-UV methodology are listed in Supplementary Material Table S5 .
4
UHPLC Peptide Purity Analysis
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For determination of purity by UHPLC, the filtered peptide solution was diluted in 10–50% acetonitrile (MeCN) in water with 0.1% TFA (500 μL) to a final concentration of approximately 0.5 mg/mL. The samples were analyzed on Agilent 1290 Infinity II Series, which was computer-controlled through Agilent OpenLab CDS and ChemStation software. For standard analysis of all peptide samples, analytical UHPLC spectra were recorded on an analytical Agilent Zorbax 300SB-C18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 μm particle size) kept at 40 °C; elution at a flow rate of 0.8 mL/min with A: MeCN/H2O (5:95) + 0.1% TFA and B: MeCN/H2O (95:5) + 0.1% TFA, isocratic 0% B for 3 min; then 0–100% B over 20 min (ca. 4.5% MeCN/min) with UV detection at 214 nm and 120 points s−1. The total method time was 23.1 min. Then, the column was re-equilibrated using a post-run method at 0% Solvent B for 2 min. Purities of the final peptides were calculated by integration of the Area Under the Curve (AUC) of desired product peak as a percentage of the AUC of all peaks (within 3–18 min) at λ = 214 nm (amide backbone).
5
Pharmacokinetics of Compound 2l in Rats
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Adult male rats (N = 3/group) were administered with compound 2l dissolved in distilled water (tween 80) at a single dose of 10 mg/kg by oral administration and 10 mg/mL by injection. Serum samples were collected each 30 min. The blood concentration of the test compounds was determined by LC-MS/MS (Agilent 1290 infinity II series equipped with on-line degasser, binary pump, thermostatted well-plate autosampler and column compartment, Waters Atlantis® HSS T3 (2.1 × 100 mm, 1.9 µm) column, and mobile phase linear gradient from 95% A (0.1% formic acid in water) /5% B (0.1% formic acid in acetonitrile) to 5% A/95% B, 6500+ QTRAP LC-MS/MS/MS system, Turbo Spray Ion Drive as ion source, and Carbamazepine as internal standard. Pharmacokinetic parameters were obtained by non-compartmental analysis of the plasma concentration–time profiles using KineticaTM 4.4.1 (Thermo Fisher Scientific, Inc., Woburn, MA, USA).
6
Liquid Chromatography-Mass Spectrometry Analysis of Phenolic Compounds in Herbal Extracts
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The HT-extracted CS was prepared using the optimal extraction conditions obtained from the OFAT approach. The liquid samples were dried using a freeze dryer and subjected to analysis by LC-quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS). Identification of phenolic compounds and derivatives in HT-extracted CS was performed at the Science and Technology Research Institute, Chiang Mai University, Thailand [13 (link)]. The samples were analyzed using an Agilent 1290 Infinity II series coupled with a 6546 LC-QTOF instrument. Electrospray ionization (ESI) was used for ionization in negative mode. The nebulizer was operated at 35 psi with 11 L/min N2 flow. The capillary temperature was kept at 350 °C, while the sample flow rate was set at 0.2 mL/min. The m/z range was scanned within 100−1700, the capillary voltage was 3500 V, and the dry heater temperature was 320 °C. Provisional compound identification was performed by matching accurate mass results to content from the METLIN Personal Compound Database and Library (PCDL).
7
HPLC Analysis of Ascorbic Acid
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Vitamin C content was determined using high performance liquid chromatography (HPLC) on an Agilent 1290 Infinity II Series (Germany) equipped with a degasser, quaternary pump, and autosampler injector set to a volume injection of 20 µL. Separation was carried out using a YMC-Triart C18 HPLC column (5 m particle size 150 4.6 mm I.D., maintained at 25°C) and a UV-Visible diode array detector (DAD). The mobile phase used were 0.015 mol/L potassium dihydrogen phosphate (KH 2 PO 4 ) buffer solution (pH: 2.6) and the ascorbic acid peaks were detected at 250 nm. The standard used in this analysis was L-ascorbic acid with five different concentration (10, 25, 50, 75, 100 mg/L) with equation y = 239.19x -294.14 and R² = 0.9984.
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