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Betasil c18

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Betasil C18 is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a silica-based stationary phase with C18 bonded ligands, providing a stable and reliable platform for diverse HPLC applications.

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Lab products found in correlation

Ultimate 3000, by Thermo Fisher Scientific (2 mentions) As500, by Agilent Technologies (1 mentions) Silica gel 60, by Merck Group (1 mentions) Exactive orbitrap high performance benchtop lc ms, by Thermo Fisher Scientific (1 mentions) Accela hplc system, by Thermo Fisher Scientific (1 mentions) Finnigan surveyor hplc system, by Thermo Fisher Scientific (1 mentions) Chromeleon 7, by Thermo Fisher Scientific (1 mentions) Ecd 3000rs, by Thermo Fisher Scientific (1 mentions)

7 protocols using betasil c18

1

HPLC Analysis of Acyl-CoA Species

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HPLC analysis of acyl-CoA species was performed similarly to previously published methods53 (link). Prior to sample runs, column (Betasil C18, 100 × 2.1 mm, 3 μm particle; Thermo Scientific) was washed with 100 mL of Solvent B (MeOH) followed by 100 mL of Solvent A (20 mM ammonium acetate:MeOH (94:6, v/v), pH 7.0). A methanol “blank” was injected at the start of each running period. The injection volume for all samples, blanks and standards was 15 μL. The flow rate for the method was 0.5 mL/min and the total duration of each individual run was 1 hr. The solvent percentages were as follows for Solvent A: 10.5 min of a gradient from 100% to 94%, 7.5 min at 94% isocratic, 27 min of a gradient from 94% to 25%, 2 min of a gradient from 25% to 100%, and 13 min of 100% isocratic. Acyl-CoA peaks were quantified by post-column UV-Vis absorbance measurements at 259 nm. Peak identities were verified using corresponding LvaE-generated standards as described above and abundance was quantified using the Chromeleon 7.2.10 software with Cobra Wizard defaults. Synthetic standards of 4-phosphoacyl-CoAs could not be synthesized, thus their elution times were confirmed by injecting reaction mixtures containing LvaE-generated 4-hydroxyacyl-CoAs and ACAD-inactive recombinant ACAD11 (A*). The presence of 4-phosphoacyl-CoA in these reactions was confirmed by LC-MS.
Rashan E.H., Bartlett A.K., Khana D.B., Zhang J., Jain R., Smith A.J., Baker Z.N., Cook T., Caldwell A., Chevalier A.R., Pfleger B.F., Yuan P., Amador-Noguez D., Simcox J.A, & Pagliarini D.J. (2024). ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism. bioRxiv.
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2

Analytical Characterization of Organic Compounds

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All chemicals and solvents were obtained from commercial sources without further purification. Mass spectra were recorded using Waters (Micromass ZQ) equipped with a Thermo Betasil C18 (150 × 2.1 mm, 3μ). 1H NMR spectra were recorded on a Varian AS500 instrument. Chromatography on silica gel columns were performed, using Merck silica gel 60 (230‐400 mesh), and analytical thin layer chromatography (TLC) was conducted on a glass plate coated with a 0.25 cm thin layer of silica gel 60F254.
Kennedy A.E., Laamanen C.A., Ross M.S., Vohra R., Boreham D.R., Scott J.A, & Ross G.M. (2017). Nerve growth factor inhibitor with novel‐binding domain demonstrates nanomolar efficacy in both cell‐based and cell‐free assay systems. Pharmacology Research & Perspectives, 5(5), e00339.
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3

Analyzing Metabolic Profiles via LC-MS

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Exactive Orbitrap High Performance Benchtop LC-MS (Thermo Fisher Scientific) with an electron spray ion source and an Accela HPLC System, C18 column (Betasil C18, 150 × 2.1 mm, Thermo Fisher Scientific), solvents: acetonitrile and distilled water (both supplemented with 0.1% formic acid), flow rate: 0.2 mL/min; program: hold 1 min at 5% acetonitrile, 1–16 min 5–99% acetonitrile, hold 15 min 99% acetonitrile, 19–20 min 99% to 5% acetonitrile, hold 11 min at 5% acetonitrile. The metabolic profiles of the acquired fractions were monitored with HR-ESI-LC/MS.
Mahler L., Niehs S.P., Martin K., Weber T., Scherlach K., Hertweck C., Roth M, & Rosenbaum M.A. (2021). Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities. eLife, 10, e64774.
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4

