Agilent 1260 binary pump

Manufactured by Agilent Technologies

Sourced in United States

The Agilent 1260 binary pump is a high-performance liquid chromatography (HPLC) pump designed to deliver precise and accurate solvent flow. It features two independent solvent channels, allowing for the blending of two solvents for gradient elution. The pump maintains a stable flow rate and pressure, ensuring reliable and reproducible results during analytical separations.

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Lab products found in correlation

Agilent 300 extend c18 column, by Agilent Technologies (4 mentions) Agilent 1290 column compartment, by Agilent Technologies (4 mentions) Agilent 1260 autosampler, by Agilent Technologies (4 mentions) Column switching valve, by Agilent Technologies (3 mentions) Qtrap 5500 mass spectrometer, by AB Sciex (2 mentions) Multiquant 2, by AB Sciex (2 mentions) Eclipse xdb c18 column, by Agilent Technologies (1 mentions) 300extend c18 column, by Agilent Technologies (1 mentions) Tracefinder software, by Thermo Fisher Scientific (1 mentions) Hss t3 column, by Waters Corporation (1 mentions) Photodiode array detector, by Thermo Fisher Scientific (1 mentions) Bioconfirm, by Agilent Technologies (1 mentions) Sequant zic chilic column, by Merck Group (1 mentions)

13 protocols using agilent 1260 binary pump

HPLC-DAD-MS Analysis of Aloe Compounds

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The system used was an Agilent 1260 binary pump (Agilent Technologies, Santa Clara, CA, USA) equipped with an Eclipse XDB C18 column (4.6 × 250 mm, 5 μm particle size) as the stationary phase, which was maintained at 30 °C. After the column, a passive “T” junction was used to split the flow to the DAD (1260 series) and to a Varian MS-500 Ion Trap Mass Spectrometer (Varian, Santa Clara, CA, USA) equipped with an electrospray ionisation (ESI) source, in order to obtain superimposable chromatograms. A gradient of acetonitrile (A) and Milli-Q water containing 0.1% formic acid (B) was used as the mobile phase. Gradient conditions were optimised in order to perform the analysis in 19 min, and to reach the best separation of aloin A, aloin B, aloe emodin, emodin, and danthron. The gradient was as follows: 0 min, 15% A; 5 min, 35% A; 12 min, 100% A; 18 min, 100% A; 19 min, 15% A. DAD was operated at λ = 350 nm and 425 nm, in order to optimise the detection of aloin A and aloin B, and aloe emodin, emodin, and danthron, respectively.
Under the proposed conditions, aloin B was eluted at 9.09 min, aloin A at 9.43 min, aloe emodin at 12.60 min, emodin at 13.95 min, and danthron at 14.62 min (Figure 1).

Loschi F., Faggian M., Sut S., Ferrarese I., Maccari E., Peron G, & Dall’Acqua S. (2022). Development of an LC–DAD–MS-Based Method for the Analysis of Hydroxyanthracene Derivatives in Food Supplements and Plant Materials. Molecules, 27(6), 1932.

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Peptide Separation and Purification Protocol

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Dried TMT labeled peptides were solubilized in buffer A (5% ACN, 10 mM ammonium bicarbonate, pH 8.0) and separated by an Agilent 300 Extend C18 column (3.5 μm particles, 4.6 mm ID x 220 mm in length). Using an Agilent 1260 binary pump coupled with a degasser and a single wavelength detector set at 220 nm, a 60-min linear gradient from 13% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8 (flow rate of 0.6 mL/min) separated the peptide mixtures into a total of 96 fractions. 96 Fractions were consolidated into 12 samples in a checkerboard manner, acidified with 10 μL of 20% formic acid and vacuum dried. Each sample was re-dissolved in 5% FA/5% ACN, desalted via StageTips, dried via vacuum centrifugation, and reconstituted for LC-MS/MS analysis.

Neurohr G.E., Terry R.L., Lengefeld J., Bonney M., Brittingham G.P., Moretto F., Miettinen T.P., Vaites L.P., Soares L.M., Paulo J.A., Harper J.W., Buratowski S., Manalis S., van Werven F.J., Holt L.J, & Amon A. (2019). Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence. Cell, 176(5), 1083-1097.e18.

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Quantification of Tryptamine and Nicotinic Acid

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200 μL of extract was dried using a vacuum concentrator (Genevac) and resuspended in 200 μL 50:50 methanol:water, clarified by centrifugation and analyzed using reverse phase chromatography coupled to TSQ Vantage triple quadupole mass spectrometer with HESI II source. LC separation was using an HSS T3 column (100 x 2.1 mm, 1.8 μm particle size, Waters) and Agilent 1260 binary pump. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The gradient was 0 min, 0% B; 2 min, 0% B; 5 min, 12% B; 7 min, 70% B; 8.5 min, 97% B, 11.5 min, 97% B with 3.5 min of re-equilibration time. LC parameters were: flow rate 300 μL/min, injection volume 15 μL and column temperature 35°C. The mass spectrometer was operated in positive ionization with transitions for tyrptamine (m/z 161.1 → 115.1, CE 30V^∗; 161.1 → 144.1, CE 4 V) and nicotinic acid (m/z 124.1 → 80.1, CE 18 V^∗; 124.1 → 78.1, CE 19V), with ^∗ indicating the primary transition used for quantitation. MS parameters were: capillary temp: 300°C; vaporizer temp: 350°C; sheath gas: 50; aux gas: 30; spray voltage 4000 V. Data was acquired and analyzed using TraceFinder software (version 4.1, Thermo Scientific) confirmed by comparison with authentic standards.

