The determination of amino acids was performed using high-performance liquid chromatography, HPLC (Agilent Technologies, mod. 1260 Infinity, Santa Clara, CA, USA), in the reverse phase by precolumn derivatization reaction with orthophthaldehyde (OPA) in the presence of mercaptoethanol. The liquid chromatograph used consisted of a 1525 pump system, a 2475 fluorescence detector, 356 nm excitation and 445 nm emission wavelength (λ), and a 717 automatic injector (Waters, Milford, Massachusetts, USA). Equipment monitoring and data acquisition and processing were performed through Waters’ Breeze program. Separations were carried out using a Nova-Pack C18 column (3.9 × 150 mm) from Waters. OPA was used as a derivatizing reagent; 750 mg of OPA was dissolved in 5 mL of methanol and 0.5 mL of 2-mercaptoethanol was added. The protocol was followed, and the chromatographic conditions used are detailed in Pripis-Nicolau et al. (2001) [26 (link)].
Nova pack c18 column
Manufactured by Waters Corporation
Sourced in United States
The Nova-Pack C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a C18 bonded stationary phase and is suitable for use in reversed-phase HPLC applications.
Automatically generated - may contain errors
Lab products found in correlation
717 automatic injector, by Waters Corporation (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Agilent 1200 series chromatograph, by Agilent Technologies (1 mentions)
Volume formic acid, by Merck Group (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
1100 series, by Agilent Technologies (1 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
Waters 717 plus autosampler, by Waters Corporation (1 mentions)
Millennium 32, by Waters Corporation (1 mentions)
Lc module 1, by Waters Corporation (1 mentions)
9 protocols using nova pack c18 column
1
HPLC Amino Acid Analysis by OPA Derivatization
2
Quantification of Diene Valepotriates via HPLC
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To obtain the VAL fraction, 100 g (dry weight) of dried and powdered plant material was submitted to supercritical CO2 (SCCO2) extraction, using a Pilot Equipment as described elsewhere [37 (link), 40 (link), 41 (link)]. The conditions of the extraction were 40°C, 90 bar, SCCO2 flow rate through the extraction vessel: 6.67 × 10−4 kg s−1. The SCCO2 extraction recovery was 2.96 g%.
The VAL fraction was dissolved in HPLC grade methanol and filtered (0.22 μm pore size, Merck) before the analysis by HPLC according to a method previously described [37 (link), 41 (link), 42 (link)], using Shimadzu HPLC system and Waters Nova-Pack C18 column (4 mm, 3.9 × 150 mm i.d. with Waters Nova-Pack C-18 guard column, 60 Å, 3.9 × 20 mm). The isocratic mobile phase consisted of acetonitrile and water (50 : 50 v/v); flow rate of 1 mL/min; UV detection at 254 nm. All diene valepotriates were quantified in terms of mg of valtrate equivalent/g extract. The VAL fraction was suspended in saline with 1% of polysorbate 80 (vehicle) prior to use.
The VAL fraction was dissolved in HPLC grade methanol and filtered (0.22 μm pore size, Merck) before the analysis by HPLC according to a method previously described [37 (link), 41 (link), 42 (link)], using Shimadzu HPLC system and Waters Nova-Pack C18 column (4 mm, 3.9 × 150 mm i.d. with Waters Nova-Pack C-18 guard column, 60 Å, 3.9 × 20 mm). The isocratic mobile phase consisted of acetonitrile and water (50 : 50 v/v); flow rate of 1 mL/min; UV detection at 254 nm. All diene valepotriates were quantified in terms of mg of valtrate equivalent/g extract. The VAL fraction was suspended in saline with 1% of polysorbate 80 (vehicle) prior to use.
