C18 reversed phase column

Manufactured by Agilent Technologies

Sourced in United States

The C18 reversed-phase column is a type of high-performance liquid chromatography (HPLC) column used for the separation and purification of a wide range of compounds. It is composed of a silica-based stationary phase with chemically bonded C18 alkyl chains, which provide a non-polar surface for the separation of analytes. The C18 column is a widely used and versatile tool in analytical and preparative applications.

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Lab products found in correlation

42 protocols using c18 reversed phase column

Esterification Reaction Monitoring by HPLC-DAD and MS

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The esterification reaction was monitored by HPLC-DAD (Merck) in a reverse-phase C18 column (Agilent) with 250 × 4.6 mm i.d., 2.7 μm at 25 °C. The eluents used were (A) 1% (v/v) formic acid in water and (B) 1% (v/v) formic acid in acetonitrile, and the elution gradient was performed from 50 to 100% B during 51 min at a flow rate of 0.4 mL/min. After 51 min, the column was washed with 100% B for 15 min and, then, it was stabilized with the initial conditions for more 10 min.
Mass spectrometry (MS) analysis of the pyranoflavylium-cinnamic derivative esters was performed using a Finnigan Surveyor series liquid chromatograph equipped with a reversed-phase C18 column (Agilent) with 250 × 4.6 mm i.d., 2.7 μm thermostatted at 25 °C). The mass detection was carried out in the positive ion mode in a Finnigan LCQ DECA XP MAX (Finnigan Corp., San José, CA, USA) mass detector with an API (Atmospheric Pressure Ionization) source of ionization and an ESI (ElectroSpray Ionization) interface. The solvents and HPLC gradient were used as the same reported above for the HPLC analysis. Spectra were recorded in the positive ion mode between m/z 300 and 1500.

Pereira A.R., de Freitas V., Mateus N, & Oliveira J. (2022). Functionalization of 7-Hydroxy-pyranoflavylium: Synthesis of New Dyes with Extended Chromatic Stability. Molecules, 27(21), 7351.

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Phytosterol Bioconversion and Analysis

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The bioconversion of phytosterols was detected as previously described [11 (link)]. The target product 9-OHAD was analyzed by high-performance liquid chromatography (Agilent Technologies, CA, USA) with an Agilent reversed-phase C18 column (250 × 4.6 mm²; 30 °C) using methanol/water (80:20, v/v) as the mobile phase at a flow rate of 1 mL/min with ultraviolet detection at 254 nm. The amounts of phytosterols were determined using a gas chromatography system 7820A (Agilent Technologies, CA, USA) with an Agilent DB-5 column [30 m × 0.25 mm (i.d.) × 0.25-μm film thickness]. The oven temperature was controlled as follows: 200 °C for 2 min, 200–280 °C within 4 min, 280 °C for 2 min, 280–305 °C within 1.5 min, and 305 °C for 10 min. The inlet and flame-ionization detector temperatures were maintained at 320 °C. The nitrogen carrier gas flow was 2 mL/min at 50 °C.

Song L., Ke J., Luo Z.K., Xiong L.B., Dong Y.G., Wei D.Z, & Wang F.Q. (2023). Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide. Biotechnology for Biofuels and Bioproducts, 16, 98.

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Quantification of Phenolic Compounds in ME

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Phenolic composition in the ME were identified and quantified by reverse phase HPLC, using the Agilent 1100 Series (Agilent, USA) separation module equipped with a reversed-phase C₁₈ column (250 mm × 4.6 mm, 5.0 μm, Kromasil) according to the method reported by Liu et al.⁵⁴. A binary solvent system was employed consisting of formic acid/water (1%, v/v) as solvent A and acetonitrile/methanol (80:20, v/v) as solvent B, and the diode array UV detector (DAD) was set at 280 nm to record the peak intensity. The gradient programme was 0–5 min with 3% solvent B, 5–12 min with 3–10% B, 12–25 min with 10–18% B, 25–35 min with 18–20% B, 35–40 min with 20–80% B, 40–45 min with 80–3% B and 45–50 min with 3% B. The flow rate of the mobile phase was 1.0 mL/min, and the injection volume was 10.0 μL. The column was operated at 30 °C. Identification was performed from the comparison with the retention time of standard, and individual phenolic content was estimated on the basis of peak area and the calibration curves of the corresponding standards.

