HPLC analysis was conducted with a Zorbax Eclipse XDB-C18 (4.6 × 12.5 mm, 5 µM) guard column and a Zorbax Eclipse XDB-C18 (4.6 × 150 mm, 5 µM) analytical column (both at 25 °C), with a flow rate of 1 ml min−1 (ddH2O with 0.1% (v/v) TFA at pump A; HPLC grade CH3CN with 0.1% (v/v) TFA at pump B). A linear gradient method was used: pre-run equilibration, 3.0% pump B; 0.00 min, 3.0% pump B; 3.00 min, 3.0% pump B; 8.00 min, 97.0% pump B; 8.01 min, 3.0% pump B; 13.00 min, 3.0% pump B. The autosampler was maintained at 37 °C. Following injection, the needle was washed with CH3CN. The photodiode array detector used a 4 nm slit width and was autobalanced pre-run and post-run. Integration events were automated with the following features: tangent skim mode = standard; tail peak skim height ratio = 0.00; front peak skim height ratio = 0.00; skim valley ratio = 20.00; baseline correction = classical; peak to valley ratio = 500.00; slope sensitivity = 0.751; peak width = 0.121; area reject = 2.536; height reject = 0.176; shoulders = off.
Eclipse xdb c18
Manufactured by Agilent Technologies
Sourced in United States, Germany, Japan, France
The Eclipse XDB-C18 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a stable, spherical silica-based stationary phase with a C18 alkyl-bonded ligand. The column is suitable for both reversed-phase and normal-phase liquid chromatography applications.
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Lab products found in correlation
1100 series, by Agilent Technologies (6 mentions)
Agilent 1260, by Agilent Technologies (5 mentions)
Chemstation software, by Agilent Technologies (5 mentions)
Agilent 1200, by Agilent Technologies (5 mentions)
1200 series, by Agilent Technologies (4 mentions)
1260 infinity, by Agilent Technologies (4 mentions)
Agilent 1200 series, by Agilent Technologies (4 mentions)
Spd 20a, by Shimadzu (3 mentions)
1200 series hplc, by Agilent Technologies (3 mentions)
1100 series hplc system, by Agilent Technologies (3 mentions)
1100 hplc system, by Agilent Technologies (3 mentions)
Hplc system, by Agilent Technologies (3 mentions)
Ms 500 mass spectrometer, by Agilent Technologies (3 mentions)
1200 series hplc system, by Agilent Technologies (3 mentions)
Methanol, by Merck Group (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (3 mentions)
Waters 1525 binary pump, by Waters Corporation (2 mentions)
Lachrom elite, by Hitachi (2 mentions)
Agilent 1100 system, by Agilent Technologies (2 mentions)
Chemstation, by Agilent Technologies (2 mentions)
287 protocols using eclipse xdb c18
1
HPLC Analysis of Organic Compounds
2
Radiolabeling of cRGD Peptides
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[125I]NaI with
a 10 mCi activity in an aqueous solution of 0.1 M NaOH was supplied
by PerkinElmer Republic of Korea. All chemicals including 4-amino-3-nitrophenol,
methyl 5-bromovalerate, zinc dust (<10 μm) N-(2-aminoethyl)maleimide trifluoroacetate, chloramine-T trihydrate,
tris(2-carboxyethyl)phosphine hydrochloride (TCEP), DIPEA, and HSA
were purchased from Sigma-Aldrich. Cysteine containing the cRGD peptide
was purchased from Peptide International. All chemicals were pure
and used without further purification step. HPLC experiments were
performed using an Agilent Technologies 1290 infinite analytical HPLC
system (Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) and 1260 infinite
preparative HPLC system (Eclipse XDB-C18, 21.2 × 150 mm, 7 μm).
