Luna c18 column

Manufactured by Agilent Technologies

Sourced in United States

The Luna C18 column is a reversed-phase liquid chromatography (RPLC) column designed for the separation and analysis of a wide range of compounds. It features a spherical silica-based stationary phase with chemically bonded C18 alkyl ligands. The Luna C18 column is commonly used for the separation and purification of small organic molecules, peptides, and other biomolecules.

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14 protocols using luna c18 column

Isolation and Purification of Compounds

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Isolation and purification were carried out by column chromatography. Agilent 1260 HPLC and thin-layer chromatography were used to monitor the separation, and thin-layer chromatography was performed on precoated silica gel 60 GF254 plates and visualized using UV illumination at 254 nm and 365 nm or by spraying with a 10% solution of sulfuric acid and 1% vanillin in ethanol. ¹H and ¹³C nuclear magnetic resonance spectra were recorded on a Bruker Avance 400 MHz spectrometer (Bruker BioSpin GmbH, Beijing, China) with tetramethylsilane as the internal standard. Chemical shifts are expressed in δ values. HPLC quantitative analysis was performed on Agilent 1260 LC Series instrument (Agilent, Santa Clara, CA, USA) equipped with a G4212B DAD using a Luna C-18 column (5 μm, 4.6 mm i.d. × 250 mm; Phenomenex, Inc., Torrance, CA, USA). Flow rate was 1.0 mL/min. The mobile phase was a mixture of 0.2% (v/v) phosphoric acid–water solution (A) and methanol (B) with a gradient elution as follows: 0–6 minutes, 0–50% B; 6–13 minutes, 50–57% B; 13–25 minutes, 57–60% B; 25–40 minutes, 60–70% B; 40–50 minutes, 70–100% B; 50–57 minutes, 100% B; 57–60 minutes, 100–0% B. The injection volume was 10 μL, and the column oven was maintained at 25°C. DAD detection wavelength was set at 295 nm for all analytes.

Cui X., Guo J., Lai C.S., Pan M.H., Ma Z., Guo S., Liu Q., Zhang L., Ho C.T, & Bai N. (2016). Analysis of bioactive constituents from the leaves of Amorpha fruticosa L. Journal of Food and Drug Analysis, 25(4), 992-999.

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Purification of SalC Assay Product

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For purification of the SalC assay product, reactions were set up as described above at a 50 mL scale in an Erlenmeyer flask. SalB-PCP loading was allowed to proceed for 12 hours, at which point approximately 1 gram of XAD7 resin and SalC (20 μM) were added to the reaction mixture and assays were allowed to proceed overnight. In the morning, resin was extracted (3×) with ethyl acetate. Organic extracts were combined and evaporated to dryness. Samples were resuspended in acetonitrile and the peak corresponding to R-17 was purified by preparative HPLC using a Phenomenex Luna C18 column (5 μm, 100 mm, 2mm i.d.), along with an Agilent Technologies system composed of a PrepStar pump, a ProStar 410 autosampler, and a ProStar UV detect (Agilent Technologies, Inc, Santa Clara, USA). The sample were eluted by a gradient from 20–100% acetonitrile over 40 min at a flow rate of 10 mL/min. The peak corresponding to R-17 was collected and dried by rotary evaporation and lyophilization. Finally, R-17 was purified using a small-scale silica column with a dichloromethane/acetonitrile solvent system. All stages of purification were monitored by LCMS analysis.

Bauman K.D., Shende V.V., Chen P.Y., Trivella D.B., Gulder T.A., Vellalath S., Romo D, & Moore B.S. (2022). Enzymatic assembly of the salinosporamide γ-lactam-β-lactone anticancer warhead. Nature chemical biology, 18(5), 538-546.

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HPLC Analysis of Antioxidant Compounds

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Each standard stock was prepared by dissolving GA, K-rol or K-ride in 100% methanol at 1 mg/mL. The solution was then filtered through a 0.2-mm syringe membrane filter (Whatman Ltd, Maidstone, UK) and the sample stock solution (50 mg in 100% methanol) was extracted using ultrasonic vibration at room temperature for 30 min. All standard and sample solutions were stored at 4 °C before analysis.
Separation was performed in a high-performance liquid chromatography (HPLC) system (Dionex Ultimate 3000; Thermo Fisher Scientific) comprising a pump, auto sampler, column oven, and diode array UV/VIS detector. Chromatograms were recorded using the Chromeleone software (version 7) system. The components of the AO extract were separated on a Luna C₁₈ column (4.6 × 250 mm, 5 μm; Agilent, CA, USA) at 40 °C, with an injection volume of 10 μL and a detective wavelength set at 330 nm. The mobile phase, which consisted of 0.05% phosphoric acid and acetonitrile, was run at a flow rate of 1.0 mL/min. The isocratic elution program was set at 35% acetonitrile for 60 min^{20 (link)}.

