During the testing (always before and after the bath change), samples for the determination of Diclofenac concentration were regularly withdrawn from test tanks. Diclofenac determination in water samples was performed by high performance liquid chromatography (HPLC) with photometric detection. Water samples were filtered through a 0.45 μm nylon filter (Millipore, Billerica, MA) and used for analysis. The sample volume injected into the HPLC system was 20 μL. Diclofenac was separated by an isocratic elution method with acetonitrile/water 50/50 (v/v) on a Polaris C18-A column (3 μm, 150 × 4.6 mm, Varian, Inc., Palo Alto, CA). The mobile phase flow rate was 1 mL min−1, the column temperature was 25°C, and UV detection was performed at 310 nm. Chromatographic analysis was accomplished by means of an Alliance 2695 chromatographic system (Waters, Milford, MA) with a PDA 2996 photodiode array detector (Waters, Milford, MA). Diclofenac was purchased from Sigma-Aldrich (St. Louis, MO). All solvents were of HPLC-grade purity (Chromservis, s.r.o., CZ). The detection limit for Diclofenac was 11 ng mL−1. The limit of quantification for Diclofenac was 37 ng mL−1. The coefficient of variation was 4.5%.
Pda 2996 photodiode array detector
Manufactured by Waters Corporation
Sourced in United States
The PDA 2996 photodiode array detector is a high-performance liquid chromatography (HPLC) detector that employs a photodiode array to collect spectral data across a wide range of wavelengths. Its core function is to provide simultaneous detection and quantification of multiple analytes in a single HPLC run.
Automatically generated - may contain errors
Lab products found in correlation
Acquity ultra performance lc system, by Waters Corporation (3 mentions)
Beh c18, by Waters Corporation (2 mentions)
C18 guard column, by Phenomenex (2 mentions)
Polaris c18 a column, by Agilent Technologies (1 mentions)
0.45 μm nylon filter, by Merck Group (1 mentions)
Diclofenac, by Merck Group (1 mentions)
Acquity ultra performance lctm system, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Autosampler, by Waters Corporation (1 mentions)
Milli q plus ultrapure water system, by Merck Group (1 mentions)
515 hplc pump, by Waters Corporation (1 mentions)
2695 separations module, by Waters Corporation (1 mentions)
Syro 1 peptide synthesizer, by Biotage (1 mentions)
Isolera one, by Biotage (1 mentions)
Empower 2, by Waters Corporation (1 mentions)
Waters alliance 2690 2695 separations module, by Waters Corporation (1 mentions)
Tskgel g3000swxl size exclusion column, by Tosoh (1 mentions)
Tskgel swxl guard column, by Tosoh (1 mentions)
Luna c18 2, by Phenomenex (1 mentions)
1525 binary pump, by Waters Corporation (1 mentions)
14 protocols using pda 2996 photodiode array detector
1
Diclofenac Quantification by HPLC
2
UPLC Analysis of Compounds
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An ACQUITY Ultra Performance LC™ system (Waters, Milford, MA, USA) linked to a PDA 2996 photodiode array detector (Waters) was used for ultra-high-performance liquid chromatography (UPLC) analyses [30 (link)]. The instruments were controlled through the Empower software EM5B01629. Before analysis, 0.45 μm of the samples and the standards (previously dissolved in methanol) was filtered. Run conditions: mobile phase 7.5 mM acetic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 250 μL min−1; gradient elution: 5% B for 0.8 min, then 5–20% B over 5.2 min, isocratic 20% B for 0.5 min, 20–30% B for 1 min, isocratic 30% B for 0.2 min, 30–50% B over 2.3 min, 50–100% B over 1 min, isocratic 100% B for 1 min, and finally 100–5% B over 0.5 min; column assessment under the initial conditions for 2.5 min; pressure from 6000 to 8000 psi; T = 30 °C; column: reversed-phase column (BEH C18 1.7 μm, 2.1 × 100 mm, Waters); LC detector (scanning range: 210–400 nm, resolution: 1.2 nm); injection volume: 5 μL.
