NMR spectra were obtained on a Bruker Avance 500 spectrometer with residual solvent peaks as references (DMSO-d6: δH 2.50, δC 39.52). High-resolution ESIMS measurements were obtained on an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in the positive ion mode. HPLC was performed using an Agilent 1200 Series separation module equipped with an Agilent 1200 Series diode array, Agilent 1200 Series fraction collector, and Agilent ZORBAX SB-C18 column (250 × 9.4 mm, 5 µm).
1200 series separation module
Manufactured by Agilent Technologies
Sourced in Spain
The 1200 Series separation module is a key component of Agilent's analytical instrumentation portfolio. It is designed to perform liquid chromatography separations, a fundamental technique in analytical chemistry and biochemistry. The module facilitates the separation of complex mixtures into their individual components, enabling their identification and quantification. The core function of the 1200 Series separation module is to provide a reliable and efficient platform for liquid chromatography analysis.
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3 protocols using 1200 series separation module
1
NMR, ESIMS, and HPLC Characterization
2
HPLC Quantification of Omeprazole
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The HPLC system consisted of a 1200 Series separation module (Agilent Technologies, Madrid, Spain) combined with Agilent MassHunter Workstation Data Acquisition software for programming samples and controlling chromatographic conditions.
Separations were carried out at 25ºC in a Zorbax Extend C18 Rapid Resolution column (4.6 mm x 50 mm [particle size 3.5 µm] Agilent Technologies, Madrid, Spain). The mobile phase consisted of a combination of ammonium acetate 1 mM in water (pH = 8.5, solution A) and ACN (solution B). It is important to maintain this pH during recording, because OME is very sensitive to acidic pH. The chromatographic run was performed under isocratic conditions at a flow rate of 0.8 mL/min with 55% solution A and 45% solution B. The elution time of each sample was 0.889 min for OME and 0.884 min for OME D3. The total run time was 1.2 min, and a re-equilibration time was not required owing to the isocratic conditions used. At the end of every day, the column was washed by increasing the percentage of ACN to 100% at a 0.8-mL/min flow rate for 20 min and then returning to the initial conditions within 5 min. Washing was then continued for a further 10 min.
Separations were carried out at 25ºC in a Zorbax Extend C18 Rapid Resolution column (4.6 mm x 50 mm [particle size 3.5 µm] Agilent Technologies, Madrid, Spain). The mobile phase consisted of a combination of ammonium acetate 1 mM in water (pH = 8.5, solution A) and ACN (solution B). It is important to maintain this pH during recording, because OME is very sensitive to acidic pH. The chromatographic run was performed under isocratic conditions at a flow rate of 0.8 mL/min with 55% solution A and 45% solution B. The elution time of each sample was 0.889 min for OME and 0.884 min for OME D3. The total run time was 1.2 min, and a re-equilibration time was not required owing to the isocratic conditions used. At the end of every day, the column was washed by increasing the percentage of ACN to 100% at a 0.8-mL/min flow rate for 20 min and then returning to the initial conditions within 5 min. Washing was then continued for a further 10 min.
3
HPLC Analysis of Chromatographic Separation
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The HPLC system consisted of a 1200 Series separation module (Agilent Technologies, Madrid, Spain) controlled by Agilent MassHunter Workstation Data Acquisition software for programming samples and chromatographic conditions.
Separations were carried out at 25ºC in an ACE C18-PFP column (3-μm, 4.6 x 100 mm; SYMTA, Madrid, Spain) at 0.6 ml/min. The mobile phase consists of ammonium formate (5 mM, solvent A)-acetonitrile (solvent B) (pH 4.0; 65:35, v/v). The chromatogram was run under gradient condition as follows: initial conditions: 65% (A) and 35% (B); 0-0.1 min, gradually increasing eluent B to 75% (B); 0.1-0.5 min, gradually increasing eluent B to 90%; 0.5-1.5 min, gradually increasing eluent B to 99% and maintain from 1.5 to 3.0 min; 3.0-3.2 min returning to the initial conditions (65% A and 35% B) and maintain from 3.2 to 5.0 min. The chromatogram was followed by a reequilibration time of 3.0 min. The volume injected into the HPLC was 5 µl.
Separations were carried out at 25ºC in an ACE C18-PFP column (3-μm, 4.6 x 100 mm; SYMTA, Madrid, Spain) at 0.6 ml/min. The mobile phase consists of ammonium formate (5 mM, solvent A)-acetonitrile (solvent B) (pH 4.0; 65:35, v/v). The chromatogram was run under gradient condition as follows: initial conditions: 65% (A) and 35% (B); 0-0.1 min, gradually increasing eluent B to 75% (B); 0.1-0.5 min, gradually increasing eluent B to 90%; 0.5-1.5 min, gradually increasing eluent B to 99% and maintain from 1.5 to 3.0 min; 3.0-3.2 min returning to the initial conditions (65% A and 35% B) and maintain from 3.2 to 5.0 min. The chromatogram was followed by a reequilibration time of 3.0 min. The volume injected into the HPLC was 5 µl.
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