SolventA (0.1% formic acid in deionized water) and solvent B (0.1% formic acid in acetonitrile) were used for the HPLC
analysis and purification. All nuclear magnetic resonance (13C NMR and 1H NMR) spectra were acquired using a JEOL 500
MHz spectrometer with DMSO-d6, acetone-d6, or chloroform-d (CDCl3) as a solvent. Chemical shifts are given as δ (ppm)
relative to tetramethylsilane (0.0 ppm) as an internal standard; multiplicities
are given as singlet (s), doublet (d), doublet-of-doubles (dd), or
multiplet (m). Agilent ESI-TOF analyzer and 4800 MALDI TOF/TOF Analyzer-(AB
SCIEX) were used for mass spectroscopy.
a 10 mCi activity in an aqueous solution of 0.1 M NaOH was supplied
by PerkinElmer Republic of Korea. All chemicals including 4-amino-3-nitrophenol,
methyl 5-bromovalerate, zinc dust (<10 μm) N-(2-aminoethyl)maleimide trifluoroacetate, chloramine-T trihydrate,
tris(2-carboxyethyl)phosphine hydrochloride (TCEP), DIPEA, and HSA
were purchased from Sigma-Aldrich. Cysteine containing the cRGD peptide
was purchased from Peptide International. All chemicals were pure
and used without further purification step. HPLC experiments were
performed using an Agilent Technologies 1290 infinite analytical HPLC
system (Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) and 1260 infinite
preparative HPLC system (Eclipse XDB-C18, 21.2 × 150 mm, 7 μm).
Solvent
analysis and purification. All nuclear magnetic resonance (13C NMR and 1H NMR) spectra were acquired using a JEOL 500
MHz spectrometer with DMSO-d6, acetone-d6, or chloroform-d (CDCl3) as a solvent. Chemical shifts are given as δ (ppm)
relative to tetramethylsilane (0.0 ppm) as an internal standard; multiplicities
are given as singlet (s), doublet (d), doublet-of-doubles (dd), or
multiplet (m). Agilent ESI-TOF analyzer and 4800 MALDI TOF/TOF Analyzer-(AB
SCIEX) were used for mass spectroscopy.
3
Chromatographic Separation of NMPs
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Chromatographic separation of nucleoside 5′-monophosphates (NMPs) was achieved on an Eclipse XDB-C18 analytical column (5.0 µm, 4.6 mm × 150 mm, Agilent) equipped with an Eclipse XDB-C18 analytical guard column (5.0 µm, 4.6 × 12.5 mm). The gradient mobile phase contained the DMH ion pair reagent (Auriola et al. 1997 (link)) and comprised two eluents: eluent A, which was 20 mM aqueous DMH adjusted to pH 4.8 with formic acid, and eluent B, which was a 1:1 (v/v) mixture of acetonitrile and aqueous 20 mM DMH adjusted to pH 4.8 with formic acid. The elution was performed at room temperature (RT) and at a flow rate of 700 µL min−1. The gradient was optimized to obtain sufficient chromatographic separation of all analytes of interest (AMP, AMPS, and GMP). The optimal gradient was 0%–100% eluent B in 15 min.
4
Comprehensive Analytical Characterization of Compounds
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Optical rotations were measured on a PerkinElmer 341-MC digital polarimeter, UV spectra were recorded on a TU-1900 spectrophotometer; A Hitachi 260-30 spectrometer was used for scanning IR spectroscopy; Experimental ECD spectra were recorded on a JASCO J-815 Circular Dichroism (CD) Spectropolarimeter; NMR spectra were performed on Bruker ARX-600 spectrometers, and on Agilent DD2-500 NMR spectrometer (500) MHz; HRESIMS were performed on a UPLC/xevo G2 Qtof spectrometer.
Preparative RP-HPLC was conducted on Agilent 1260 Infinity Series equipped with quaternary pump with Eclipse XDB-C18 (5 μm 9.4 × 250 mm) column at flow rate of 2.5 mL/min, at 210 nm UV detection using single wavelength detector. While the separation conditions were optimized on semi-preparative Agilent 1260 HPLC equipped with DAD detector by using Eclipse XDB-C18 (5 μm 4.6 × 250 mm) at flow rate of 1 mL/min. Thin layer Chromatography was performed on TLC aluminum sheets pre-coated with silica gel GF254 (EMD Chemicals, Merck KGaA, Dermstadt, Germany), visualized under UV light of 254 and 365 nm followed by 5% vanilline-H2SO4 reagent, and heat.
