Analytical protein A chromatography was performed using a TSK 5PW protein A column (Tosoh) with an inner diameter of 4.6 mm and a length of 3.5 cm. The column was connected to a Dionex U3000 (Thermo Fisher) equipped with a Dionex Ultimate 3000 DAD detector (10 mm pathlength) and a Dionex WPS‐3000 TSL Micro Autosampler. The column was equilibrated (30 mM potassium phosphate, pH 7.5, 150 mM NaCl) at 2 mL/min and 10 or 50 μL of filtered (0.2 μm, Millipore) sample was injected. After a wash step, the bound protein was eluted using 0.01 M HCl. The UV absorbance was monitored at 280 and 300 nm. For antibody peak determination, the 280 or 300 nm antibody peak was integrated and compared with a calibration curve to calculate mAb concentration. For purity determination, the flow through signal at 280 nm and the antibody peak were used to calculate the percentage of signal corresponding to the mAb.
Dionex u3000
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The Dionex U3000 is a high-performance liquid chromatography (HPLC) system designed for routine analytical applications. It features a modular design, allowing for flexible configuration to meet specific laboratory requirements. The Dionex U3000 provides reliable and reproducible separation and analysis of a wide range of compounds, including small molecules, peptides, and proteins.
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Lab products found in correlation
Sequant, by Merck Group (2 mentions)
Dionex ultimate 3000 dad detector, by Thermo Fisher Scientific (1 mentions)
Chromeleon v 6, by Thermo Fisher Scientific (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Nanospray flex ion source, by Thermo Fisher Scientific (1 mentions)
Zic philic column, by Merck Group (1 mentions)
Hpx 87h, by Bio-Rad (1 mentions)
Q exactive orbitrap, by Thermo Fisher Scientific (1 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (1 mentions)
9 protocols using dionex u3000
1
Protein A Chromatography for mAb Analysis
2
Quantitative HPLC Analysis of Fisetin
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Fisetin was assayed by high-performance liquid chromatography after purification using a reverse phase HPLC system (Dionex U3000, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a polymeric PRP-1 250 × 4.6 mm, 5 µm (Hamilton Company, Reno, NV, USA). The injected volume was 10 µL. Eluent A consisted of 2% acetic acid in deionized water and eluent B was ACN. The flow rate was 1 mL/min. The elution gradient started with 15% B for 3 min. From 3 to 6 min, the percentage of eluent B was increased to achieve 50%, which was maintained for 6 min, to turn back to 15% in 2 min, followed by an equilibrium phase of 2 min. The UV detection of the drugs was carried out at 360 nm as it is the maximum absorbance of fisetin determined by spectrophotometry. Data were processed using the Chromeleon® (v6.8) software (Thermo Fisher Scientific, Waltham, MA, USA).
The calibration curve was prepared by dissolving fisetin in methanol. This method was validated according to ICH Q2 (R1) (Peak area = 0.177 × Concentration + 0.851, R² = 0.999 ± 0.001). The purified fisetin-loaded or liposomes co-encapsulating fisetin and cisplatin were diluted in methanol prior to the injection to range in the calibration curve (5–100 µg/mL).
The calibration curve was prepared by dissolving fisetin in methanol. This method was validated according to ICH Q2 (R1) (Peak area = 0.177 × Concentration + 0.851, R² = 0.999 ± 0.001). The purified fisetin-loaded or liposomes co-encapsulating fisetin and cisplatin were diluted in methanol prior to the injection to range in the calibration curve (5–100 µg/mL).
3
Nano-LC-MS/MS Peptide Separation and Analysis
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Peptides were separated with a linear gradient of 5–50% buffer B (95% ACN and 0.2% formic acid) at a flow rate of 200 nL/min on a C18-reversed phase column (75 mm × 15 cm) packed in-house with Waters YMC-ODS C18-AQ 5 mm resin. A Dionex U3000 nano-LC chromatography system (ThermoFisher Scientific) was coupled on-line to an Orbitrap Elite instrument (ThermoFisher Scientific) via a Nanospray Flex Ion Source (ThermoFisher Scientific). MS data was acquired with a data-dependent strategy selecting the fragmentation events based on the precursor abundance in the survey scan (400-1700 Th). The resolution of the survey scan was 60,000 at m/z 400 Th with a target value of 1e6 ions and 1 microscan. Low resolution CID MS/MS spectra were acquired with a target value of 5000 ions in normal CID scan mode. MS/MS acquisition in the linear ion trap was partially carried out in parallel to the survey scan in the Orbitrap analyzer by using the preview mode (first 192 ms of the MS transient). The maximum injection time for MS/MS was 100 ms. Dynamic exclusion was 120 s and early expiration was enabled. The isolation window for MS/MS fragmentation was set to 2.
