Analysis was performed on a C18 column (Waters Sunfire, 5 μm, 4.6 × 150 mm). 0.1% formic acid added in Water (A) and acetonitrile (B) was used as mobile phases. The gradient elution was used for analysis and the program was as follows: 0–20 min, 10–100% B; 20–24 min, 100% B. The flow rate of the analysis was 1.0 mL/min. UV-Vis spectra were obtained with the wavelength range of 200–500 nm with 10 scans per second. The splitted eluent was at a ratio of 1:5 before the mass spectrometer. Both positive and negative ion modes were used for ESIMS recording. The capillary voltage was 4000 V, the capillary exit voltage was 140.0 V, and the skimmer voltage was 40 V. The nebulizer gas pressure was set to 40 psi, the dry gas flow to 10.0 L/min and the dry temperature to 320°C. Mass range was set from 120 to 1500 m/z. Data acquisition and processing were achieved with MassLynxTM 4.0 software (Waters, Milford, MA, USA).
Sunfire
Manufactured by Waters Corporation
Sourced in United States
The SunFire is a high-performance liquid chromatography (HPLC) system designed by Waters Corporation. It is a modular instrument that can be configured to meet diverse analytical requirements. The SunFire system provides reliable and consistent separation of complex mixtures, enabling accurate quantification and identification of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Waters 2487 instrument, by Waters Corporation (2 mentions)
Dual λ absorbance detector, by Waters Corporation (1 mentions)
Binary hplc pump, by Waters Corporation (1 mentions)
Chrompass software, by Jasco (1 mentions)
Prep c18, by Waters Corporation (1 mentions)
2695 separation module, by Waters Corporation (1 mentions)
Dnapac pa 100, by Thermo Fisher Scientific (1 mentions)
Mercury 300 nmr spectrometer, by Agilent Technologies (1 mentions)
Agilent 1100 series system, by Agilent Technologies (1 mentions)
Cell disruptor, by Constant Systems (1 mentions)
1220 infinity lc, by Agilent Technologies (1 mentions)
Binary hplc system, by Waters Corporation (1 mentions)
Lc 20ad, by Shimadzu (1 mentions)
C18 column, by Waters Corporation (1 mentions)
18 protocols using sunfire
1
LC-MS Analysis of Organic Compounds
2
HPLC Analysis of Organic Compounds
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HPLC analysis was carried out on an Agilent system (Santa Clara, CA, USA) using an analytical C18 column, (SunFire, Waters Corporation, Milford, MA, USA) (5 µm, 150 × 4.6 mm). A gradient system was run from 5:95 (v/v) acetonitrile/water as eluent A and acetonitrile as eluent B. Both eluents were adjusted by the addition of 0.25% acetic acid. The gradient was run from 45% A to 60% B in 10 min. The injection volume was 20 µL, the flow rate was 1.2 mL/min, and the detection wavelength was 425 nm.
3
Quantification of Rice Seed ABA
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After imbibed in control, 2.0 g · L−1 of eugenol and eugenol + GA3 combined solution for 3 h, 9 h, 18 h and 24 h, seed samples were collected and quickly frozen in liquid nitrogen and stored at −80 °C. Approximately, 500 mg of powdered rice seeds were used for ABA measurement with HPLC following the method described by Qin (2013)25 and Tombesi et al.26 (link) with some modification25 , 26 (link), 20 μL of extraction for each sample was injected into a High Performance Liquid Chromatography system consisting of Binary HPLC Pump (Model 1525, Waters) and Dual λ absorbance detector (Model 2487, Waters). Sample was separated through a C18 reversed-phase chromatographic column (SunFire, 5 μm, 46 × 250 mm, Waters) with flow velocity of 0.8 mL/min. ABA content were calculated according to the standard curve.
4
Quantifying Drug Loading in Polymeric Micelles
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Drug loading was determined by high-performance liquid chromatography (HPLC), which consisted of a pump (G1311G; Agilent Technologies, Palo Alto, CA, USA), a UV detector (G4212B; Agilent Technologies) set at 260 nm, an autosampler (G1329B; Agilent Technologies), and a reversed-phase C18 column (4.6×250 mm, 5 μm, SunFire; Waters Co., Milford, MA, USA). The mobile phase, with a flow rate of 1.0 mL/min, consisted of a mixture of acetonitrile and 0.1% phosphoric acid solution (50:50, v/v), which was freshly prepared and filtered with a 0.45 μm membrane. BCA-loaded dried micelles were obtained after freeze-drying. Weighed amounts of dried micelles were then dissolved in methanol and fragmented by ultrasonication. The entrapment efficiency (EE) and drug-loading (DL) content of BCA were calculated using the following equations:
5
HPLC Analysis of Maltol
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Analysis of the maltol was conducted in a Jasco (Tokyo, Japan) HPLC system with PU-2089 Plus gradient pump equipped with a degasser, an AS-2075 Plus autosampler, and a MD-2010 Plus DAD. Data were collected with the Jasco Chrompass Software. Comparative analysis was carried out using a SunFire (Waters) C18 column (particle size: 5 μm, id: 4.6 mm, length: 250 mm). The mobile phase consisting of eluent of A (2% acetic acid in water) and B (0.5% acetic acid in acetonitrile) was run at 1.2 mL/min. The linear gradient elution program was set as follows: 100% A at 0–20 min, 100–97% A at 20–24 min, and 10% A at 24–30 min. The eluted maltol was detected at 274 nm. The injection volume was 10 μL, and the column temperature was maintained at 40°C.
