Chromatographic analyses were carried out according to the method described by Ferreres et al. (2014) [20 (link)], with some modifications. In this case, the chromatographic column used was a Zorbax SB-C18 (5 µm, 4.6 × 250 mm; Agilent, Santa Clara, CA, USA) with a Security Guard ULTRA Cartridges (Zorbax SB-C18, 4.6 × 125 mm, 5-µm pore size; Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of acidified water (formic acid, 0.05%) (A) and acetonitrile (B); the gradient of the eluents started with 5% B, reaching 15% B at 10 min, 25% B at 30 min, 30% B at 35 min, 55% B at 50 min and 90% B at 60 min, with a flow rate of 1 mL/min. The UV detection was set at 280 and 340 nm. The injection volume was 20 µL, and the full scan mass covered the range from m/z 100 to 1000.
Zorbax sb c18
Manufactured by Agilent Technologies
Sourced in United States, Germany, China, Japan
Zorbax SB-C18 is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with a C18 bonded ligand, which provides efficient and reproducible separations.
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Lab products found in correlation
Zorbax sb c18 column, by Agilent Technologies (8 mentions)
Agilent 1200, by Agilent Technologies (7 mentions)
1260 infinity, by Agilent Technologies (5 mentions)
1200 series, by Agilent Technologies (4 mentions)
Agilent 1260, by Agilent Technologies (4 mentions)
Agilent 1200 rr hplc, by Agilent Technologies (4 mentions)
Co 2060 plus, by Jasco (4 mentions)
As 2057 plus, by Jasco (4 mentions)
Zorbax sb c8, by Agilent Technologies (3 mentions)
1100 hplc system, by Agilent Technologies (3 mentions)
Hplc system, by Agilent Technologies (3 mentions)
Mass spectrum lc 2020, by Shimadzu (3 mentions)
Mass workstation software, by Shimadzu (3 mentions)
Lc 20at system, by Shimadzu (3 mentions)
1200 series hplc, by Agilent Technologies (3 mentions)
High performance liquid chromatography (hplc), by Shimadzu (3 mentions)
Masshunter workstation software, by Agilent Technologies (3 mentions)
Md 2018 plus, by Jasco (3 mentions)
1260 infinity hplc system, by Agilent Technologies (3 mentions)
1100 hplc, by Agilent Technologies (2 mentions)
341 protocols using zorbax sb c18
1
Chromatographic Analysis of Compounds
2
Synthesis and Characterization of pH-Responsive pHLIP Peptides
3
HPLC Analysis of Chlorambucil Derivatives
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The analytical and biological samples were injected into a reversed-phase C18 analytical column Zorbax SB-C18 (3 × 150 mm, 3.5 µm) coupled with guard-column Zorbax SB-C18 (Agilent Technologies, Little Falls Wilmington, DE, USA). The solutions were eluted in isocratic mode with 50% acetonitrile and deionized water (with 0.1% formic acid) as eluents at a flow rate of 0.8 mL/min. The sample peaks were monitored by Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Karlsruhe, Germany) with UV detection at 254 nm. The retention time of chlorambucil derivatives (1 and 2 ) and chlorambucil (3 ) were 2.4, 1.6, and 5.0 min, respectively.
4
HPLC Analysis of Deoxynivalenol in Oat Samples
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Analyses were performed on an HPLC system of Agilent Technologies 1200 Series, equipped with an auto-sampler and the analytical column Zorbax SB-C18, 4.6 × 250 mm i.d. with the sorbent particle size of 5 μm, connected to a guard column Zorbax SB-C18, 12.5 × 4.6 mm i.d., with the sorbent particle size of 5 μm (both from Agilent Technologies). A diode-array detector (DAD) was set at following wavelengths: sample/bandwidth: 220/16 nm, reference/bandwidth: 360/80 nm. As a mobile phase, the mixture of acetonitrile: deionized water (10:90, v/v) was used at a flow rate of 1 mL·min−1. The injected volume of the sample was 50 μL and was performed by the auto-sampler. The column temperature was adjusted to 25 °C and was automatically controlled by the Agilent ChemStation (Agilent Technologies). The final concentration of DON in the oat samples was calculated using the equation C [μg·kg−1] = C*·F (1000/m), where C* is the concentration of the mycotoxin estimated from the calibration curve in μg·ml−1 (injection concentration); F is the conversion factor, which includes the initial and final volumes of the sample taken into analysis, as well as the factor 1000, which presents a conversion from μg·g−1 to μg·kg−1; and m is the sample weight in grams.