Lee J.J., Lee J.H., Yim N.H., Han J.H, & Ma J.Y. (2017). Application of galangin, an active component of Alpinia officinarum Hance (Zingiberaceae), for use in drug-eluting stents. Scientific Reports, 7, 8207.

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LC-MS Analysis of HMF Degradation Products

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To perform a LC‐MS analysis, samples of hydrothermally treated HMF solutions are separated on a Luna C18 column (150×4.6 mm I.D.) using an Agilent 1290 system and a 0.2 % w/v formic acid (solvent A), 0.1 % w/v formic acid in methanol (solvent B) gradient with the following profile: 0.0–2.5 min: 90 % A; 2.5–8.0 min: 67 % A; 13.0–18.0 min: 40 % A; 18.0–22.0 min: 10 % A; 22.0–26.0 min: 90 % A. The flow rate is 0.5 ml/min at an oven temperature of 40 °C.
For mass detection a Q‐Exactive Plus mass spectrometer is used under the control of the software XCalibur 3.0.63. The MS and MS/MS spectra are detected at the parameters detailed in Table 1 in negative and positive mode.

Körner P., Jung D, & Kruse A. (2019). Influence of the pH Value on the Hydrothermal Degradation of Fructose. ChemistryOpen, 8(8), 1109-1120.

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Quantification of Bioactive Compounds in PMC-12

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For analysis of quality and quantity for PMC-12, sample of 0.5 g dry weight was sonicated in 10 ml MeOH, filtered through a 0.45 μm membrane filter before HPLC analysis. HPLC using G1100 systems (Agilent Technologies, Waldbronn, Germany) was performed on a Luna C₁₈ column (5 μm, 150 mm × 3.0 mm i.d. Phenomenex, Torrance, CA, USA) with a mobile phase gradient of acetonitrile–water (0 to 100) for 35 min. The injection volume was 10 μl of sample and mobile phase flow rate 0.4 ml/min with UV detection at 254 nm for 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THS) and 3′,6-disinapoyl sucrose (DISS) and at 203 nm for catalpol. Acquisition and analysis of chromatographic data were performed using Agilent chromatographic Work Station software (Agilent Technologies). Stock solutions of THS, DISS, and catalpol were prepared for quantification of PMC-12. The contents of PMC-12 were determined by regression equations, calculated in the form of y = ax + b, where x and y were peak area and contents of the compound. The limits of detection (LOD) and quantification (LOQ) under the current chromatographic conditions were determined at a signal-to-noise ratio of 3 and 10, respectively.

Ahn S.M., Kim Y.R., Kim H.N., Choi Y.W., Lee J.W., Kim C.M., Baek J.U., Shin H.K, & Choi B.T. (2015). Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia. BMC Complementary and Alternative Medicine, 15, 202.

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CmnC Activity Assay and Hydroxylation Reaction

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The CmnC activity assay was followed by the procedures carried out in OrfP (Chang et al., 2014 (link)). The hydroxylation reaction was performed in a final volume of 200 μL containing a given substrate (1 mM), α-KG (1 mM), FeCl₂ (0.05 mM), ascorbic acid (0.2 mM) and purified enzyme (5 μM) in a MOPS buffer (50 mM, pH 7.0). The reaction was incubated at 26°C for 5 h. Protein was precipitated with HCl, and the supernatant was collected for dansyl chloride (DNS-Cl) derivatization. For dansylation, the reaction mixture (100 μl) was added sequentially with solutions of 80 mM Li₂CO₃ (100 μl, in water) and 3 mM DNS-Cl (100 μl, in acetonitrile). The reaction mixture was then filtered and subjected to HPLC-ESI-LTQ analysis (Phenomenex Luna-C18 column, 3 μm; 4.6 × 250 mm, Agilent 1200 Series HPLC interface with an ESI source coupled to a Thermo-Finnigan LTQ-XL ion trap spectrometer) at a flow rate of 1 ml/min with three-stage elution gradient (% = A/B × 100%, A: deionized water with 0.1% trifluoroacetic acid, B: acetonitrile with 0.1% trifluoroacetic acid): 98% for 5 min, a linear gradient to 70% in 27 mins, a second linear gradient to 2% in 5 min. The UV wavelength was set at 254 nm.

