For the quantification of free amino acids, the raw extracts used for phytohormone analysis were diluted 1:10 with water containing an algal amino acid mix (10 μg mL−1 U-13C, U-15N isotopically labelled amino acid mix; Isotec, Miamisburg, OH, USA) as internal standard. The extracts were analyzed on an Agilent 1260 Infinity high-performance liquid chromatography system (Agilent Technologies) coupled to an API 6500 ESI-Triple Quad mass spectrometer (AB Sciex, Darmstadt, Germany). Separation was achieved on a Zorbax Eclipse XDB-C18 column (50 mm × 4.6 mm, 1.8 μm, Agilent Technologies). Both 0.05% formic acid in H2O and acetonitrile were employed as mobile phases A and B, respectively. The elution profile was 0–1 min 3% B; 1.0–2.7 min 3–100% B; 2.7–3.0 min 100% B; 3.1–6.0 min 3% B. Column temperature was maintained at 20°C, the flow rate was constant at 1.1 mL/min. The mass spectrometer was operated in the positive ionization mode with multiple reaction monitoring (MRM) as described by Jander et al. (2004) (link). The Analyst 1.6.3 software (Applied Biosystems) was used for data acquisition and processing. Individual amino acids in the sample were quantified by the respective 13C, 15N-labelled amino acid internal standard, except for tryptophan that was quantified using 13C, 15N-Phe applying a response factor of 0.42.
Agilent 1260 infinity high
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system is a versatile and reliable laboratory instrument designed for advanced analytical applications. It features a modular design, allowing for customization and expansion to meet the specific needs of various research and testing environments.
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Lab products found in correlation
Zorbax eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
30 m j w capillary column, by Agilent Technologies (1 mentions)
Hplc grade syringe filters, by Pall Corporation (1 mentions)
Ultrahydrogel 120 column, by Waters Corporation (1 mentions)
5 protocols using agilent 1260 infinity high
1
Quantification of Free Amino Acids
2
Quantification of Chlorinated Ethenes and Arsenic
3
Molecular Mass Determination of PHMG and PHMB
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The weight-average molecular mass (Mw) was determined using gel permeation chromatography (GPC) using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA), equipped with a refractive index (RI) detector. A tandem of two Ultrahydrogel 120 columns (7.8 × 300 mm; 6 μm; Waters, Milford, MA, USA) was employed. HPLC grade water was eluted at a flow rate of 0.5 mL min−1 at 30 °C. Polyethylene glycol samples were used as mass calibrants. Aqueous solutions of PHMG and PHMB were prepared at 1000 mg L−1 for characterization.
4
HPLC Analysis of Phenolic Compounds
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HPLC quantification was carried out according to a reported method (Zeb 2015). Briefly, 1-g powdered sample of each of the whole and fractionated extract was added to water and methanol in equal ratios, and the mixture was subject to heating in a water bath at 70°C for 1 h. The mixture was then filtered through a non-pyogenic 0.4 μm CA syringe filter.
To identify and quantify phenolic compounds the Agilent-1260 infinity High-performance liquid chromatography (HPLC) system was used. The HPLC system’s essential parts were a quaternary pump, an auto-sampler, a degasser, and a C18 column (Agilent-Zorbax-Eclipse column). The solution (B and C) gradient was such that solvent B was a mixture of acetic acid: methanol: deionized water (20: 100: 180 v/v), and solvent C was a mixture of acetic acid deionized water: methanol (20: 80: 900) v/v. The solvents were provided as a gradient such that they started and gradually decreased the solvent in concentration. Solvent B was given in volume 100, 85, 50, and 30% at 0, 5, 20, and 25 min, finally giving way to 100% solvent C from 30 min onwards till 40 min. The ultraviolet array detector (UVAD) was set at wavelength 250 nm to analyze phenolic compounds, and the chromatograms were recorded. Phenolic compounds were identified by comparing the retention times of obtained HPLC chromatogram with that of the standards.
To identify and quantify phenolic compounds the Agilent-1260 infinity High-performance liquid chromatography (HPLC) system was used. The HPLC system’s essential parts were a quaternary pump, an auto-sampler, a degasser, and a C18 column (Agilent-Zorbax-Eclipse column). The solution (B and C) gradient was such that solvent B was a mixture of acetic acid: methanol: deionized water (20: 100: 180 v/v), and solvent C was a mixture of acetic acid deionized water: methanol (20: 80: 900) v/v. The solvents were provided as a gradient such that they started and gradually decreased the solvent in concentration. Solvent B was given in volume 100, 85, 50, and 30% at 0, 5, 20, and 25 min, finally giving way to 100% solvent C from 30 min onwards till 40 min. The ultraviolet array detector (UVAD) was set at wavelength 250 nm to analyze phenolic compounds, and the chromatograms were recorded. Phenolic compounds were identified by comparing the retention times of obtained HPLC chromatogram with that of the standards.
5
Tissue Elemental Analysis by HPLC-ICP-MS
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Agilent1260 Infinity high performance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA) with anion exchange Hamilton PRP-X100 (250 mm × 4.6 mm, particle size 10 µm) column (Hamilton, Reno, NV, USA) coupled with polyetheretherketone (PEEK) tubing to Elan 6100 DRC ICP–MS system (Perkin Elmer Sciex, QC, Canada), was used for tissue extract analysis.
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