3
Quantification of Free Amino Acids in Cheese
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The water-soluble fraction of the cheeses was prepared according to the method of Kuchroo and Fox (1982) . Total FAAs from the water-soluble extracts were determined by a Biochrom series 30 Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK) as described by Siragusa et al.(2007) (link) by reversed-phase high-performance liquid chromatography (HPLC, 1200 Series Agilent chromatograph, Agilent Technologies, USA) using a Waters Nova Pack C18 column (3.9 × 300 mm, Waters Corporation, USA) and UV detection (254 nm).
4
HPLC Analysis of Phenolic Compounds
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Chromatographic analysis was carried out on a Prominence HPLC system (Shimadzu, Kyoto, Japan), equipped with an on-line degassing unit (DGU-20A), a quaternary pump (LC--20A), an auto sampler (SIL-20A), a column oven (CTO-20A) and a photodiode array detector (SPD-M20A). Separation was performed on a Waters Nova-Pack C18 column (150 mm×3.9 mm, 4 μm particle size; Milford, CT, USA) at ambient temperature (25 °C) with gradient elution (solution A: 0.1% by volume formic acid in 5% methanol (Merck) and solution B: 0.1% by volume formic acid (Merck) in 100% methanol). The gradient programme was the following: 100% A at 0 min, 75% A for 15 min, 65% A for 40 min, 55% A for 60 min, 50% A for 65 min, 0% A for 90–95 min and 100% A for 110–120 min. The flow rate of the mobile phase was 1 mL/min and the injection volume was 20 μL (23 (link)).
Identification of the individual phenolic compounds was based on the comparison of the retention times and the UV spectra of unknown peaks to those of standard compounds. Quantitative analysis was based on calibration curves constructed at specific wavelengths of reference compounds using the external standard method.
Identification of the individual phenolic compounds was based on the comparison of the retention times and the UV spectra of unknown peaks to those of standard compounds. Quantitative analysis was based on calibration curves constructed at specific wavelengths of reference compounds using the external standard method.
5
HPLC Quantification of Etofenamate and Ibuprofen
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Quantification of etofenamate and ibuprofen was performed by HPLC using the following chromatographic conditions: Waters Nova-Pack C18 Column, 250x4.6 mm, 4 μm; the mobile phases consisted, for etofenamate, of a mixture of methanol:acetonitrile:water (45:35:20) containing 1% concentrated phosphoric acid (pH 6.0), and, for ibuprofen, of a mixture of methanol:acetonitrile:water (45:30:25) containing 1% concentrated phosphoric acid (pH 6.5); the flow rate used was 1 mL/min, with a running time of 10 min, and the injection volume was 20 μL. Detection by UV absorption was at 285 nm for etofenamate and 200 nm for ibuprofen.
6
Amino Acid Analysis by RP-HPLC
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A similar procedure as followed by Flores et al. (1997) [17 (link)] was performed, with several modifications. After filtration at 0.45 µm with nylon filters of deproteinized samples, 150 μL of each one was derivatized. Amino acid chromatographic separation was achieved by using a reversed-phase high-performance liquid chromatography (RP-HPLC) system (Series 1100; Agilent, Santa Clara, CA, USA) counting with a Waters Nova Pack® C18 column (3.9 × 300 mm; Waters Corporation, Milford, MA, USA). A gradient was generated between solvent A (70 mM sodium acetate pH 6.55 and 2.5 acetonitrile) and solvent B (4.5:4.0:1.5 of acetonitrile, water, and methanol, respectively). Temperature was set at 52 °C and the amino acids were detected at 254 nm. The concentrations were predicted by introducing the peak areas into standard curves.
7
Quantification of Free Amino Acids
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The abundance
of free amino acids released from the proteolytic activity in the
liquid sample was measured following the methodology described by
Aristoy and Toldrá,19 (link) which includes
the deproteinization and derivatization of the sample. Norleucine
(10 mM in 0.01 M HCl) was used as an internal standard. The separation
of free amino acids was performed by reversed-phase HPLC chromatography
in an Agilent Series 1100 equipment (Agilent, Santa Clara, CA, U.S.A.)
equipped with a Waters Nova Pack C18 column (3.9 × 300 mm, Waters
Corporation, Milford, MA, U.S.A.) at 52 °C using a photodiode
array detector.20 (link) The separated amino acids
were detected at 254 nm. Each medium supernatant was analyzed in triplicate.