Colosimo D.A., Kohn J.A., Luo P.M., Piscotta F.J., Han S.M., Pickard A.J., Rao A., Cross J.R., Cohen L.J, & Brady S.F. (2019). Mapping Interactions of Microbial Metabolites with Human G-Protein-Coupled Receptors. Cell Host & Microbe, 26(2), 273-282.e7.

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Quantitative Peptide Separation and Analysis

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TMT labeled peptides were solubilized in 5% ACN/10 mM ammonium bicarbonate, pH 8.0 and separated by an Agilent 300 Extend C18 column (3.5μm particles, 4.6 mm ID and 250 mm in length). An Agilent 1260 binary pump coupled with a photodiode array (PDA) detector (Thermo Scientific) was used to separate the peptides. A 45 minute linear gradient from 10% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8.0 (flow rate of 0.6 mL/min) separated the peptide mixtures into a total of 96 fractions (36 seconds). A total of 96 Fractions were consolidated into 24 samples in a checkerboard fashion, acidified with 20 μL of 10% formic acid and vacuum dried to completion. Each sample was desalted via Stage Tips and re-dissolved in 5% FA/ 5% ACN for LC-MS3 analysis.

Ast T., Itoh Y., Sadre S., McCoy J.G., Namkoong G., Wengrod J.C., Chicherin I., Joshi P.R., Kamenski P., Suess D.L., Amunts A, & Mootha V.K. (2024). METTL17 is an Fe-S cluster checkpoint for mitochondrial translation. Molecular cell, 84(2), 359-374.e8.

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Peptide Fractionation and Analysis Using TMT

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Tandem mass tag (TMT)–labeled peptides were solubilized in 5% acetonitrile (ACN)/10 mM ammonium bicarbonate, pH 8.0, and 300 μg of TMT-labeled peptides was separated by an Agilent 300 Extend C18 column (3.5 μm particles, 4.6 mm ID and 250 mm in length). An Agilent 1260 binary pump coupled with a photodiode array detector (Thermo Scientific) was used to separate the peptides. A 45 min linear gradient from 10% to 40% ACN in 10 mM ammonium bicarbonate pH 8.0 (flow rate of 0.6 ml/min) separated the peptide mixtures into a total of 96 fractions (36 s). A total of 96 fractions were consolidated into 24 samples in a checkerboard fashion, acidified with 20 μl of 10% formic acid, and vacuum dried to completion. Each sample was desalted via Stage Tips and redissolved in 5% formic acid/5% ACN for LC-MS3 analysis.

Joshi P.R., Sadre S., Guo X.A., McCoy J.G, & Mootha V.K. (2023). Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells. The Journal of Biological Chemistry, 299(9), 105075.

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Multiplexed Peptide Fractionation for Mass Spectrometry

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TMT labeled peptides were solubilized in 500 l solution containing 5% ACN/10 mM ammonium bicarbonate, pH 8.0 and 300 μg of TMT labeled peptides was separated by an Agilent 300 Extend C18 column (3.5 mm particles, 4.6 mm ID and 250 mm in length). An Agilent 1260 binary pump coupled with a photodiode array (PDA) detector (Thermo Scientific) was used to separate the peptides. A 45 minute linear gradient from 10% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8 (flow rate of 0.6 mL/min) separated the peptide mixtures into a total of 96 fractions (36 seconds). A total of 96 fractions were consolidated into 24 samples in a checkerboard fashion, acidified with 20 μl of 10% formic acid and vacuum dried. Each sample was desalted via StageTips and re-dissolved in 12 μl 5% FA/ 5% ACN, prior to LC–MS/MS analysis.

Dumesic P.A., Egan D.F., Gut P., Tran M.T., Parisi A., Chatterjee N., Jedrychowski M., Paschini M., Kazak L., Wilensky S.E., Dou F., Bogoslavski D., Cartier J.A., Perrimon N., Kajimura S., Parikh S.M, & Spiegelman B.M. (2019). An evolutionarily conserved uORF regulates PGC1α and oxidative metabolism in mice, flies, and bluefin tuna. Cell metabolism, 30(1), 190-200.e6.

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TMT Peptide Fractionation and Separation

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TMT labeled peptides were solubilized in 5% acetonitrile/10 mM ammonium bicarbonate, pH 8.0 and ~300 μg of TMT labeled peptides were separated by an Agilent 300 Extend C18 column (3.5 μm particles, 4.6 mm ID and 250 mm in length). An Agilent 1260 binary pump coupled with a photodiode array (PDA) detector (ThermoFisher Scientific) was used to separate the peptides. A 45-minute linear gradient from 10% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8.0 (flow rate of 0.25 mL/min) separated the peptide mixtures into a total of 96 fractions (36 seconds). A total of 96 Fractions were consolidated into 24 samples in a checkerboard fashion and vacuum dried to completion.