6
Quantification of GSH and GSSG by LC-MS
7
Synthesis of Peptide SEQ ID NO: 4
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Example 1
Synthesis of SEQ ID NO: 4
The solid phase synthesis was carried out on Novabiochem Rink-Amide resin (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.34 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Boc)-OH were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. Hereafter Palm-Glu(γOSu)-OtBu was coupled to the liberated amino-group. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.1% TFA).
Finally, the molecular mass of the purified peptide was confirmed by LC-MS.
8
HPLC and NMR Characterization of Compounds
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Example 1
Analytical Methods
High-performance liquid chromatography (HPLC) analysis was done with a C-18 reversed-phase narrow bore column (3 mm i.d., 150 mm length, 5 μm; SunFire; Waters) on a Waters 2695 separation module equipped with 996 photodiode array detector. The column was eluted with a gradient of (A) aqueous 25 mM triethylammonium acetate and (B) 100% acetonitrile. The elution program created a linear gradient started from 100% (by volume) A to 85% A at 10 min with flow rate of 0.5 mL/min. Peak detection and integration were conducted with the signal at 260 nm. Full UV spectra (230˜400 nm) were also obtained. Preparative HPLC purification was done using an ion exchange column (22 mm id, 250 mm length, 5 μm; DNAPac PA-100; Thermo Fisher Scientific) on a Waters Delta 600 module. The column was eluted with a gradient of (A) water and (B) 1 M ammonium bicarbonate. The elution program created a linear gradient started from 100% A to 70% A at 15 min with flow rate of 10 ml/min. Peak detection was conducted using the 260 nm absorbance. 1H NMR spectra were recorded in deuterium oxide on Varian Mercury 300 NMR spectrometer. High resolution mass spectrometry was conducted on Agilent 6220 Time-of-Flight connected with Agilent 1100 series system consisting of G13793 degasser and G1312B binary pump with Electro spray ionization in negative mode.
9
Extraction and Quantification of Macamides from Maca
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Dried hypocotyls of maca were obtained from Lijiang BaiSuiFang Biotechnology Development Co. Ltd. (Yunnan, China). They were ground into a powder and passed through an 80-mesh sieve to obtain maca powder. Dried maca powder (10 g) was soaked in 100 ml of 95% ethanol (1/10, w/v) and reflux-extracted at 70 °C for 2 h twice. The extract was collected, filtered, and evaporated with a rotary evaporator at 50 °C to yield 1.97 g of EEM. To identify the active ingredients in EEM, we used a chromatographic technique. HPLC experiments comprised an UltiMate3000 system equipped with a UV-vis detector. Separation was performed using a C18 column (250 mm × 4.6 mm × 5 μm, Waters Sunfire) at 30 °C. The mobile phase was 90% acetonitrile with a flow rate of 0.6 mL min−1. EEM was dissolved using an appropriate volume of methanol and was filtered through a 0.45 μm syringe filter. The extract with an injection volume of 20 μL was added to HPLC vials and injected into the automatic sampler, and was detected at 210 nm. Macamides were qualitatively identified by comparing the retention times with the standards, and their contents were calculated based on the peak area. By HPLC analysis, the extract contained 750.23 mg/100 g total macamides.
10
Characterization of Polyphenol-rich Extracts
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The characterization of Polyphenol-rich extracts was carried out using high performance liquid chromatography (HPLC). Samples were suspended in LC-MS metanol and filtered through a membrane filter (pore size, 0.45 mm). The separation was achieved on a SunFire (Waters, Milford, MA, USA) C18 5 μm 4.6 × 150 mm column at ambient temperature using a Waters 2487 instrument (Waters, Milford, MA, USA). The mobile phase consisted of acetonitrile with 3% Acetic Acid (solvent A), water with 3% Acetic Acid (solvent B). The gradient used to separate Solieria filiformis extract was: 100% A at 0 min, 90% A and 10% B at 3.5 min, 50% A and 50% B at 5 min, and 100% B at 10–20 min. To separate Ecklonia arborea extract we used the following gradient: 100% A at 0 min, 50% A and 50% B at 2 min, 25% A and 75% B at 5 min, and 100% B at 7–20 min. The flow rate was 1.5 mL/min for 20 min at an injection volume of 10 μL. Once collected, the fractions were, dried and resuspended in methanol for mass analysis. Liquid chromatography/mass spectra (LC-MS) [+ESI] were taken on a JEOL AccuTOF TC-100 Mass Spectrometer (JEOL Ltd., Tokyo, Japan).
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