Kinetex c18 column

Manufactured by Agilent Technologies

Sourced in United States

The Kinetex C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a core-shell particle technology that provides efficient separation and fast analysis times.

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Kinetex C18 C18 column

7 protocols using kinetex c18 column

Quantification of Coral Prenylquinones

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Coral nubbins for prenylquinone extraction were stored in liquid nitrogen for a maximum of 48 hours. Nubbins were extracted using a 1:1 mixture of isopropanol and ethyl acetate containing 0.1 μM CoQ₉ (internal standard). Coral CoQ₁₀ and Symbiodinium PQ₉ pools were quantified by LC-MS using a slightly modified method of Lutz et al. [25 (link)]. In brief: prenylquinones were resolved using a Phenomenex Kinetex C18 column (150 mm × 4.6 mm, 2.6 μm particle size) on an Agilent 1100 series HPLC (Agilent, USA) coupled to a Bruker Esquire 3000 (Bruker Daltonics, USA). Absolute quantities of the prenylquinones were calculated from calibration plots obtained from standard compounds containing 0.1 μM CoQ₉ (internal standard). CoQ and PQ redox states (%PQH₂ and %CoQH₂) were expressed as the proportion of reduced to total (oxidised + reduced) prenylquinone. Coral CoQ data could potentially be biased by CoQ of Symbiodinium; however, symbiont CoQ was not detected with the method applied here, either because Symbiodinium type C2 contains a different isoform than the host CoQ₁₀ or because concentrations are below the detection limit [37 (link)].

Lutz A., Raina J.B., Motti C.A., Miller D.J, & van Oppen M.J. (2015). Host Coenzyme Q Redox State Is an Early Biomarker of Thermal Stress in the Coral Acropora millepora. PLoS ONE, 10(10), e0139290.

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UHPLC Analysis of Botanical Compounds

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UHPLC analysis was performed using an Agilent UHPLC system (1290 Infinity, Waldbronn, Germany) consisting of a binary pump VL (G4220B), a diode array detector (G4212A, DAD), a sampler (G4226A), a thermostatted column compartment (G1316A), and a thermostat (G1330B). The system was operated by OpenLAB CDS (ChemStation Edition) software (Agilent Technologies, Santa Clara, CA, USA). HPLC grade acetonitrile, methanol, acetic acid (J.T. Baker, Center Valley, PA, USA), and ultrapure water (Millipore RiOs & Milli-Q-Gradient water purification system, Millipore, Bedford, MA, USA) were used for the analyses. A Kinetex C18 column (50 × 2.1 mm, id, 1.7 μm, Agilent) with a mobile phase consisting of acetonitrile and 0.1 % acetic acid in water was used. The mobile phase gradient elution was programmed as follows: acetonitrile 1–5 % (0–7 min), 5–20 % (7–27 min), and 20–60 % (27–40 min). The flow rate of the mobile phase was set to 0.3 mL/min. The sample injection volume was set to 2.0 μL. The column temperature was maintained at 40 °C, and the UV detector was set to 254 and 220 nm. The sample solutions for the UHPLC analyses, including the EEDS (2,000 μg/mL, 80 % methanol), ethyl acetate (EtOAc) fraction (2,000 μg/mL, 100 % methanol), and helveticoside (100 μg/mL 100 % methanol) were filtered (Millex-FG 0.2 μ m, Millipore) prior to the injections.

Kim B.Y., Lee J, & Kim N.S. (2015). Helveticoside is a biologically active component of the seed extract of Descurainia sophia and induces reciprocal gene regulation in A549 human lung cancer cells. BMC Genomics, 16(1), 713.

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Quantifying Lasso Peptide Expression

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The level of expression of each lasso peptide was evaluated using reverse-phase HPLC. After expression, 25 μL of each variant was assessed using Phenomenex Kinetex C18 column (100 Å; 50 × 3.0 mm; 5 μm particle size) on an Agilent 1260 Infinity HPLC instrument operating at a flow rate of 1.0 mL/min using mobile phases ddH₂O/0.1% formic acid (mobile phase A) and acetonitrile/0.1% formic acid (mobile phase B). Samples were separated using a linear gradient beginning with 85% A/15% B and ending with 30% A/70% B over 20 mins. Samples were analyzed using a UV-Vis detector monitoring 220 nm, and the relative expression level was determined from the area of the peak corresponding to that variant.

Hills E., Woodward T.J., Fields S, & Brandsen B.M. (2022). Comprehensive mutational analysis of the lasso peptide klebsidin. ACS chemical biology, 17(4), 998-1010.

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HPLC Analysis of Phenolic Compounds

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In order to identify and characterize the phenolic compounds, a sample of 10 μL of the extracts was analyzed using a Shimadzu LC-20AT HPLC system equipped with a quaternary pump, solvent degasser, autosampler and a photodiode (PDA) detector. The separation of the compounds was performed on a 150 × 4.6 mm, 5 μm Kinetex C18 column, (Agilent Technologies, Santa Clara, CA, USA) using mobile phases A (water and phosphoric acid, pH 2.3) and B (acetonitrile) in a gradient mode (between 5 and 90% component B) for 45 min at a temperature of 35 °C, with a flow rate of 0.8 mL/min. The components of the mobile phase were filtered and degassed before starting the HPLC daily run, through Macherey-Nagel (MV) filters, 0.20 μm pore size and using an Elma-Elmasonic P sonication bath. Detection was performed by scanning from 190 to 800 nm and quantification was assessed at three specific wavelengths (280, 320 and 360 nm). The acquisition of data and the interpretation of the results occurred using the LC Solution software. Concentrations of standard compounds in extracts were determined from the peak areas using the equation for linear regression obtained from the calibration curves.