5
UPLC Chromatographic Separation Protocol
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For all the chromatographic experiments, a Waters UPLC (Waters Corp., Milford, MA, USA), Agilent ZORBAX-SB C18 (100 mm × 4.6 mm i.d: 1.8 μm) was used, for which the mobile phases were 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B). The linear gradient pro-gram parameters were as follows: 0/5, 2/5, 25/50, 33/95 (min/B%); sample injection volume = 10 μL; column oven temperature = 30°C; flow rate = 0.8 mL min−1; the UV detector was set to 254 nm.
6
High-Sensitivity Proteomic Workflow
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Chromatography was performed on an Agilent 1260 quaternary HPLC with constant flow rate of ~600 nL min -1 achieved via a splitter. A Sutter P-2000 laser puller was used to generate sharp nanospray tips from 200 µm ID fused silica capillary. Columns were all inhouse packed on a Next Advance pressure cell using 200 µm ID capillary. All samples were loaded into a 10 cm capillary column packed with 5 μM Zorbax SB-C18 (Agilent)
and then connected to a 5 cm-long strong cationic exchange (SCX) column packed with 5µM PolySulfoethyl. The SCX column was then connected to a 20 cm long nanospray tip, packed with 2.5 µm C18 (Waters). For global protein abundance, 45 µg of labeled peptides were fractionated online using 27 ammonium acetate salt steps. For phosphoproteomics, 25 µg of enriched peptides and 14 salt steps were used. For MAKS, 30 µg of enriched peptides and 17 salt steps were used. Each salt step was then separated using a 150 min reverse-phase gradient (Zhang et al., 2019) .
Eluted peptides were analyzed using a Thermo Scientific Q-Exactive Plus high-resolution quadrupole Orbitrap mass spectrometer, which was directly coupled to the HPLC. Data Charge exclusion was set to unassigned, 1, 5-8, and >8. MS1 that triggered MS2 scans were dynamically excluded for 25 s for global proteome and 45 s for phosphoproteome.
and then connected to a 5 cm-long strong cationic exchange (SCX) column packed with 5µM PolySulfoethyl. The SCX column was then connected to a 20 cm long nanospray tip, packed with 2.5 µm C18 (Waters). For global protein abundance, 45 µg of labeled peptides were fractionated online using 27 ammonium acetate salt steps. For phosphoproteomics, 25 µg of enriched peptides and 14 salt steps were used. For MAKS, 30 µg of enriched peptides and 17 salt steps were used. Each salt step was then separated using a 150 min reverse-phase gradient (Zhang et al., 2019) .
Eluted peptides were analyzed using a Thermo Scientific Q-Exactive Plus high-resolution quadrupole Orbitrap mass spectrometer, which was directly coupled to the HPLC. Data Charge exclusion was set to unassigned, 1, 5-8, and >8. MS1 that triggered MS2 scans were dynamically excluded for 25 s for global proteome and 45 s for phosphoproteome.
7
Stability Evaluation of Amorphous Compound 1-(S)
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Example 5
Stability of Amorphous Compound 1-(S)
Amorphous Compound 1-(S) has been found to be physically stable under elevated temperature and humidity. Table 1 indicates the conditions/time to which the amorphous form was subjected and provides the measured water content (Karl-Fischer analysis) and purity (by HPLC/UV). The HPLC/UV instrument was Agilent HPLC 1100 with an Agilent Zorbax SB-C18, 3.5 μm, 4.6×150 mm column. The HPLC conditions were as follows: column temperature: 40° C.; mobile phase A (MPA): 0.05% (v/v) trifluoroacetic acid (TFA) in water; mobile phase B (MPB): 0.05% (v/v) TFA in acetonitrile; and flow rate: 1 mL/min.
