The LC-MS/MS determination of PA, IB, and CF in the wastewater sample was carried out in a HP 1100 chromatograph (Agilent Technologies, Palo Alto, CA, USA) attached to a 4000 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a turbospray electrospray (ESI) interface and was based on the method described in Garcia–Galan et al. [29 (link)]. The chromatographic separation was achieved using an Atlantis C18 (Waters, 150 mm × 2.1 mm, 6 µm) LC-column. The mobile phase consisted of HPLC-grade water and acetonitrile, both 0.1% in formic acid. MS/MS data acquisition was performed in the selected reaction monitoring (SRM) mode. Instrument control and data acquisition were carried out using the Analyst 1.4.2 software package Applied Biosystems.
Atlantis c18
Manufactured by Waters Corporation
Sourced in United States
The Atlantis C18 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C18 stationary phase that is well-suited for the retention and separation of both polar and non-polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
717 plus autosampler, by Waters Corporation (2 mentions)
Delta 600, by Waters Corporation (2 mentions)
4000 qtrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Analyst 1, by Thermo Fisher Scientific (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
515 hplc pump, by Waters Corporation (1 mentions)
Waters micromass zq, by Waters Corporation (1 mentions)
Waters 1525, by Waters Corporation (1 mentions)
Uv vis 2487 detector, by Waters Corporation (1 mentions)
Waters 515 hplc pump, by Waters Corporation (1 mentions)
Avance 600, by Bruker (1 mentions)
Autopurification system, by Waters Corporation (1 mentions)
2545 pump gradient module, by Waters Corporation (1 mentions)
Prominence hplc system, by Waters Corporation (1 mentions)
9 protocols using atlantis c18
1
Quantitative LC-MS/MS Analysis of Wastewater Analytes
2
Isolation and Characterization of Marker Compound from Taraxacum Koreanum Extract
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Dried TTK (4 kg) was extracted with 70% ethanol (40 L) for 3 days at 25–30 °C and then filtered. After concentrating the 70% ethanol layers and lyophilization, the TTK extracts were stored at −70 °C until use. Final yield of the 70% ethanol extract was 22.43% w/w (992.6 g). A quantitative analysis was performed using an 1100 series high-performance liquid chromatographδ system (HPLC, Agilent Technologies, Santa Clara, CA, USA). The analytical column with an Atlantis C18 (4.6 × 250 nm, 5 μm, Waters, MA, USA) was maintained at 30 °C during the experiment. The mobile phase included distilled water (DW) with 0.1% trifluoroacetic acid (A) and acetonitrile (B). The gradient flow was as follows: 0–25 min, 10–15% (v/v) B; 25–50 min, 15–30% (v/v) B; 50–60 min, and 30–100% (v/v) B. The analytes were detected at 330 nm and operated at a mobile phase flow rate of 1.0 mL/min. The injection volume was 10 μL. The data were acquired and processed by ChemStation software (Agilent Technologies). We isolated marker compound 1 from TTK, and the compound were identified as 6-methoxykaempferol-3-O-β-d -glucosyl(1′′′→2″)-β-D-gluco-pyranosyl-(6″″-(E)-caffeoyl)-7-O-β-d -glucopyranoside via nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) experiments.
3
HPLC Analysis of Prenylated Flavanones
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The HPLC system consisted of a Waters 515 HPLC pump, a 717 Plus autosampler, and a dual λ absorbance UV-vis 2487 detector (Waters, Milford, MA, USA). The analytical column was Atlantis® C18 5 μm 250 mm × 4.6 mm, Waters. The analyte separation was performed with 10 μL sample injection volume. The separations were done in isocratic mode at room temperature. The mobile phase with a flow rate of 1 mL/min comprised of W-water and AcN-acetonitrile (%W: %AcN) with a different composition for each prenylated flavanone studied: 1 (30:70), A (20:80), B (40:60), C (20:80) and D (10:90). The detection wavelengths determined by spectrum scan were 300 nm for 1 , B , C , D , and 320 nm for A . The Peak area was used to quantify each analyte.
