1200 system

Manufactured by Agilent Technologies

Sourced in Germany, United States

The Agilent 1200 system is a high-performance liquid chromatography (HPLC) instrument. It is designed to perform analytical separations and quantification of chemical compounds. The 1200 system provides reliable and consistent performance for a variety of applications.

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59 protocols using 1200 system

HPLC Analysis of Organic Compounds

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The HPLC system consisted of an Agilent 1200 system consisting of an on-line degasser, a binary pump G1312B, an autosampler G1367C, a column oven G1316B, and a DAD detector G1315C. The analytical separation column was a Kinetex XB-C18 (150 x 2.1 mm) with 2.6 μm particles. The gradient elution was performed using mobile phase A: 0.2 M ammonium formate buffer pH 4.0 and water (10:90 v/v) and mobile phase B: acetonitrile and methanol (40:60 v/v) at a flow rate of 0.2 ml/min. The gradient was 18% B from 0-10 min, then changed from 18% B to 36% B from 10-25 min returning to 18% B after 26 min with a total run time of 35 min. For UV detection, the wavelength at 330 nm was used.

Hansen S.H., Holmfred E., Cornett C., Maldonado C, & Rønsted N. (2015). An Efficient, Robust, and Inexpensive Grinding Device for Herbal Samples like Cinchona Bark. Scientia Pharmaceutica, 83(2), 369-376.

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Quantification of Propranolol in Biofluids

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Marrow was prepared for extraction by homogenization at a 1:1 ratio with 50 mM potassium phosphate buffer pH 7.4. A volume of 20 μL of serum or marrow homogenate was combined with 100 μL of HPLC grade acetonitrile and vortex mixed for two minutes. Subsequent to centrifugation at 14000 rpm for five minutes, the supernatant was transferred to a 96-well plate for liquid chromatograph tandem mass spectrometry (LC/MS-MS) analysis. A calibration curve was formed in mouse plasma from 1.00–200 nM by serial dilution and extracted via the same methodology. An Agilent 1200 system consisting of a binary pump, column compartment and autosampler was used for solvent delivery and sample introduction. Chromatographic separation was performed on an Agilent Zorbax SB C18 2.1 × 50 mm 2.7 μm column via a gradient using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Gradient elution was 95% A ramping to 5% A from 0.0–3.0 minutes, with re-equilibration at initial conditions from 3.0 to 4.0 minutes. The flow rate was 0.75 mL/min, and column temperature was 30°C. Detection of propranolol was obtained using an Agilent 6460 triple quadrupole mass spectrometer, monitoring the transition 260.0 → 116.0 with a fragmentor of 86 V and a collision energy of 21 V. The retention time of propranolol was 1.89 minutes.

Treyball A., Bergeron A.C., Brooks D.J., Langlais A.L., Hashmi H., Nagano K., Barlow D., Neilson R.J., Roy T.A., Nevola K.T., Houseknecht K.L., Baron R., Bouxsein M.L., Guntur A.R, & Motyl K.J. (2022). Propranolol promotes bone formation and limits resorption through novel mechanisms during anabolic parathyroid hormone treatment in female C57BL/6J mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 37(5), 954-971.

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Multimodal Characterization of Biomolecules

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Size-exclusion chromatography was conducted using a HiPrep 16/60 Sephadex S-200 column connected to an ÅKTA fast pro-tein liquid chromatography (FPLC) system in a Coy anaerobic chamber (Grass Lake, MI). UV-visible spectra were recorded on a Cary 50 spectrometer from Varian (now Agilent Technologies) using the WinUV software package to control the instrument. High performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (LC-MS/MS) was conducted on an Agilent Technologies (Santa Clara, CA) 1200 system coupled to an Agilent Technologies 6410 QQQ mass spectrometer. The system was operated with the associated MassHunter software package, which was also used for data collection and analysis. High-resolution ESI mass spectra were acquired in on a Waters LCT Premier instrument at the Penn State Huck Institute of Life Sciences proteomics and mass spectrometry core facility. DNA sequencing was conducted at the Huck Institutes of the Life Sciences genomics core facility. NMR spectra were recorded on a Bruker AV-3-HD-500 instrument and calibrated using residual solvent peaks as an internal reference. Multiplicities are recorded as: s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet, q = quartet, dt = doublet of triplets.