Hsiao Y.H., Huang S.J., Lin E.C., Hsiao P.Y., Toh S.I., Chen I.H., Xu Z., Lin Y.P., Liu H.J, & Chang C.Y. (2022). Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer. Frontiers in Chemistry, 10, 1001311.

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HPLC Analysis of Avenanthramides

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HPLC analysis of avenanthramides was carried out on an Agilent 1100 high pressure liquid chromatography (HPLC) instrument equipped with a 3 µm, 4.6 × 150 mm Phenomenex Luna C18 column (Torrance, CA, USA) and an Agilent photodiode array detector (Santa Clara, CA, USA) at 340 nm. The mobile phase consisted of solvent A (H₂O with 5% acetonitrile and 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). A gradient of 13% to 30% buffer B over 20 min at a flow rate of 1.0 mL/min was applied. The avenanthramides were identified by comparison of chromatography retention times and mass spectra to authentic standards (avenanthramide A, B and C) (Sigma, St. Louis, MO, USA). Mass spectrometry analysis was performed following [34 (link)].

Li Z., Chen Y., Meesapyodsuk D, & Qiu X. (2019). The Biosynthetic Pathway of Major Avenanthramides in Oat. Metabolites, 9(8), 163.

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HPLC Analysis of Rutin in Wampee

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The identification of rutin in different wampee parts was evaluated by high-performance liquid chromatography (HPLC). The HPLC analysis was carried out on an Agilent Technologies 1260 instrument (Santa Clara, CA, USA) equipped with a Luna C18 column (ø 250 mm × 4.6 mm) and UV detection. Gradient programs were used with the mobile phase consisted of solvent A (water) and solvent B (acetonitrile) as follows: 5% (initial), 5%–30% (30 min), 30%–80% (20 min), and 100% (5 min). At a flow rate of 1.0 ml/min, typically 20 µL portions were injected. The detected wavelength was 254 nm, and the column temperature was maintained at 25°C.

Zhu T., Zuo W., Yan J., Wen P., Pei Z., Lian H, & Yang H.C. (2020). Comparative Assessment of the Antioxidant Activities among the Extracts of Different Parts of Clausena lansium (Lour.) Skeels in Human Gingival Fibroblast Cells. Evidence-based Complementary and Alternative Medicine : eCAM, 2020, 3958098.

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Prep-HPLC Analysis of Organic Compounds

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A prep-HPLC was carried out using an Agilent 1100 instrument with a Luna C18 column (10 × 250 mm, ø = 5 µm). The mobile phase was a mixture of acetonitrile/methanol/water (v/v 30:50:20), and the flow rate and injection volume were 3.0 mL/min and 50 µL, respectively. The eluents were detected at 230 nm, using a photodiode array detector. The samples were dissolved in isopropanol.

Shin S.Y., Lee D.H., Gil H.N., Kim B.S., Choe J.S., Kim J.B., Lee Y.H, & Lim Y. (2017). Agerarin, identified from Ageratum houstonianum, stimulates circadian CLOCK-mediated aquaporin-3 gene expression in HaCaT keratinocytes. Scientific Reports, 7, 11175.

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Rifampicin Release from Functionalized IONPs

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An aliquot of 40 mg of the Rif@IONPs-PAA-PEG-MAN were put into a dialysis bag (MWCO: 8000–14,000). The dialysis bag (MWCO: 8000–14,000) was then put into 10 ml of PBS solution at different pH (7.4 or 5.5) with constant shaking at 37 ℃ in a tube. At predetermined time intervals, 200 µl of solution was taken out from the vial (outside the dialysis bag) with pipet and the same volume of fresh PBS solution was added. The released rifampicin was measured by high performance liquid chromatography (HPLC, Agilent, Santa Clara, CA) equipped with a Luna C18 column (250 mm × 4.6 mm × 5 μm) and wavelength of 480 nm using 25 mM ammonium acetate/acetonitrile (45%/55%) as the mobile phase.

Shen L., Liao K., Yang E., Yang F., Lin W., Wang J., Fan S., Huang X., Chen L., Shen H., Jin H., Ruan Y., Liu X., Zeng G., Xu J.F, & Pi J. (2023). Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance. Journal of Nanobiotechnology, 21, 369.

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