Identification of amino acids was achieved by comparison against a
solution of mixed standards (Sigma, Merck, Germany), and quantification
was based on the calculated response factors. They were calculated
using five amino acid standard levels in the presence of the added
internal standard (norleucine). The final results were expressed as
milligrams of free amino acid per gram of protein in the model, and
the differences in released free amino acids depending upon the protein
source, animal or vegetal, were determined.
of free amino acids released from the proteolytic activity in the
liquid sample was measured following the methodology described by
Aristoy and Toldrá,19 (link) which includes
the deproteinization and derivatization of the sample. Norleucine
(10 mM in 0.01 M HCl) was used as an internal standard. The separation
of free amino acids was performed by reversed-phase HPLC chromatography
in an Agilent Series 1100 equipment (Agilent, Santa Clara, CA, U.S.A.)
equipped with a Waters Nova Pack C18 column (3.9 × 300 mm, Waters
Corporation, Milford, MA, U.S.A.) at 52 °C using a photodiode
array detector.20 (link) The separated amino acids
were detected at 254 nm. Each medium supernatant was analyzed in triplicate.
Identification of amino acids was achieved by comparison against a
solution of mixed standards (Sigma, Merck, Germany), and quantification
was based on the calculated response factors. They were calculated
using five amino acid standard levels in the presence of the added
internal standard (norleucine). The final results were expressed as
milligrams of free amino acid per gram of protein in the model, and
the differences in released free amino acids depending upon the protein
source, animal or vegetal, were determined.
8
HPLC Quantification of Ophthalmic Drugs
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Concentrations of brimonidine tartrate in the samples were obtained by HPLC with a Waters LC Module I by using a Waters Spherisorb 5 μm ODS‐2 (4.6 mm×150 mm) column. Concentrations of triamcinolone acetonide and betamethasone 17‐valerate in the samples were obtained by using an HPLC with a Waters 717 plus autosampler and a 600 controller pump equipped with a 2996 photodiode array detector by using a 4 μm (3.9 mm×150) Nova‐Pack C18 column. To quantify brimonidine tartrate, the mobile phase consisted of acetonitrile/water (50:50, acidified to pH 2.4 with acetic acid) with a flow rate of 1 mL min−1 setting a detection wavelength of 316 nm. To quantify betamethasone 17‐valerate, the mobile phase consisted of acetonitrile/water following a gradient that consisted of 35:65 at t=0 min, 100:0 at t=10 min, 35:65 at t=11 min, and 35:65 at t=16 min with a flow rate of 1 mL min−1. The detection wavelength was set to 239 nm. Each sample had a run time of 16 min. To quantify triamcinolone acetonide, the mobile phase consisted of acetonitrile/water following a gradient that consisted of 40:60 at t=0 min, 85:15 at t=8 min, 40:60 at t=9 min, and 40:60 at t=14 min with a flow rate of 1 mL min−1. The detection wavelength was set to 239 nm. Each sample had a run time of 14 min. The data were collected by using Millennium32 version 4.0.0 software from Waters Corporation.
9
Amino Acid Profiling by RP-HPLC
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Amino acids were analyzed by RP-HPLC after derivatization with diethyl ethoxymethylenemanolate, and determined according to the method described by Alaiz, Navarro, Giron, & Vioque (1992), using D, L aminobutyric acid as internal standard and a Novapack C 18 column (300 x 3.9 mm i.d., 4 m, Waters). Electro-sprayionization high-resolution mass spectra were recorded with a micrOTOF-QII High Resolution-of-Flight mass spectrometer (UHR-TOF) with qQ-TOF geometry (Bruker Daltonic, Bremen, Germany).
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