Preparative RP-HPLC was conducted on Agilent 1260 Infinity Series equipped with quaternary pump with Eclipse XDB-C18 (5 μm 9.4 × 250 mm) column at flow rate of 2.5 mL/min, at 210 nm UV detection using single wavelength detector. While the separation conditions were optimized on semi-preparative Agilent 1260 HPLC equipped with DAD detector by using Eclipse XDB-C18 (5 μm 4.6 × 250 mm) at flow rate of 1 mL/min. Thin layer Chromatography was performed on TLC aluminum sheets pre-coated with silica gel GF254 (EMD Chemicals, Merck KGaA, Dermstadt, Germany), visualized under UV light of 254 and 365 nm followed by 5% vanilline-H2SO4 reagent, and heat.
5
HPLC Analysis of 1-Deoxynojirimycin
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HPLC analysis was performed using an HP-1100 system (Hewlett Packard, Palo Alto, CA, USA). A reversed phase column (Eclipse XDB-C18, 150 mm × 4.6 mm id, 5 μm; Agilent, Palo Alto, CA, USA) was connected with a guard column (Eclipse XDB-C18, 4.0 mm × 4.6 mm id, 5 μm; Agilent, Palo Alto, CA, USA). An isocratic mobile phase system composed of acetonitrile and 0.1% v/v trifluoroacetic acid at a ratio of 45:55 was used to elute the sample at a flow rate of 1 mL/min. All samples were detected for UV absorbance (Hewlett Packard, Palo Alto, CA, USA) at 254 nm. 1-Deoxynojirimycin was used as a marker for quantitative analysis. All experiments were performed in triplicate.
6
HPLC Separation of Complex Mixtures
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Chromatographic separation was performed on an Agilent HPLC 1200 system with a C18 column (Agilent Eclipse XDB C18, 4.5 mm × 150 mm, 5 μm) equipped with a guard column (Agilent Eclipse XDB C18, 4.5 mm × 12.5 mm, 5 μm), a G1311A quaternary pump, a G1329A autosampler, and a G1316A column oven.
The mobile phase was composed of solvent A (2 mM ammonium formate and 0.05% formic acid in water) and solvent B (2 mM ammonium formate and 0.05% formic acid in acetonitrile). The column was maintained at 45 °C and eluted with a gradient of 10% B (0–1 min), 10–30% B (1–2 min), 30–50% B (2–6 min), 50–70% B (6–13 min), and 70–95% B (13–13.5 min), and the column was then flushed with 95% B (13.5–16.5 min), 95–10% B (16.5–18 min). The total run time was 18 min at a flow rate of 0.20 mL min−1. The temperature of the auto–sampler prior to analysis was maintained at 8 °C. The injection volume was fixed at 5 μL in the partial loop with the needle overfill mode.
The mobile phase was composed of solvent A (2 mM ammonium formate and 0.05% formic acid in water) and solvent B (2 mM ammonium formate and 0.05% formic acid in acetonitrile). The column was maintained at 45 °C and eluted with a gradient of 10% B (0–1 min), 10–30% B (1–2 min), 30–50% B (2–6 min), 50–70% B (6–13 min), and 70–95% B (13–13.5 min), and the column was then flushed with 95% B (13.5–16.5 min), 95–10% B (16.5–18 min). The total run time was 18 min at a flow rate of 0.20 mL min−1. The temperature of the auto–sampler prior to analysis was maintained at 8 °C. The injection volume was fixed at 5 μL in the partial loop with the needle overfill mode.
