1260 infinity series hplc

Melting points were measured on SGW X-4B melting point apparatus (Shenguang, Shanghai, China). ¹H-NMR spectra were recorded on Avance 300 (300 MHz) and 400 (400 MHz) spectrometers (Bruker, Karlsruhe, Germany). Chemical shifts were reported in ppm from tetramethylsilane with the solvent resonance resulting from incomplete deuterium incorporation as the internal standard (CDCl₃: δ 7.26 ppm). ¹³C-NMR spectra were recorded on Bruker Avance 300 (75 MHz) and 400 (100 MHz) spectrometers with complete proton decoupling. Chemical shifts were reported in ppm from tetramethylsilane with the solvent resonance as the internal standard. High-resolution mass spectrometry was performed on a Thermo Orbitrap Elite, instrument (Agilent, Palo Alto, CA, USA). Optical rotations were measured on an Autopol IV (d = 589 nm, Hg lamp, 50 mm cell) instrument (Rudolph, NJ, USA). The enantiomeric excess was determined by a 1260 infinity series HPLC (Agilent, Palo Alto, CA, USA) equipped with Chiralpak OD-H, AD-H and IA columns (4.6 mm × 250 mm, Daicel Chiral Technologies, Shanghai, China). Chemicals and solvents were purchased from Linfeng (Shanghai) and Annaiji (Shanghai) in China, and used as received. Purification of the products was carried out by flash column chromatography using silica gel (Yantai Jiangyou Company, Shandong, China, particle size 0.100–0.075 mm).

SEC-MALS experiments were performed by loading ∼10 μg of protein sample onto a Phenomenex Yarra 3 µm SEC-2000 column (Phenomenex, Torrance, CA, USA) at a flow-rate of 0.5 ml/min using an Agilent 1260 Infinity series HPLC. The mobile phase was PBS buffer at pH 7.4. The elution was detected with a UV detector (Agilent, USA a miniDAWN triple-angle light-scattering detector (Wyatt Technology, USA) and with an Optilab rEX differential refractometer (Wyatt Technology) connected in series. The isotropic scatterer for detector normalisation was bovine serum albumin. Since the light scattered by a protein is directly proportional to its weight-average molecular mass and concentration, molecular masses were calculated from the light-scattering and interferometric refractometer data using ASTRA 6.1 software.

All analytes were assayed using HPLC. Equipment consisted of an Agilent 1260 Infinity series HPLC with a quaternary pump (G1311B), a column compartment (G1316A), a variable wavelength and fluorescence detector (G1314B and G1321B) and an autosampler system (G1329A) (Agilent Technologies, Santa Clara, CA, USA). Peak separation was accomplished using a 5 mm Agilent Zorbax Eclipse C₁₈ column (3.0 × 150 mm, 5 µm) except for T-2/HT-2 toxin analyses for which a Luna^® Phenyl-Hexyl column (4.6 × 150 mm, 5 µm) was used (Phenomenex, Torrance, CA, USA). All analytes, except AFM₁, were extracted using Immunoaffinity columns (R-biopharm Rhöne Ltd, Darmstadt, Germany).

Typical assays containing 10 mM pyrrole-2-carboxylate, 50 mM KCl,
and 50 mM NaPi, pH 6, were incubated with and without enzyme at 30
°C overnight. The sample was centrifuged at 16 100g to remove precipitate, and 50 μL was added to 450
μL of 50% v/v H₂O/acetonitrile. Sample analysis was
performed using an Agilent 1260 Infinity Series HPLC equipped with
a UV detector. The stationary phase was a Kinetex 5 μm C18 100A
column, 250 × 4.6 mm. The mobile phase was acetonitrile/water
(50/50) with 0.1% TFA at a flow rate of 1 mL/min, and unless otherwise
stated, detection was performed at a wavelength of 210 nm.

Following the method described by Marra et al. [73 (link)], the spectrometric analysis of the plant extracts was performed by an LC-MS Q-TOF Agilent Technologies (Santa Clara, CA, USA), equipped with a 1260 Infinity series HPLC with DAD detector, and a mass spectrometer Q-TOF (model G6540) with Dual ESI source. The plant extracts were separated with a reverse-phase analytical Ascentis ^® Express C18 column (2.7 μm, 50 mm × 3.0 mm id, Supelco ©, Bellefonte, PA, USA). The identification and quantification of the three phenolic acids p-coumaric, caffeic and rosmarinic were obtained by comparing the mass and the retention time (RT) to standard compounds (Sigma-Aldrich, St. Louis, MO, USA). These standards were analyzed with the same LC-MS method and the quantification was obtained by interpolating the averaged data with a previously constructed calibration curve.

A 1260 Infinity series HPLC (Agilent Technologies, Richmond, VA, USA) equipped with an Agilent Porshell HC-C18 column (Cat. # 518905-902, 5 µm 4.6 × 250 mm) was used to analyze DHM. The flow rate was set at 0.6 mL/min using a binary mobile-phase composed of methanol/water [32:68 (v/v)] with the use of 0.1% trifluoroacetic acid (TFA) as a solvent modifier. The DAD detector (Agilent) was programed to scan the entire UV range (190 to 400 nm) whereas the 291 nm wavelength was used for DHM detection. The heights of detected peaks were used for DHM quantification after establishing a standard analytical-curve using the commercially obtained DHM.

SEC–MALS experiments involved loading 10 µg of each protein sample onto a stationary phase comprised of a TSKgel Super SW mAb HTP column (Tosoh Biosciences, King of Prussia, Pennsylvania, USA) at a flow rate of 0.25 ml min⁻¹ with an Agilent 1260 Infinity series HPLC. The mobile phase buffer was phosphate-buffered saline pH 7.4. Each experiment was performed in triplicate. The detector system consisted of a UV detector (Agilent) and a miniDAWN triple-angle light-scattering detector (Wyatt Technology) with an Optilab rEX differential refractometer (Wyatt Technology) connected in series. The refractometer provides a continuous index of protein concentration using a dn/dc (refractive-index increment) value of 0.185 ml mg⁻¹. Bovine serum albumin was used as an isotropic scatterer for detector normalization. Since the light scattered by a protein is directly proportional to its weight-average molecular mass and concentration, molecular masses were calculated from the light-scattering and interferometric refractometer data using the ASTRA 6.0 software.