Zhang L.L., Zhang L.F, & Xu J.G. (2020). Chemical composition, antibacterial activity and action mechanism of different extracts from hawthorn (Crataegus pinnatifida Bge.). Scientific Reports, 10, 8876.

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Spectroscopic Characterization of Compounds

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Optical
rotations were measured using a JASCO P-2000 polarimeter with a 10
cm cell. UV spectra were obtained using Varian Cary 50 Bio UV–visible
spectrophotometer. IR spectra were acquired on a PerkinElmer 1600
series FTIR spectrometer. ¹H, ¹³C, and 2D NMR
spectral data were obtained on a Varian Inova 500 MHz NMR spectrometer.
High-resolution mass spectra were recorded on a ThermoFinnigan MAT900XL
instrument with an Agilent ESI-TOF detector at The Scripps Research
Institute, La Jolla, CA. Low-resolution LC/MS spectra were obtained
on a Hewlett-Packard HP1100 integrated LC-MS system with a reversed-phase
C18 column (Agilent, 4.6 mm × 100 mm, 5 μm) at a flow rate
of 0.7 mL/min. Reversed-phase HPLC separations were performed using
a semipreparative C18 Phenomenex Luna (2) 5 μm (10 mm ×
250 mm) column with a CH₃CN/H₂O gradient solvent
system. Preparative HPLC was performed using a Waters model 4000 system
with a UV variable-wavelength detector monitoring at 210 nm using
a C18 Nova-Pak 6 μm 60 Å, (40 mm × 300 mm) column.

Kim M.C., Winter J.M., Asolkar R.N., Boonlarppradab C., Cullum R, & Fenical W. (2021). Marinoterpins A–C: Rare Linear Merosesterterpenoids from Marine-Derived Actinomycete Bacteria of the Family Streptomycetaceae. The Journal of Organic Chemistry, 86(16), 11140-11148.

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Quantitation of Drug Nanocrystals by HPLC

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The amount of MTK suspended in the preparations was determined by HPLC assay. Nanocrystal suspension (1 mL) was ultra-centrifuged at 13,000 rpm for 10 min to settle the nanocrystals in the aqueous vehicle. Methanol was then added to the precipitate and vortexed for 30 min to dissolve the drug nanocrystals. The concentration of MTK in the organic solvent or in the supernatant was analyzed using Waters HPLC system that is composed of a pump (Model 515 pump), a UV–VIS (ultraviolet–visible) detector (Model 486), and an autosampler (Model 717 plus). The mobile phase comprised acetonitrile and distilled water at a volume ratio of 4:6 with 0.15% v/v of trifluoroacetic acid, was flowed through the reversed-phase C18 column (4.6 mm × 50 mm, 1.8 µm, Agilent, Santa Clara, CA, USA) at a flow rate of 1.2 mL/min. The column temperature was set to 25 °C. A 20 µL aliquot was injected and the column eluent was monitored at a wavelength of 238 nm. The sharp peak of MTK was detected at 10.7 min. The least-square linear regression was linear in the MTK range from 1.0–100 μg/mL, with a coefficient of determination (r2) value of 0.997.

Im S.H., Jung H.T., Ho M.J., Lee J.E., Kim H.T., Kim D.Y., Lee H.C., Choi Y.S, & Kang M.J. (2019). Montelukast Nanocrystals for Transdermal Delivery with Improved Chemical Stability. Pharmaceutics, 12(1), 18.