LaMattina J.W., Wang B., Badding E.D., Gadsby L.K., Grove T.L, & Booker S.J. (2017). The Radical S-Adenosylmethionine Methylase NosN Catalyzes both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide. Journal of the American Chemical Society, 139(48), 17438-17445.

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Analytical Techniques for Microbial Growth and Metabolite Quantification

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Growth was monitored by measuring turbidity at 600 nm (OD₆₀₀), cell counts, and biomass dry weight (DW). Cells were counted in a Bürker chamber; the DW was determined with a thermo-balance (MB 64 M, VWR, Radnor, PA, USA) (20).
Arabitol and residual glycerol in culture supernatants—clarified by centrifugation (10,000 rpm for 5 min at 4 °C) and filtration at 0.22 μm—were analyzed in HPLC with refractive index detector (1200 System, Agilent Technologies, Waldbronn, Germany). Isocratic elution was carried out at 60 °C with 0.8 mL/min of 5 mM H₂SO₄ through an ion exclusion column (Aminex HPX-87 H, Bio-Rad, Hercules, CA, USA) [27 (link)]. ¹ H- spectra were recorded at 298 K on Bruker FT-NMR Advance 400 (400.13 MHz). Chemical shift values are given in ppm relative to TMS and were determined by taking as reference the isotopic impurity signals DMSO-d6 (2.50 ppm). Prior to NMR analysis, the supernatant of the improved fed-batch cultures was lyophilized overnight with a Alpha 1–2 LD Laboratory (Christ, Germany). Polarimetric analysis was carried out using a Polax-2 L polarimeter (Atago, Japan).

Raimondi S., Foca G., Ulrici A., Destro L., Leonardi A., Buzzi R., Candeliere F., Rossi M, & Amaretti A. (2022). Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of d-arabitol from pure glycerol. Microbial Cell Factories, 21, 179.

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Optimized Organic Synthesis Protocol

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All reactions were carried out under nitrogen unless indicated otherwise. All reagents were obtained from available commercial sources and were used without further purification unless otherwise noted. The silica gel used in flash chromatography was 60 Å, 230–400 mesh. Analytical TLC was performed on Uniplate 250 µm silica gel plates with detection by UV light. NMR spectra were acquired on a JEOL ECS-400 400 MHz NMR spectrometer with multinuclear capability and 24-sample autosampler, with solvent as internal reference; the chemical shifts are reported in ppm, in δ units. Infrared spectra were acquired on a Nicolet Avatar FTIR. Ultraviolet-visible spectroscopy experiments were conducted on a Shimadzu UV-2450 spectrometer fitted with a TCC-240A temperature-controlled cell chamber. HPLC experiments were conducted using an Agilent 1200 system with a degasser, photodiode array detector, and temperature-controlled autosampler. High resolution mass spectroscopic data were obtained at the Mass Spectrometry & Analytical Proteomics Laboratory at the University of Kansas (Lawrence, KS). Regression analyses were completed using the GraphPad Prism 6 software suite.

Klingaman C.A., Wagner M.J., Brown J.R., Klecker J.B., Pauley E.H., Noldner C.J, & Mays J.R. (2016). HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation. Analytical biochemistry, 516, 37-47.

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HPLC Analysis of Bilirubin

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The HPLC analyses were performed using an Agilent 1200 system (CA, USA) with a diode-array detector. The method was a modification of that by McDonagh et al. [28 (link),29 ]. The mobile phase consisted of 0.1 M di-n-octylamine acetate in methanol and water; the stationary phase was represented by a Poroshell 120, SB-C18 column (4.6 x 100 mm, 2.7 μm; Agilent, CA, USA). Samples were prepared by mixing 20 μl of bilirubin solution with 180 μl of ice-cold 0.1 M di-n-octylamine acetate in methanol, then vortexed and centrifuged to eliminate proteins. Twenty μl of the prepared sample was injected onto the column.

Jasprova J., Dal Ben M., Vianello E., Goncharova I., Urbanova M., Vyroubalova K., Gazzin S., Tiribelli C., Sticha M., Cerna M, & Vitek L. (2016). The Biological Effects of Bilirubin Photoisomers. PLoS ONE, 11(2), e0148126.