4
Targeted Metabolomics Analysis by LC-MS
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Metabolite samples were analyzed on a Q-exactive Orbitrap mass spectrometer coupled with a Dionex U3000 high-performance LC (HPLC; Thermo Fisher) with a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm; SeQuant, Merck). The MS system was operated at 35,000 resolution in both positive and negative electrospray ionization modes with a detection range of 85–1,275 m/z. Column temperature was maintained at 25 C, and the mobile phase consisted of 20 mM of ammonium carbonate (solvent A) and acetonitrile (solvent B). Metabolites were eluted in a step gradient, starting with 80% solvent B at a flow rate of 0.3 ml/min followed by a linear gradient to 50% solvent B over 15 min (Maifiah et al., 2016 (link); Maifiah et al., 2017 (link)). All samples were randomized and analyzed in a single LC-MS batch. Analytical reproducibility was monitored on the basis of pooled QC samples throughout the batch, which were periodically analyzed after groups of six samples. Analyses of a mixture of pure standards containing >250 metabolites were performed to assist in the identification of metabolites.
5
Liquid Product Characterization by HPLC
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The liquid products
obtained were analyzed by high-performance liquid chromatography (DIONEX
U3000, Thermo Fisher Scientific), equipped with a refractive index
detector (RI-101, Shodex) and an aminex column (HPX-87H, 300 mm ×
7.8 mm, Bio-Rad). The eluent was dilute H2SO4 (5 mM) flowing at a rate of 0.60 mL·min–1 with the column temperature maintained at 50 °C. The concentrations
of all components were determined by comparison to standard calibration
curves.
obtained were analyzed by high-performance liquid chromatography (DIONEX
U3000, Thermo Fisher Scientific), equipped with a refractive index
detector (RI-101, Shodex) and an aminex column (HPX-87H, 300 mm ×
7.8 mm, Bio-Rad). The eluent was dilute H2SO4 (5 mM) flowing at a rate of 0.60 mL·min–1 with the column temperature maintained at 50 °C. The concentrations
of all components were determined by comparison to standard calibration
curves.
6
High-resolution HILIC-MS for Metabolite Profiling
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The above metabolite samples were analysed on a Dionex U3000 high-performance liquid chromatography (HPLC) system in tandem with a Q-Exactive Orbitrap high-resolution mass spectrometer (Thermo Fisher) in both positive and negative mode with a mass range of 85–1275 m/z. A previously described hydrophilic interaction liquid chromatography (HILIC) method was employed [32] (link). Briefly, samples (10 μL, maintained at 6 °C) were eluted through a ZIC-pHILIC column (5 μm, polymeric, 150 × 4.6 mm; SeQuant, Merck) by mobile phases containing 20 mM ammonium carbonate in lane A and acetonitrile in lane B (flow rate, 0.3 mL/min). A linear gradient from 80% mobile phase B to 50% of mobile phase B was employed over the first 15 min, then to 5% mobile phase B at 18 min, and followed by a washing step containing 5% mobile phase B for 3 min. An 8-min re-equilibration step with 80% mobile phase B was conducted prior to the next injection. All samples were analysed within the same LC-MS batch to minimise analytical variations. A pooled quality control sample consisting of 10 μL of each sample was analysed periodically throughout the batch to assess chromatographic peaks, signal reproducibility and analyte stability. A total of 8 external standard samples containing a mixture of >300 authentic standards were also analysed within the batch to assist metabolite identification.
7
Purification and Monosaccharide Analysis of Astragalus Polysaccharides
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The APS was extracted from Astragalus membranaceus by boiling water extraction, and then the supernatant was deproteinized using Sevag reagent (Sevag et al., 1938 (link)). After removing Sevag reagent, the liquid was mixed with 95% ethanol to a final concentration of 80% (v/v). Twenty-four hours later, the precipitation was dried to get the crude APS. The crude polysaccharides were eluted by water using DEAE-cellulose column to obtain the purified APS.