Quantitative Analysis of Coenzyme Q Levels

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Samples were injected into HPLC (Ultimate 3000, Thermo Scientific) with an electrochemical detector (ECD-3000RS). The first electrode (6020RS) was set to +600 mV and placed before the column (Thermo Scientific, Betasil C18, 100 × 2.1 mm, 3 μM particle) to oxidize all quinones. The second electrode (6011RS) was set to −600 mV to reduce all quinones exiting the column, and the third electrode was set at +600 mV to measure redox active species. Peaks were quantified with Chromeleon 7.2.10 software. To measure CoQ4 levels, extracted lipids were resuspended in methanol, and the mobile phase was 95% methanol 5% 1M ammonium acetate pH 4.4 in water. To measure CoQ10 levels, extracted lipids were resuspended in isopropanol, and the mobile phase was 78% methanol, 20% isopropanol, and 2% 1M ammonium acetate pH 4.4 in water.
Steenberge L.H., Rogers S., Sung A.Y., Fan J, & Pagliarini D.J. (2024). Coenzyme Q4 is a functional substitute for coenzyme Q10 and can be targeted to the mitochondria. The Journal of Biological Chemistry, 300(5), 107269.
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5

HPLC Analysis of Aceclofenac Content

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Measurements were performed on a Thermo Scientific Finnigan Surveyor HPLC system, consisting of a quaternary pump, an autosampler with a temperature-controlled column compartment, and a diode array detector. A Thermo Scientific Betasil C18 (150 × 4.6 mm, 5 μm) chromatographic column was employed throughout the test, thermostatted at 55 °C. The mobile phase consisted of 0.1% (v/v) aqueous phosphoric acid solution: acetonitrile (45:55 v/v%), delivered with a 1.5 mL/min flow rate (retention time of aceclofenac was ~3.0 min). Quantitative determinations were performed at 270 nm, based on a five-point calibration curve (methanolic solution, at 10–100 μg/mL range). To determine the aceclofenac content, the microfibrous samples (n = 3) were dissolved in methanol and diluted 10-fold with the same solvent.
Bitay E., Gergely A.L, & Szabó Z.I. (2023). Optimization and Production of Aceclofenac-Loaded Microfiber Solid Dispersion by Centrifugal Spinning. Pharmaceutics, 15(9), 2256.
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6

Quantification of Lipid-Soluble Quinones via HPLC-ECD

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Extracted dried lipids were resuspended in 50 μL of mobile phase (78% methanol. 20% isopropanol, 2% 1 M ammonium acetate pH 4.4 in water and transferred to amber glass vials with inserts. Vials were inserted into HPLC (Ultimate 3000, Thermo Scientific) with electrochemical detector (ECD-3000RS) and 10 μL of each sample was injected. The flow rate was set to 0.3 mL/min (LPG-3400RS pump). The first electrode (6020RS) was set to +600 mV and placed before the column (Thermo Scientific, Betasil C18, 100 × 2.1 mm, 3um particle) to oxidize all the quinones and ensure their simultaneous elution. Second electrode (6011RS) was set to −600 mV to reduce the quinones exiting the column, and then the third electrode was set to +600 mV to make final recordings. A CoQ6 standard (Avanti Polar Lipids) was used to identify corresponding peak in the obtained data. Peaks were then quantified with Chromeleon 7.2.10 software using cobra wizard option.
Manicki M., Aydin H., Abriata L.A., Overmyer K.A., Guerra R.M., Coon J.J., Peraro M.D., Frost A, & Pagliarini D.J. (2022). Structure and functionality of a multimeric human COQ7:COQ9 complex. Molecular cell, 82(22), 4307-4323.e10.
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7

Quantifying Coenzyme Q Levels

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Extracted lipids were resuspended in 2-propanol (50 μl) and transferred to an amber vial. Sodium borohydride (2 μl of 10 mM in 2-propanol) was added to each vial, followed by brief vortexing and incubation (10 min, r.t.) to reduce the CoQ. Methanol (50 μl) was then added to each sample to remove excess sodium borohydride and the vials were flushed with argon gas. CoQ measurements were performed using reverse-phase HPLC-ECD66 . Separation was performed using a C18 column (Thermo Scientific, Betasil C18, 100 × 2.1 mm, particle size 3 μm) at a flow rate of 0.3 ml min−1 with a mobile phase of 78% methanol, 10% 2-propanol, 10% acetonitrile and 2% ammonium acetate (1 M, pH 4.4). Electrochemical detection was performed using an ECD detector (Thermo Scientific ECD3000-RS) containing a 6020RS omni Coulometric Guarding Cell (E1) set to −200 mV and two 6011RS ultra Analytical Cells (E2 and E3) set to 600 mV. CoQ measurements were made on the analytical E2 electrode. For each experiment, CoQ6 and CoQ8 standards (Avanti) were prepared in the same manner as the experimental samples. Peak areas were quantified using Chromeleon 7.2.10 software (Thermo) and normalized to the CoQ8 internal standard. CoQ6 levels were further normalized to OD600 at collection.
Tai J., Guerra R.M., Rogers S.W., Fang Z., Muehlbauer L.K., Shishkova E., Overmyer K.A., Coon J.J, & Pagliarini D.J. (2023). Hem25p is required for mitochondrial IPP transport in fungi. Nature cell biology, 25(11), 1616-1624.
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