The gradient conditions were as follows:
8
HPLC Analysis of Sweetener Compound in Carbonated Beverages
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The HPLC analysis was performed on an Agilent 1260 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) and controlled by the Agilent ChemStation software. All separations were carried out on an Agilent ZORBAX SB-C18 column (250 × 4.6 mm i.d., 5 µm) and a guard column (ZORBAX SB-C18, 12.5 × 4.6 mm i.d., 5 µm) at a column temperature of 35 °C. The detection wavelength was set at 280 nm and the injection volume of all samples was 5.0 µL. The mobile phase consisting of solvent A (methanol) and solvent B (0.1% formic acid solution) was eluted with the following gradient elution program: 0–3 min, 35% A; 3–9 min, 35%–100% A; 9–12 min, 100%–50% A. The flow rate was at 1.0 mL/min. The samples were filtered through a 0.22 µm membrane filter (Navigator Lab Instrument, Tianjin, China) before being injected into HPLC for analysis.
The SBA was prepared with different concentrations of 6.25–1000 ppm in 10.0 mM PBS (pH = 6.0), and a calibration curve for the determination of SBA by HPLC analysis was established. Then, the spiked recovery tests of the carbonated beverage samples (Cola, Sprite, and Fanta) were performed by the HPLC method after spiking with three different concentrations of SBA (final concentrations of 50, 100, and 200 ppm).
The SBA was prepared with different concentrations of 6.25–1000 ppm in 10.0 mM PBS (pH = 6.0), and a calibration curve for the determination of SBA by HPLC analysis was established. Then, the spiked recovery tests of the carbonated beverage samples (Cola, Sprite, and Fanta) were performed by the HPLC method after spiking with three different concentrations of SBA (final concentrations of 50, 100, and 200 ppm).
9
UHPLC-based CPF Quantification
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A UHPLC instrument (Agilent 6410, Agilent Technologies Co., Ltd., Santa Clara, CA, USA) equipped with a column thermostat, an autosampler, a diode array detector and a degasser unit was used to measure CPF samples to validate the SERS method. The analytical column (Agilent ZORBAX SB-C18, 150 mm × 2.1 mm × 3.5 μm) was kept at 30 °C and the elution was operated at 300 nm with a mixture of methanol and water at a ratio of 1:1 and at a flow rate of 0.3 mL/min.
10
Silymarin Components Analysis by HPLC
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An Agilent 1260 Infinity High Performance Liquid Chromatography (HPLC) system equipped with an Agilent 1260 Infinity pump (G1361A), Agilent 1260 diode array detector VL (G1315D), Agilent ZORBAX SB-C18 and Agilent 1260 Infinity autosampler (G2260A) were used to analyze the content of individual silymarin components according to the reported method by (AbouZid et al., 2016b ). The mobile phase was composed of methanol/0.1% formic acid (Phase A) and water/0.1% formic acid (Phase B); 0–5 min gradient 40–45% phase A; 5–10 min isocratic 45% phase A; 10–20 min gradient 45–50% phase A; 20–25 min isocratic 50% phase A. The flow rate was 1.5 ml/min, and ambient temperature was used. The auto sampler was adjusted to inject 10 µl and quantified at 280 nm. The method offered the following retention times (minutes) for the major silymarin compounds: silychristin A (Rt = 8.9), silydianin (Rt = 10.6), silybin A (Rt = 19.6), silybin B (Rt = 21.3), isosilybin A (Rt = 25.4), and isosilybin B (Rt = 26.6). Flavonolignans identification and quantification was obtained using reference standards (Sigma-Aldrich). Total flavonolignan content is the result of the sum of the single constituents derived from HPLC analysis.
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