4
CML Quantification via HPLC-MS
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Each fraction collected from FPLC separation was hydrolyzed in 6 M HCl at 110 °C for 24 h and was then vacuum dried (40 °C).31 (link) After drying, each fraction was dissolved in 0.5 ml of aqueous methanol (10%) and filtered through a membrane (0.46 μm) before HPLC-MS analysis.
The HPLC-MS method was the same as that of Han, with some modifications:32 (link) CML was analyzed via HPLC (Waters 1525, Waters, USA) in tandem with a single quadrupole mass spectrometer (Waters Micromass ZQ, Waters, USA). An Atlantis C18 (Waters, USA) column was selected for the HPLC separations, with aqueous methanol (10%) as the mobile phase at a flow rate of 0.5 ml min−1. The measurements were collected under the following conditions: a capillary voltage of 3.0 kV; a cone voltage of 20 V; a source temperature of 100 °C; and a desolvation temperature of 300 °C. Electrospray ionization (ESI) in positive mode and single ion recording (SIR) were applied to the +205.1 (CML), +166.2 (Phe), +182.2 (Tyr), +132.2 (Leu), +118.1 (Val), +120.1 (Thr), +116.1 (Pro), and +133.1 (Asn) channels. The CML/Lys content in digested fractions was expressed in relative amounts, considering the ion suppression from the salts of the SEC mobile phase on the MS signal, and assuming that 100% was equal to the sum of CML or Lys in all fractions.
The HPLC-MS method was the same as that of Han, with some modifications:32 (link) CML was analyzed via HPLC (Waters 1525, Waters, USA) in tandem with a single quadrupole mass spectrometer (Waters Micromass ZQ, Waters, USA). An Atlantis C18 (Waters, USA) column was selected for the HPLC separations, with aqueous methanol (10%) as the mobile phase at a flow rate of 0.5 ml min−1. The measurements were collected under the following conditions: a capillary voltage of 3.0 kV; a cone voltage of 20 V; a source temperature of 100 °C; and a desolvation temperature of 300 °C. Electrospray ionization (ESI) in positive mode and single ion recording (SIR) were applied to the +205.1 (CML), +166.2 (Phe), +182.2 (Tyr), +132.2 (Leu), +118.1 (Val), +120.1 (Thr), +116.1 (Pro), and +133.1 (Asn) channels. The CML/Lys content in digested fractions was expressed in relative amounts, considering the ion suppression from the salts of the SEC mobile phase on the MS signal, and assuming that 100% was equal to the sum of CML or Lys in all fractions.
5
HPLC Quantification of Flavanone Compounds
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The flavanone solutions (FS1 , FS1a –FS1d ) were prepared by dissolving 5.0 mg of the respective flavanone (1 , 1a , 1b , 1c and 1d ) with 1 mL of the mixture of EtOH/H2O (7:3). The purity and chemical stability of dissolutions were examined after 5 days of their preparation storage at 4 ± 1 °C using the validated analytic method described before in terms of linearity, accuracy, and precision [60 (link)]. Briefly, the flavanones were determined by high performance liquid chromatography (HPLC) on a Waters system, equipped with a Waters 515 HPLC pump, a 717 Plus autosampler, and a dual λ absorbance UV-vis 2487 detector (Waters, Milford, Worcester, MA, USA). The chromatographic column used was a reversed phase column Atlantis® C18 (5 µm 250 mm × 4.6 mm, Waters). The separations were carried out with a flow rate of 1 mL/min under isocratic elution. The mobile phase was W-water and AcN-acetonitrile (% W: % AcN) with a different composition for each flavanone: 1 (30:70), 1a (20:80), 1b (40:60), 1c (20:80) and 1d (10:90) and detection wavelength at 300 nm for 1 , 1b , 1c , 1d , and at 320 nm for 1a . The Peak area was used to quantify each analyte.