Gradinaru L.M., Barbalata-Mandru M., Enache A.A., Rimbu C.M., Badea G.I, & Aflori M. (2023). Chitosan Membranes Containing Plant Extracts: Preparation, Characterization and Antimicrobial Properties. International Journal of Molecular Sciences, 24(10), 8673.

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Characterization of Organic Compounds

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Optical rotations were measured on a Rudolph Research Analytical AUTOPOL IV digital polarimeter at 589 nm. UV absorptions were acquired with an Agilent Cary 60 UV-vis spectrophotometer. IR spectra were recorded with an Agilent Cary FTIR 630 spectrometer and PerkinElmer Spectrum Two equipped with a UATR (single reflection diamond) sample introduction system. NMR spectra were recorded on Varian Direct Drive 500 MHz and Varian Inova 500 MHz spectrometers. Chemical shifts are reported with the use of the residual CDCl₃ signals (δ_H 7.27 ppm; δ_C 77.0 ppm) as internal standards for ¹H and ¹³C NMR spectra, respectively. COSY, HSQC, HMBC, and ROESY experiments corroborated the ¹H and ¹³C NMR assignments. Analytical LC/MS with a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 μm) on an Agilent 6230 LC/TOF-MS with electrospray ionization detection provided the high-resolution masses. Semi-preparative and analytical HPLC separations were performed on a Shimadzu LC-20 AT system equipped with an ultraviolet (UV) detector using a Luna silica column (5 μm, 250 × 10 mm), and a YMC C-18 column (10 μm, 150 × 4 mm). MPLC was performed on a Teledyne Isco CombiFlash Rf 200i equipped with an evaporative light-scattering detector (ELSD) and a multiwavelength UV detector using a RediSep Rf silica 80 g flash column, and silica gel 230–400 mesh was used to load samples.

Limon A.C., Patabendige H.M., Azhari A., Sun X., Kyle D.E., Wilson N.G, & Baker B.J. (2022). Chemistry and Bioactivity of the Deep-Water Antarctic Octocoral Alcyonium sp.. Marine Drugs, 20(9), 576.

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Spectroscopic Characterization of Compounds

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Optical rotations were measured on a Rudolph Research Analytical AUTOPOL IV digital polarimeter. UV absorptions were measured by an Agilent Cary 60 UV–vis spectrophotometer in MeOH or MeCN, while IR spectra were recorded with an Agilent Cary 630 FTIR. All NMR spectra were acquired in CDCl₃ or CD₃CN with residual solvent referenced as an internal standard (7.27 and 1.94 ppm for ¹H and 77.0 and 1.29 ppm for ¹³C, respectively). All ¹H NMR spectra were recorded on a Varian 500 or 600 MHz Direct Drive instrument equipped with cold-probe detection, and ¹³C NMR spectra were recorded at 125 or 150 MHz, respectively. Analytical LC/MS was performed on a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 μm) on either an Agilent 6120 single quadrupole, an Agilent 6230 LC/ToF-MS, or an Agilent 6540 LC/QToF-MS with electrospray ionization detection, the latter two of which were utilized for HRESIMS. All HPLC analysis was performed on a Shimadzu LC20-AT system equipped with a photodiode array detector (M20A) using semipreparative [Phenomenex Luna C18 (250 × 10 mm, 5 μm)] or analytical [Phenomenex Luna C18 (250 × 4.6 mm, 5 μm) and Phenomenex Luna Silica (250 × 4.6 mm, 5 μm)] conditions. All solvents were obtained from Fisher Scientific and were HPLC grade (>99% purity) unless otherwise stated.

Shilling A.J., Witowski C.G., Maschek J.A., Azhari A., Vesely B.A., Kyle D.E., Amsler C.D., McClintock J.B, & Baker B.J. (2020). Spongian Diterpenoids Derived from the Antarctic Sponge Dendrilla antarctica Are Potent Inhibitors of the Leishmania Parasite. Journal of natural products, 83(5), 1553-1562.

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LCMS Methods for Analytical Separations

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One Agilent 6520 and two Agilent 6550 LCMS QTOF instruments were used in the course of these experiments. LCMS methods were carried out as described below in Supplementary Figures 1–43, all with A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) gradients as follows:
Method 1) Agilent 6550–1 (1290 Infinity HPLC system with iFunnel QTOF MS run in positive ionization mode with a low m/z range 100–1700) with a Phenomenex Jupiter C4 column, 150 × 1.0 mm, 5 μm, 300 Å silica; flow rate 100 μL/minute, 1–61% B gradient over 10 minutes, MS on from 4–12 minutes
Method 2) Agilent 6550–1 (as above) with a Phenomenex Luna C18(2 ) column, 150 ×5 mm, 3 μm, 100 Å silica; flow rate 50 μL/min, 1–61% B gradient over 12 minutes, MS on from 4–14 minutes
Method 3) Agilent 6550–2 (as in 6550–1 but with m/z range 100–3000) with an Agilent Zorbax 300SB C3 column, 150 × 2.1 mm, 5 pm, 300 Å silica; flow rate 500 pL/minute, 1–61% B gradient from 2–12 minutes, MS on from 4–12 minutes
Method 4) Agilent 6550–2 (as above) with a Phenomenex Kinetex C18 column; not reported in Supplementary Figures 1–43.
Method 5) Agilent 6520 (1290 Infinity HPLC system with QTOF MS run in positive ionization mode with m/z range 100–3000) with an Agilent Zorbax 300SB C3 column, 150 × 2.1 mm, 5 μm, 300 Å silica; flow rate 800 μL/minute, 1–61% B gradient over 9 minutes, MS on from 4–11 minutes.

Albin J.S, & Pentelute B.L. (2020). Efficient flow synthesis of human antimicrobial peptides. Australian journal of chemistry, 73(4), 380-388.

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