Reversed-phase HPLC analysis of GLA was performed on an Agilent 1200 series HPLC (Agilent Technologies). Separation was performed on a C18 Atlantis T3 reversed-phase column (Waters, Milford, MA, USA) with a gradient over 30 minutes. Mobile phase A contained 75:15:10 (v/v/v) methanol:chloroform:water, 1% (v/v) acetic acid, and 20 mM ammonium acetate. Mobile phase B contained 50:50 (v/v) methanol:chloroform, 1% (v/v) acetic acid, and 20 mM ammonium acetate. Column temperature was held constant at 30°C, and GLA was detected with a Corona Charged Aerosol Detector (CAD) (ESA Biosciences, Chelmsford, MA, USA). Triplicate samples were diluted 1:50 in mobile phase B and analyzed using a nine-point GLA standard curve prepared in mobile phase B. The peak areas from the standards were determined by integration, and the peak areas were fit with a second-order polynomial. GLA content of samples was calculated by interpolation.
Atlantis t3 c18
Manufactured by Waters Corporation
Sourced in United States
The Atlantis T3 C18 is a reverse-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with a C18 bonded ligand, which provides reliable and consistent performance for a variety of applications.
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Lab products found in correlation
Agilent 1200 series hplc, by Agilent Technologies (1 mentions)
Glcnac 1 p, by Merck Group (1 mentions)
Ltq orbitrap xl system, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Bodipy fl nhs ester, by Thermo Fisher Scientific (1 mentions)
Xterra c18, by Waters Corporation (1 mentions)
Breeze 2 hplc system, by Waters Corporation (1 mentions)
D2000, by Hitachi (1 mentions)
8 protocols using atlantis t3 c18
1
Reversed-phase HPLC Analysis of GLA
2
HPLC-Based GLA Quantification
3
Enzymatic Assay for UDP-GlcNAc Production
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The activity and substrate specificity of the enzyme Ccan_15070 was assessed by incubating 30 µg of purified recombinant Ccan_15070 protein with 1,245 µg of UTP (catalog number U6625; Sigma) and 500 µg of GlcNAc-1-P (A2142; Sigma) in 1 ml 100 mM Tris-HCl (pH 7.5) and 10 mM MgCl2 solution for 16 h at 37°C. As a control, the same amount of substrate was incubated in the absence of recombinant purified Ccan_15070. UDP-GlcNAc production was detected by HPLC using a Waters 600 E device with a C18 Atlantis T3, 5 µm, 4.60- by 250-mm column. Elution was done with 50 mM triethylimine acetic acid, pH 6.8, 0.5% CH3CN; detection was at 262 nm and at a flow rate of 1 ml/min.
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HPLC-MS Metabolite Separation Workflow
5
Synthesis and Characterization of PARPi-FL
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The green fluorescent dye BODIPY-FL NHS-ester (Invitrogen, Carlsbad, CA) was conjugated to 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one and purified by preparative HPLC (Waters’ XTerra C-18 5 μm column, 7 mL/min, 5 to 95% of acetonitrile in 15 min) to afford PARPi-FL in 70–79% yield as a red solid. Analytical HPLC analysis (Waters’ Atlantis T3 C18 5 μm 4.6 × 250 mm column) showed high purity (>97%) of the imaging agent. The identity of PARPi-FL was confirmed using ESI-MS (MS(+) m/z = 663.4 [M + Na]+).
6
Reversed-Phase HPLC Determination
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The Agilent 1200 HPLC system was equipped with a reversed-phase column (Atlantis T3C18, 2.1×150 mm i.d., 5 µm; Waters CA) protected by a 4.0×3.0 mm i.d. Security Guard (5 µm) C18 guard column (Phenomenex, Torrance, CA). Mobile phases A and B consisted of 0.1% (volume fraction) formic acid in water and methanol, respectively. Gradient elution at a flow rate of 0.2 ml/min was with the following program: initial 70% B for 3 min, increased to 95% in 14 min, maintaining this condition for 8 min, ramping back down to 70% B in 1 min and holding at this condition for a further 4 min, to give a total runtime of 30 min.
7
Peptide Fractionation via HPLC
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Reconstituted peptide extracts were fractionated using either a Breeze 2 HPLC system (Waters, Milford, MA) equipped with a reversed-phase column (Atlantis T3, C18, 2.1-mm inner diameter × 150 mm length, 5-μm particle size, 100 Å pore size; Waters), or a Magic 2002 analytical LC system (Michrom Bioresources Inc., Auburn, CA), also using a reversed-phase column (Vydac, C18, 2.1-mm inner diameter × 150 mm length, 5-μm particle size, 300 Å pore size; W. R. Grace & Co., Columbia, MD). Solvent gradients were established by mixing solvents A and B. Fractions were collected with an analytical gradient of ∼10–50% solvent B ramped over a period of 35–45 min. For the Breeze 2 HPLC system, solvent A was water + 1% acetonitrile (ACN) + 0.1% FA, and solvent B was ACN + 0.1% FA. For the Magic 2002 analytical LC system, solvent A was 5% ACN + 0.1% FA + 0.01% trifluoroacetic acid (TFA), and solvent B was 95% ACN + 0.1% FA + 0.01% TFA.
8
Aflatoxin Quantification in Chili Peppers
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We used high-performance liquid chromatography (HPLC; D-2000, Hitachi, Tokyo) to measure the AFB1, AFB2, AFG1, and aflatoxin G2 (AFG2) concentrations in the red chili peppers by our previously described method (Asai et al., 2012) . Briefly, we used the following operation conditions: column: Atlantis T3 C18 (5-μm particle size, 250 mm × 3.0 mm, Waters, Milford, MA, USA); column temperature: 40°C; mobile phase: acetonitrile : methanol : water (1 : 3 : 6, v/v/v); flow rate: 0.4 mL/min; detection wavelength: excitation wavelength 365 nm/emission wavelength 450 nm; injection volume: 20 μL.
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