6
HPLC-MS Characterization of Organic Compounds
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Chemicals were
purchased from Sigma Aldrich Co. NMR spectra were
recorded at 310 K on a Bruker AVANCE 600 spectrometer operating at
14 T (corresponding to 600 and 150 MHz 1H and 13C Larmor frequencies, respectively). Analytical and preparative HPLC–MS
was carried out on a Waters Auto Purification system (3100 Mass Detector,
2545 Pump Gradient Module, 2767 Sample Manager, and 2998 PDA detector).
The purity was double checked by analytical HPLC using an Atlantis
C18, 3.5 μm, 4.6 mm × 150 mm column and 0.1% TFA in water
(solvent A) and acetonitrile (solvent B); applying a gradient of CH3CN in H2O (0.1%TFA) from 5 to 80% in 15 min and
from 80 to 100% in 5 min, flow 1 mL min–1 (method
1). pH measurements were made by using an AS pH meter equipped with
a glass electrode.
purchased from Sigma Aldrich Co. NMR spectra were
recorded at 310 K on a Bruker AVANCE 600 spectrometer operating at
14 T (corresponding to 600 and 150 MHz 1H and 13C Larmor frequencies, respectively). Analytical and preparative HPLC–MS
was carried out on a Waters Auto Purification system (3100 Mass Detector,
2545 Pump Gradient Module, 2767 Sample Manager, and 2998 PDA detector).
The purity was double checked by analytical HPLC using an Atlantis
C18, 3.5 μm, 4.6 mm × 150 mm column and 0.1% TFA in water
(solvent A) and acetonitrile (solvent B); applying a gradient of CH3CN in H2O (0.1%TFA) from 5 to 80% in 15 min and
from 80 to 100% in 5 min, flow 1 mL min–1 (method
1). pH measurements were made by using an AS pH meter equipped with
a glass electrode.
7
Synthesis and Characterization of TTF Peptides
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All chemical reagents and solvents are purchased from Sigma-Aldrich, Fisher, AnaSpec, Alfa-Aesar, and Cambridge Isotope Laboratories without further purification. All chemical reactions are performed under inert N2 atmospheres unless otherwise noted. All NMR experiments are performed on INOVA 400 MHz or INOVA 600 MHz NMR spectrometers.
H–TTFTTF–NH2 (TTF)2 and H–TTFTTFTTF–NH2 (TTF)3 peptides are synthesized using standard Fmoc solid-phase synthesis protocols on CEM Liberty Microwave Automated Peptide Synthesizer on rink amide resin. The peptides are cleaved from dried resin (resulting in C-terminal amine capped peptide) by addition of cleavage cocktail (90 vol% TFA, 5 vol% thioanisole, 3 vol% ethanedithiol and 2 vol% anisole) for 4 h at room temperature, precipitated and washed in cold diethyl ether then purified by reverse-phase HPLC (Waters Delta 600) using a Waters Atlantis C-18 preparative column (19 × 250 mm) with acetonitrile–water gradient and lyophilized. The molecular identities of purified peptides are confirmed by MALDI-TOF mass spectrometry. Structural models were constructed by energy minimizing (MacroModel v9.9. Schrodinger, LLC63 (link)) H-bonded peptides (OPLS 2005 force field64 (link)) arranged as β-strands.