Van Vranken J.G., Li J., Mintseris J., Gadzuk-Shea M., Gygi S.P, & Schweppe D.K. (2024). Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays. bioRxiv.

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TMT-labeled Peptide Fractionation

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TMT-labeled peptides were solubilized in 5% ACN/10 mM ammonium bicarbonate, pH 8.0, and 300 μg of TMT-labeled peptides were separated by an Agilent 300 Extend C18 column (3.5 μm particles, 4.6 mm ID and 250 mm in length). An Agilent 1260 binary pump coupled with a photodiode array (PDA) detector (Thermo Scientific) was used to separate the peptides. A 45-min linear gradient from 10% to 40% acetonitrile in 10 mM ammonium bicarbonate pH 8.0 (flow rate of 0.6 mL/min) separated the peptide mixtures into a total of 96 fractions (36 s). A total of 96 Fractions were consolidated into 24 samples in a checkerboard fashion, acidified with 20 μL of 10% formic acid, and vacuum dried to completion. Each sample was desalted via Stage Tips and re-dissolved in 5% FA/5% ACN for LC-MS3 analysis.

Shin S., Han M.J., Jedrychowski M.P., Zhang Z., Shokat K.M., Plas D.R., Dephoure N, & Yoon S.O. (2023). mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching. Cell reports, 42(8), 112868.

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Serum Analysis by Electrospray TOF MS

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Serum (2 L) was diluted in 20 L water, 0.25 L formic acid added (then microfuged) and 2 L injected into an electrospray time-of-flight (TOF) MS system, consisting of an Agilent 1260 binary pump connected to an Agilent 6230 Accurate-Mass TOF liquid chromatography MS instrument (Agilent Technologies, Santa Clara, CA, USA) operating under MassHunter software. Chromatography was performed on a Luna C-8 5 m (20 Â 2 mm) column (Phenomenex, Torrance, CA, USA) with an acetonitrile/water solvent system (solvent A: 5% acetonitrile, 0.1% formic acid; solvent B: 85% acetonitrile, 0.1% formic acid; solvent gradient was run (300 L/min) from 45 to 50% solvent B over 3 min, then to 100% solvent B over 1 min). 5, 6 Instrumental conditions: source gas temperature 300 C, capillary 3500 V, fragmentor 250 V, skimmer 50 V. Profile data were collected and multicharged spectral envelopes deconvoluted from 1050 to 1600 m/z using maximum entropy processing and BioConfirm (Agilent Technologies) software with an isotope width of 16.1 Da. 6 Peptide m/z mapping Serum (20 L) was precipitated with 14 L of saturated ammonium sulphate and the supernatant dialysed against water before precipitation with an equal volume of acetone. The protein (800 g) was digested with trypsin and taken up in 50% acetonitrile, 0.5% formic acid before direct injection (1 g) into the TOF ion source at 50 L/min. 6

Ryan J.B., Brennan S.O., Potter H., Wolmarans L., Florkowski C.M, & George P.M. (2016). Familial dysalbuminaemic hyperthyroxinaemia: a rapid and novel mass spectrometry approach to diagnosis. Annals of clinical biochemistry, 53(Pt 4).

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Cardiac Metabolite Profiling by LC-MS

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Pulverized cardiac tissues were resuspended in 80:20 methanol:water and soluble extracts were collected and dried by speed vac. Extracts were reconstituted in 5 mM ammonium acetate in 95% water/5% acetonitrile + 0.5% acetic acid and filtered prior to LC-MS analysis. The filtered samples were injected to the LC system which was composed of two Agilent 1260 binary pumps, an Agilent 1260 auto-sampler and an Agilent 1290 column compartment containing a column-switching valve (Agilent Technologies, Santa Clara, CA). The chromatography was performed using Solvents A (5 mM ammonium acetate in water + 0.5% acetic acid + 0.5% acetonitrile) and B (acetonitrile + 0.5% acetic acid + 0.5% water), with 5% B for 2 min, 5% B to 80% B in 3 min, 80% B for 3 min, 80% B to 5% B in 3 min, and 5% B for 7 min.
After the chromatographic separation, MS ionization and data acquisition was performed using AB Sciex QTrap 5500 mass spectrometer (AB Sciex, Toronto, ON, Canada) equipped with electrospray ionization (ESI) source. Multiple-reaction-monitoring (MRM) mode was used for targeted data acquisition. The extracted MRM peaks were integrated using MultiQuant 2.1 software (AB Sciex, Toronto, ON, Canada).

Dai D.F., Karunadharma P.P., Chiao Y.A., Basisty N., Crispin D., Hsieh E.J., Chen T., Gu H., Djukovic D., Raftery D., Beyer R.P., MacCoss M.J, & Rabinovitch P.S. (2014). Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart. Aging Cell, 13(3), 529-539.

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