The monosaccharides composition of APS was analyzed with High-Performance Liquid Chromatography (HPLC; Dionex U3000, ThermoFisher) equipped with ZORBAX EclipseXDB-C18 (Agilent) column. The mobile phase was acetonitrile and phosphate-buffered saline (12 g/L of KH2PO4, 2 M NaOH, and adjust the pH to 6.8) in a proportion of 17:83, the flow rate was 0.8 ml/min and the wavelength was 250 nm. Five point 2 mg of the purified APS was dissolved in trifluoroacetic acid (TFA) solution at 121°C for 2 h and dried with termovap sample concentrator. Then methanol was used to wash the sample till the TFA was completely removed. The sample and standard monosaccharides were reacted with PMP reagent (1-phenyl-3-methyl-5-pyrazolone) before HPLC analysis.
The monosaccharides composition of APS was analyzed with High-Performance Liquid Chromatography (HPLC; Dionex U3000, ThermoFisher) equipped with ZORBAX EclipseXDB-C18 (Agilent) column. The mobile phase was acetonitrile and phosphate-buffered saline (12 g/L of KH2PO4, 2 M NaOH, and adjust the pH to 6.8) in a proportion of 17:83, the flow rate was 0.8 ml/min and the wavelength was 250 nm. Five point 2 mg of the purified APS was dissolved in trifluoroacetic acid (TFA) solution at 121°C for 2 h and dried with termovap sample concentrator. Then methanol was used to wash the sample till the TFA was completely removed. The sample and standard monosaccharides were reacted with PMP reagent (1-phenyl-3-methyl-5-pyrazolone) before HPLC analysis.
8
HPLC-DAD and HPLC-MS Analysis of Phenolic Compounds
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HPLC-DAD analyses were carried out using Thermo Scientific Dionex U3000 apparatus (Les Ulis, France), as described by Chin et al. [66 (link)] with certain modifications. The stationary phase used for compounds separation is a phenyl-hexyl column (X select CSH, 4.6 × 250 mm, 5 μm). The two systems of solvents used as a mobile phase are; (A) MeCN/H2O/HCOOH with a proportion of 50:49.95:0.05 (v/v/v) and (B) H2O/HCOOH with a proportion of 99.9:0.1 (v/v). The gradient of solvent was starting from 15% of (A) and increasing to 72% during 15 min, then isocratic at 72% of (A) during 13 min, from 72 to 30 of (A) during 5 min, from 30 to 20% of (A) during 2 min, and finally from 20 to 15% of (A) during 10 min. The volume of sample injection was 20 µL and the flow rate was 0.7 mL/min. The chromatograms of phenolic compounds were recorded at 280 nm.
The same method of separation was operated for HPLC-MS analyses. The mass spectrum was acquired in the negative ionization mode using the following operating conditions: ionic excitation voltage 3 kV, cone voltage 50 V, Q energy 70 V, and desolvation temperature 500 °C. The m/z was ranged from 100 to 1000. Chromeleon® software ®, version 6.8, provided by Thermo Scientific Dionex (Les Ulis, France) was used for the treatment of results.
The same method of separation was operated for HPLC-MS analyses. The mass spectrum was acquired in the negative ionization mode using the following operating conditions: ionic excitation voltage 3 kV, cone voltage 50 V, Q energy 70 V, and desolvation temperature 500 °C. The m/z was ranged from 100 to 1000. Chromeleon® software ®, version 6.8, provided by Thermo Scientific Dionex (Les Ulis, France) was used for the treatment of results.
9
Quantifying Microbial Fermentation Products
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The fermentation broth was sampled every 6 h and centrifuged at 4,500 g for 10 min. The resulting precipitate was washed twice with sterile water and dried to a constant weight at 105 °C to measure the dry cell weight (DCW) of the culture, and the supernatant was used to determine the concentration of ε-PL and glycerol. ε-PL concentration in various solutions was determined according to the method described by Itzhaki (1972) . The concentration of glycerol was detected by an HPLC system (Dionex U-3000, Thermo Fisher Scientific, Sunnyvale, CA) with an exchange column (Aminex HPX-87H, Bio-Rad, Hercules, CA) and a refraction index detector (Shodex RI-101, Tokyo, Japan). The elution of the ion exchange column was performed at 60 °C with 5 mM H 2 SO 4 , and a flow rate of 0.6 mL/min.
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