H–TTFTTF–NH2 (TTF)2 and H–TTFTTFTTF–NH2 (TTF)3 peptides are synthesized using standard Fmoc solid-phase synthesis protocols on CEM Liberty Microwave Automated Peptide Synthesizer on rink amide resin. The peptides are cleaved from dried resin (resulting in C-terminal amine capped peptide) by addition of cleavage cocktail (90 vol% TFA, 5 vol% thioanisole, 3 vol% ethanedithiol and 2 vol% anisole) for 4 h at room temperature, precipitated and washed in cold diethyl ether then purified by reverse-phase HPLC (Waters Delta 600) using a Waters Atlantis C-18 preparative column (19 × 250 mm) with acetonitrile–water gradient and lyophilized. The molecular identities of purified peptides are confirmed by MALDI-TOF mass spectrometry. Structural models were constructed by energy minimizing (MacroModel v9.9. Schrodinger, LLC63 (link)) H-bonded peptides (OPLS 2005 force field64 (link)) arranged as β-strands.
8
Extracting and Analyzing Plant Pigments
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Pigments were extracted from cotyledons and upper parts (∼2–4 mm) of seedling hypocotyls (n ≈ 200) in 1 mL acetone:ethyl acetate 3:2 (v/v) as described in Cuttriss et al., 2007 (link). Extracted pigments were separated using the Shimadzu Prominence HPLC System with PDA detector on Atlantis C-18 (4.6 × 250 mm, 5 μm, 100 Å) column (Waters). Elution was performed using ethyl acetate gradient in acetonitrile:water:triethylamine 9:1:0.01 (v/v) at 1 mL min–1 for 40 min according to the following timetable (0–2 min, 0% ethyl acetate; 2–32 min 0–66.7%, 32.2–37 min 66.7–100%). Compounds were identified, and the HPLC peak areas were integrated based on absorption spectra and retention times, according to Cuttriss et al. (2007) (link); peak areas were integrated at 456 nm.
9
Synthesis and Characterization of TTF Peptides
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All chemical reagents and solvents are purchased from Sigma-Aldrich, Fisher, AnaSpec, Alfa-Aesar, and Cambridge Isotope Laboratories without further purification. All chemical reactions are performed under inert N2 atmospheres unless otherwise noted. All NMR experiments are performed on INOVA 400 MHz or INOVA 600 MHz NMR spectrometers.
H-TTFTTF-NH2 (TTF)2 and H-TTFTTFTTF-NH2 (TTF)3 peptides are synthesized using standard Fmoc solid-phase synthesis protocols on CEM Liberty Microwave Automated Peptide Synthesizer on rink amide resin. The peptides are cleaved from dried resin (resulting in C-terminal amine capped peptide) by addition of cleavage cocktail (90 vol% TFA, 5 vol% thioanisole, 3 vol% ethanedithiol and 2 vol% anisole) for 4 h at room temperature, precipitated and washed in cold diethyl ether then purified by reverse-phase HPLC (Waters Delta 600) using a Waters Atlantis C-18 preparative column (19 × 250 mm) with acetonitrile-water gradient and lyophilized. The molecular identities of purified peptides are confirmed by MALDI-TOF mass spectrometry. Structural models were constructed by energy minimizing (MacroModel v9.9. Schrodinger, LLC 62 ) H-bonded peptides (OPLS 2005 force field 63 ) arranged as β-strands.
H-TTFTTF-NH2 (TTF)2 and H-TTFTTFTTF-NH2 (TTF)3 peptides are synthesized using standard Fmoc solid-phase synthesis protocols on CEM Liberty Microwave Automated Peptide Synthesizer on rink amide resin. The peptides are cleaved from dried resin (resulting in C-terminal amine capped peptide) by addition of cleavage cocktail (90 vol% TFA, 5 vol% thioanisole, 3 vol% ethanedithiol and 2 vol% anisole) for 4 h at room temperature, precipitated and washed in cold diethyl ether then purified by reverse-phase HPLC (Waters Delta 600) using a Waters Atlantis C-18 preparative column (19 × 250 mm) with acetonitrile-water gradient and lyophilized. The molecular identities of purified peptides are confirmed by MALDI-TOF mass spectrometry. Structural models were constructed by energy minimizing (MacroModel v9.9. Schrodinger, LLC 62 ) H-bonded peptides (OPLS 2005 force field 63 ) arranged as β-strands.
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