3
Ultra-Performance LC Analysis of Extracts
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An ACQUITY Ultra-Performance LC system (Waters) linked to a PDA 2996 photodiode array detector (Waters) was used for ultra-performance liquid chromatography analyses. The Empower software controlled the instruments and acquired and processed the data. The extracts and standards (previously dissolved in methanol) were filtered (0.45 μm; Waters) before analysis. The analyses were carried at 30°C using a reversed phase column (BEH C18, 1.7 μm, 2.1 100 mm; Waters) following the method of Fratianni et al. [17 (link)]. The mobile phase consisted of solvent A (7.5 mM acetic acid) and solvent B (acetonitrile) at a flow rate of 250 μL min−1. Gradient elution was employed, starting with 5% B for 0.8 min; then 5–20% B over 5.2 min; isocratic 20% B for 0.5 min; 20–30% B for 1 min; isocratic 30% B for 0.2 min; 30–50% B over 2.3 min; 50–100% B over 1 min; isocratic 100% B for 1 min; and 100–5% B over 0.5 min. At the end of this process, the system equilibrated the column under the initial conditions for 2.5 min.
4
UPLC-PDA Analysis of Phytochemicals
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An ACQUITY Ultra Performance LCTM system (Waters, Milford, MA, USA) linked to a PDA 2996 photodiode array detector (Waters) was used for ultra-high-performance liquid chromatography (UPLC) analysis, following the method of Pane et al. [27 (link)]. The acquisition and processing of the relative data, as well as the control of the instruments was performed through the Empower software. The extracts and standards (which were previously dissolved in methanol to have different concentrations ranging from 0.001 to 5 mM) were filtered (0.45 μm; Waters, Milford, MA, USA) before analysis. The analyses were carried at 30 °C using a reversed phase column (BEH C18, 1.7 μm, 2.1 × 100 mm; Waters). The mobile phase consisted of solvent A (7.5 mM acetic acid) and solvent B (acetonitrile) at a flow rate of 250 μL min‒1. A gradient elution was employed, starting with 5% B for 0.8 min, then 5–20% B over 5.2 min, isocratic 20% B for 0.5 min, 2%30% B for 1 min, isocratic 30% B for 0.2 min, 30–50% B over 2.3 min, 50–100% B over 1 min, and isocratic 100% B for 1 min, 5–100% B over 0.5 min, and finally the column was equilibrated under the initial conditions for 2.5 min. The pressure ranged from 6000 to 8000 psi. The injection volume was 5 μL. The effluent was introduced into an LC detector (scanning range: 210–400 nm, resolution: 1.2 nm).
5
UPLC/MS Analysis of Compounds
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UPLC/MS analyses
were carried out on an ACQUITY Ultra Performance LC system (Waters,
Milford, MA) linked simultaneously to a photodiode array detector
(PDA) 2996 photodiode array detector (Waters, Milford, MA) and an
ACQUITY TQ Detector (Waters MS Technologies, Manchester, U.K.), equipped
with a Z-spray electrospray ionization (ESI) source operating in positive
and negative modes. MassLynx software (version 4.1, Waters, Milford,
MA) was used to control the instruments, as well as for data acquisition
and processing. Diluted sample solutions (3 μL; 0.5 mg/mL; 0.58
nmol) were injected into a reversed-phase column (BEHC18, 1.7 μm, 1 × 50 mm2, Waters, Milford, MA),
which was maintained at 40 °C. The mobile phase consisted of
solvent A (H2O/0.1 HCOOH) and solvent B (acetonitrile/0.1
HCOOH) at a flow rate of 300 μL/min, eluted in a linear gradient,
as follows: T = 0 min, 5% B; T =
10 min, 95% B; T = 11 min, 5% B; T = 13 min, 5% B. The effluent was introduced into a PDA detector
(scanning range 210–400 nm, resolution 1.2 nm) and subsequently
into an electrospray source (source block temperature 120 °C,
desolvation temperature 350 °C, capillary voltage 3.5 kV, cone
voltage 30 V) and nitrogen as the desolvation gas (600 L/h). Mass
chromatograms were recorded in the positive and negative ionization
modes.
were carried out on an ACQUITY Ultra Performance LC system (Waters,
Milford, MA) linked simultaneously to a photodiode array detector
(PDA) 2996 photodiode array detector (Waters, Milford, MA) and an
ACQUITY TQ Detector (Waters MS Technologies, Manchester, U.K.), equipped
with a Z-spray electrospray ionization (ESI) source operating in positive
and negative modes. MassLynx software (version 4.1, Waters, Milford,
MA) was used to control the instruments, as well as for data acquisition
and processing. Diluted sample solutions (3 μL; 0.5 mg/mL; 0.58
nmol) were injected into a reversed-phase column (BEHC18, 1.7 μm, 1 × 50 mm2, Waters, Milford, MA),
which was maintained at 40 °C. The mobile phase consisted of
solvent A (H2O/0.1 HCOOH) and solvent B (acetonitrile/0.1
HCOOH) at a flow rate of 300 μL/min, eluted in a linear gradient,
as follows: T = 0 min, 5% B; T =
10 min, 95% B; T = 11 min, 5% B; T = 13 min, 5% B. The effluent was introduced into a PDA detector
(scanning range 210–400 nm, resolution 1.2 nm) and subsequently
into an electrospray source (source block temperature 120 °C,
desolvation temperature 350 °C, capillary voltage 3.5 kV, cone
voltage 30 V) and nitrogen as the desolvation gas (600 L/h). Mass
chromatograms were recorded in the positive and negative ionization
modes.