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HPLC Analysis of Environmental Pollutants

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The adsorbed target compounds were analyzed by high-performance liquid chromatography (HPLC). Chromatographic measurement was performed using an Agilent 1200 system with a Diode array detector (DAD). The DAD detection wavelength were 205 nm for CA, IB, GEM, OPP, ASP, 260 nm for KF, IDM and 280 nm for CP, MA, TCS, BPA respectively. Separation was conducted on an Agilent Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm). Gradient separation was performed using a 0.1% H₃PO₄ aqueous solution and ACN as the A and B solvents see (Table S1). Concentration and velocity gradients were taken to separate the 11 target compounds. The linear gradient profile was as follows: the concentration of B was initially maintained at 35%, the concentration of B was linearly increased to 55% over a period of 5.5 min, and the concentration of B was maintained over a period of 33 min. The flow rate was initially maintained at 1 mL/min, the flow rate was linearly decreased to 0.5 mL/min at 6 min and maintained over a period of 22.5 min, the increased to 1 mL/min again at 23.5 min. The column temperature is set as 30 °C.

Yao Z., Zhao Q., Ma Y., Wang W., Zhou Q, & Li A. (2017). Magnetic microsphere-based portable solid phase extraction device for on-site pre-concentration of organics from large-volume water samples. Scientific Reports, 7, 8069.

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Liquid Chromatography-Mass Spectrometry Analysis

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Liquid chromatography was performed on an Agilent 1200 system coupled with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) maintained at 35 °C. Elution was performed with a mobile phase of A (0.1 % formic acid in acetonitrile) and B (0.1 % formic acid in water). A gradient elution of 12 % A at 0–2 min, 12–25 % A at 2–5 min, 25–40 % A at 5–7.5 min, 40–65 % A at 7.5−10 min, 65–80 % A at 10–13 min and 80−12 % A at 13–17 min was employed. The flow rate was set at 0.35 ml/min. The injection volume was 5 μl.
Mass spectrometric detection was carried out on an Agilent 6540 Q-TOF mass spectrometer (Hewlett Packard, Agilent, USA) with electrospray ionization (ESI) interface. The negative ion mode was used with the mass range set at m/z 100–1700. The conditions of ESI source were as follows: gas temperature, 300°C; drying gas (N₂) flow rate, 8 L/min; nebulizer, 45 psi; sheath gas temperature, 350 °C; sheath gas flow, 10 L/min; capillary voltage, 4000 V; fragmentor, 140 V; skimmer voltage, 65 V; OctopoleRFPeak, 750 V. Data were collected with the LC-MS-QTOF MassHunter Data Acquisition Software Ver. A.01.00 (Agilent Technologies) and analyzed with the Agilent MassHunter Qualitative Analysis Software B.06.00, respectively.

Su T., Cheng B.C., Fu X.Q., Li T., Guo H., Cao H.H., Kwan H.Y., Tse A.K., Yu H., Cao H, & Yu Z.L. (2016). Comparison of the toxicities, activities and chemical profiles of raw and processed Xanthii Fructus. BMC Complementary and Alternative Medicine, 16, 24.

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Analytical Characterization of Compounds

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UV–visible spectra were recorded
on a Varian Cary 50 spectrometer (Agilent, Walnut Creek, CA) using
the WinUV software package to control the instrument. High-performance
liquid chromatography (HPLC) with detection by tandem mass spectrometry
(LC-MS/MS) was conducted on an Agilent Technologies 1200 system coupled
to an Agilent Technologies 6410 QQQ mass spectrometer. The system
was operated with the associated MassHunter software package, which
was also used for data collection and analysis. HPLC was also conducted
on an Agilent 1100 Series system coupled to an Agilent 1100 Series
variable wavelength detector and quaternary pump.

Neti S.S., Sil D., Warui D.M., Esakova O.A., Solinski A.E., Serrano D.A., Krebs C, & Booker S.J. (2022). Characterization of LipS1 and LipS2 from Thermococcus kodakarensis: Proteins Annotated as Biotin Synthases, which Together Catalyze Formation of the Lipoyl Cofactor. ACS Bio & Med Chem Au, 2(5), 509-520.

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Optimized Organic Synthesis Protocol

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Vastenhout K.J., Tornberg R.H., Johnson A.L., Amolins M.W, & Mays J.R. (2014). HPLC-Based Method to Evaluate Kinetics of Glucosinolate Hydrolysis by Sinapis alba Myrosinase. Analytical biochemistry, 465, 105-113.

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