7
Phenolic Profiling of Oat Grain
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The identification of phenolic compounds in the grain of A. sativa was done using the HPLC system (consisting of a vacuum degasser, an autosampler, and a binary pump with a maximum pressure of 400 bar; Agilent 1260, Agilent Technologies, Germany) equipped with a reversed-phase C18 analytical column of 4.6 × 100 mm and 3.5 μm particle size (Zorbax Eclipse XDB C18). The DAD detector was set to a scanning range of 200-400 nm. Column temperature was maintained at 25°C. The injected sample volume was 2 μl, and the flow rate of mobile phase was 0.4 ml/min (mobile phase B consisted of 0.1% formic acid and mobile phase A was methanol). The optimized gradient elution was illustrated as follows: 0-5 min, 10-20% A; 5-10 min, 20-30% A; 10-15 min, 30-50% A; 15-20 min, 50-70% A; 20-25 min, 70-90% A; 25-30 min, 90-50% A; and 30-35 min, return to initial conditions. Identification analysis was done by comparison of their retention time with those obtained from the extract. For the quantitative analysis, a calibration curve was obtained by plotting the peak area against different concentrations for each identified compound at 280 nm [24 (link)].
8
Enrofloxacin Quantification by HPLC
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Chromatographic conditions: column (ZORBAX Eclipse XDB-C18 150 mm × 4.6 mm); Column temperature 303 K; injection volume 20 μL; Determination of enrofloxacin mobile phase was methanol: water = 30:70 (V/V) The flow rate was 1.0 mL·L−1, the UV detection wavelength was 275 nm, and the retention time was 4.6 min.
9
Isolation and Purification of Shikonin Derivatives
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Petroleum ether-methylene chloride extract of the O. visianii roots was prepared as previously described in our paper (Vukic et al., 2017[47 (link)]). Obtained extract was fractionated using preparative TLC eluted by mobile phases consisting of petroleum ether:chloroform:ethyl acetate:acetic acid (5:2:2.5:0.5) to give eight fractions (F1-F8). Preliminary TLC analysis was conducted, and retention times were compared to those of previously isolated compounds. Out of eight obtained fractions, presence of α-methylbutyrylshikon (1 ), acetylshikonin (2 ) and β-hydroxyisovalerylshikonin (3 ) was confirmed in three fractions (F4, F6 and F7) which were further subjected to Sephadex LH20 column chromatography with methanol as eluent. After column chromatography, another TLC examination was performed using petroleum ether:ethyl acetate (90:10) to confirm isolated compounds. With the aim of obtaining high purity, isolated α-methylbutyrylshikon (1 ), acetylshikonin (2 ) and β-hydroxyisovalerylshikonin (3 ) were subjected to semi preparative HPLC on Zorbax Eclipse XDB C18 reversed phase column with isocratic elution of mixture water and methanol (40:60). Obtained spectra (UV, IR, 1H NMR and 13C NMR) were in agreement with previously published data (Vukic et al., 2017[47 (link)]).
10
Quantification of VPA and VPAG in Biological Fluids
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Quantification of VPA and VPAG in serum and urine were determined by HPLC coupled with mass spectrometry. To a 100 μL plasma/urine sample, 100 μL of IS and 800 μL of acetonitrile were added. The resulting solution was vortexed for 30 sec and centrifuged at 13,000 × g for 20 min. Next, the supernatant was transferred to an HPLC vial for injection. HPLC separation was performed on an Agilent 1100 system (Agilent, Palo Alto, CA, USA). Chromatography separation was performed using a Zorbax Eclipse XDB-C18 (150 × 2.1 mm internal diameter, 3.5 μm particle size). The mobile phase was composed of solvent A, 10 mM ammonium formate, and solvent B, acetonitrile. VPA, VPAG, and IS quantifications were achieved using negative ion-mode electrospray ionization with multiple reaction monitoring (MRM). The MRM transitions of m/z 319 to 174.8 for VPAG, m/z 143 to 143, and m/z 157 to 157 were used for analysis.
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