6
HPLC-PDA and LC-MS/MS Analysis
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A high performance liquid chromatography (HPLC) system with a 2996 photodiode-array detector (PDA) and an auto-sampler was used (Waters Technologies, USA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed with an Agilent 1290 ultra-high performance liquid chromatography (UPLC) combined with an AB Sciex Triple time of flight (TOF) 4600 MS system. A SpectraMax Plus384 multimode micro-plate reader (Molecular Devices, USA) and a Milli-Q-Plus ultra-pure water system (Millipore, Bedford, MA, USA) were used.
7
Quantification of FEB by RP-HPLC
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The amount of FEB was established with slight modification by RP-HPLC method using the earlier reported method [1 (link)]. Briefly, chromatography was performed using column, princetonSHER Ultima C-18 as a stationary phase with a dimension of (250 mm × 4.6 mm × 5 μm). The mobile phase consisting of Methanol: Potassium Dihydrogen Phosphate 10 mM Buffer, pH: 6.8 (70:30) was pumped with a flow rate of 1.2 mL/min. The sample of volume 20 μL was injected into the HPLC system and detected by Waters 2996 Photodiode Array Detector (PDA) at 315 nm. The calibration curve was constructed using a linearity range 10–50 μg/mL and obtained the regression coefficient (r2 = 0.999). The estimated and developed method was validated in accordance with guidelines rendered by ICH Q2 (R1) (2005) [22 ].
8
In Vitro Chuanxiong Release from Alginate Microparticles
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The in vitro release study was carried out with the chuanxiong-loaded alginate microparticles in water and simulated intestinal fluid. Microparticles (0.2 g) were placed in a 15 mL tube containing 2 mL of water or simulated intestinal fluid and incubated at room temperature with shaking at 100 rpm. The solution (0.5 mL) was sampled for determination of released chuanxiong by measuring ferulic acid using HPLC at time intervals. HPLC was equipped with Waters 515 HPLC Pump, Waters 2996 Photodiode Array Detector (PDA), and Waters SunFire™ C18 5 μm 4.6 × 250 mm column. The mobile phase consisted of 2% acetic acid in water/acetonitrile and the flow rate was set at 0.6 mL/min. A gradient elution was performed with 0–100% acetonitrile in 0~50 min and 100–0% acetonitrile in 55~60 min. The eluate was detected at 320 nm. The encapsulation and release efficiency of chuanxiong by alginate microparticles were calculated as follows:
9
Synthesis and Purification of SET1 Peptide Ligands
10
Protein Stability Analysis by SEC-LC-MS
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The selected formulations stressed at 40°C were analyzed by size-exclusion chromatography (SEC) using a Waters Alliance 2690/2695 Separations Module (Waters Corporation, Milford, MA) equipped with a TSK Gel G3000SW XL Size-Exclusion Column (7.8 mm×30 cm, 5 μm Tosoh Bioscience), a TSK Gel SW XL Guard Column (6.0 mm×4 cm, 7 μm Tosoh Bioscience), and a Waters 2996 Photodiode Array Detector (PDA). The mobile phase was a solution containing 10 mM phosphate, 27 mM potassium chloride, 137 mM sodium chloride, and 1.76 mM potassium phosphate with a flow rate of 0.85 mL/min. Chromatograms were monitored at 215 nm, and the data was processed using Empower 2 Software (Waters). The fractions eluted from the column were collected at retention times of 9.5 to 10.9 min for the native monomer, 10.9 to 11.6 min for the shoulder of the monomer, and 12.3 to 13.5 min for fragments. Each collected fraction was injected back into SEC column to confirm the content. The confirmed fractions were then further analyzed using liquid chromatography mass spectrometry (LC-MS) and hydrophobic interaction chromatography (HIC).
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