Agilent 1260 infinity II high performance liquid chromatography (HPLC) system was applied to remove interfering substances. Buffer A: 10 mM HCOONH4, 5% ACN, pH 10.0; buffer B: 85% ACN, 10 mM HCOONH4, pH 10.0. The peptides were eluted at a flow rate of 1 ml/min with a gradient of buffer B: 0% for 25 min, 0–7% for 5 min, 7–40% for 35 min, 40–100% for 5 min, and a final hold in 100% for 15 min. Elution was supervised by examining the absorbance at 214 nm, and the components were obtained per min (about 36 components). The samples were stored at − 80 °C [12 (link), 13 (link)].
1260 infinity 2 high
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 Infinity II is a high-performance liquid chromatography (HPLC) system designed for efficient and reliable analytical separations. The core function of this system is to provide precise and reproducible chromatographic analysis across a wide range of applications.
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5 protocols using 1260 infinity 2 high
1
HPLC-based Peptide Purification and Analysis
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Quantification of 11 PFAS compounds by HPLC-MS/MS
3
Lutein Extraction and Quantification from Microalgae
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Lutein was extracted using a method modified from that of Xie et al. [50 (link)]. The lutein content of the extract was analyzed using 1260 Infinity II high-performance liquid chromatography (Agilent, Santa Clara, CA, USA), as proposed by Taucher et al. [51 (link)]. A reversed-phase YMC carotenoid C30 column (150 × 4.6 mm, 3 μm) operated at 22 °C with mobile phase 1 mL/min. The mobile phase consists of two solvent mixtures: (A solvent) methanol, methyl tert-butyl ether (MTBE), and water: 81:15:4; and (B solvent) methanol, MTBE, and water: 8:89:3 (v:v:v). The mobile phase is a gradient condition, first flowing at 98% (A solvent) for 11 min, then decreasing to 60% (A solvent) over the next 7 min. This is maintained for 6.5 min, then decreases to 0% (A solvent) in 2.5 min and held for 3 min; it then increases to 98% (A solvent) over the next 3 min to re-equilibrate the mobile phase and is held for 7 min. Lutein was detected by measuring the absorbance at the wavelength 445 nm. The lutein standard was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The lutein content (mg/g) and lutein productivity (mg/L/d) of microalgae were calculated as shown in Equations (3) and (4).
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HPLC Analysis of Caraway Extract
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For the C. carvi extract analysis, An 1260 infinity II high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) was used. Fitted with a quaternary pump, the apparatus was coupled with a UV detector. The mobile phase was composed of two solvents, A: 0.1 percent acidified water and B: acetonitrile. A C18 Zorbax eclipse plus C18 column (5 μm, 4.6 × 150 mm) was used for the separation. The temperature of the column furnace was set at 35 °C. The sample injection volume and flow rates were set at 10 μL (100 μg/mL of sample concentration) and 1 mL/min. The concentration was calculated according to the following Equation (2), depending on the retention time (RT) and spectral correspondence of each compound:
5
Synaptosome Peptide Fractionation by bRP
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Four mg of S-trap digested synaptosome peptides were fractionated by basic reversed-phase (bRP) chromatography. bRP solvent A was made in an aqueous solution of 5mM of Ammonium Formate and 2% ACN (pH 10). bRP Solvent B was made in a solution of 5 mM Ammonium Formate and 90% ACN (pH 10). A 4.6 mm inner diameter 250 mm long Agilent Zorbax 300 Extend-C18 (Agilent) was used as the bRP column.
Chromatography occurred over a 96-minute gradient with fractions collected in a Whatman 2ml 96 well plate (GE Healthcare). A 1260 Infinity II high-performance liquid chromatography (HPLC) instrument was used (Agilent). The chromatography gradient proceeded with a solvent B composition scheme of: 0% to 5 minutes, 5% at 7.66 minutes, 28.5% at 64.98 minutes, 34% at 70.48 minutes, 60% at 83.48 minutes, and 60% solvent B continuing for the remainder of the gradient. Peptides were concatenated into a 12-fraction scheme as previously described (34) . Peptides were then frozen and dried by vacuum centrifugation in preparation for O-GlcNAc enrichment.
Chromatography occurred over a 96-minute gradient with fractions collected in a Whatman 2ml 96 well plate (GE Healthcare). A 1260 Infinity II high-performance liquid chromatography (HPLC) instrument was used (Agilent). The chromatography gradient proceeded with a solvent B composition scheme of: 0% to 5 minutes, 5% at 7.66 minutes, 28.5% at 64.98 minutes, 34% at 70.48 minutes, 60% at 83.48 minutes, and 60% solvent B continuing for the remainder of the gradient. Peptides were concatenated into a 12-fraction scheme as previously described (34) . Peptides were then frozen and dried by vacuum centrifugation in preparation for O-GlcNAc enrichment.
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