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Quantitative PCA Extraction and HPLC Detection

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PCA must be extracted from the fermentation broth and then detected and quantified by high-performance liquid chromatography (HPLC). We first extracted PCA from the fermentation broth using ethyl acetate and subjected it to reversed-phase C18 column (Agilent Technologies, 5 μm, 4.6*250 mm). Both 2-OH-PHZ and PCN are phenazine derivatives and exhibit similar material properties; the specific extraction and detection steps of these samples are described in our previous studies (Liu et al., 2016 (link); Li et al., 2020 (link)).

Liu K., Li Z., Liang X., Xu Y., Cao Y., Wang R., Li P, & Li L. (2023). Biosynthesis and genetic engineering of phenazine-1-carboxylic acid in Pseudomonas chlororaphis Lzh-T5. Frontiers in Microbiology, 14, 1186052.

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HPLC Quantification of Analyte X

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First, tissue homogenates were prepared by homogenization of 250 mg tissue in 1 mL normal saline (0.9%) using a Tissue Tearor (BioSpec Products, Bartlesville, OK, USA). Upon 200 µL plasma or tissue homogenates, 200 µL of acetonitrile was added and vortexed for 1 minute. After standing for 5 minutes, the mixture was centrifuged at 3,500 rpm for 5 minutes. A 50 µL of supernatant filtered earlier through 0.45 µm filters was automatically injected concurrently with the standard solutions into an Agilent HPLC system. The HPLC instrument (1260 Infinity LC systems; Agilent Technologies, Santa Clara, CA, USA) was equipped with a reversed-phase C18 column (25 cm ×4.6 mm; PS =5 µm) and 1260 Infinity Diode Array Detector. The system was also equipped with Agilent ChemStation^® Software. A previously validated HPLC method was utilized for the determination of AE, with slight modification.7 The samples were eluted isocratically using acetonitrile–water–phosphoric acid (64:36:0.1, v/v/v) at a flow rate of 1 mL/min at column temperature of 30°C and wavelength of 254 nm. The calibration curve of peak area against AE concentration was y =188.45x +39.105, R²=0.9961, under concentration range of 0.15–50 µg/mL. Retention time was 5.8±0.1 minutes.

Freag M.S., Elnaggar Y.S., Abdelmonsif D.A, & Abdallah O.Y. (2016). Stealth, biocompatible monoolein-based lyotropic liquid crystalline nanoparticles for enhanced aloe-emodin delivery to breast cancer cells: in vitro and in vivo studies. International Journal of Nanomedicine, 11, 4799-4818.

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HPLC-DAD Purity and Stability Analysis

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Compounds purity and stability were checked by HPLC-DAD (Merck) in a reversed-phase C18 column (Agilent) with 250 × 4.6 mm i.d., particle size 2.7 μm and at 25 °C (Fig. S1). The eluents used were (A) 1% (v/v) formic acid in water and (B) 0.5% (v/v) formic acid in 80% (v/v) acetonitrile and the elution gradient consisted in 40–85% B during 50 min at a flow rate of 0.4 mL/min. After 50 min, the column was washed with 100% B during 10 min and then it was stabilized with the initial conditions for more 10 min.

Oliveira H., Araújo P., Pereira A.R., Mateus N., de Freitas V., Oliveira J, & Fernandes I. (2021). Photoactivated cell-killing amino-based flavylium compounds. Scientific Reports, 11, 22005.

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Quantification of Drug Loading in Micelles

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The EE was measured according to previously described approach [21 (link)]. Briefly, DS-M diluted 10 fold with distilled water was filtered through a 0.22 μm nylon (polyamide) membrane and 0.1 mL of the filtered liquid was accurately transferred into a volumetric flask containing 2 mL of methanol. After the structure of micelle was destroyed (ultrasound for 20 min), the sample was diluted to 5 mL with methanol and the DS containing in the micelle was determined using a HPLC method. The quantification of DS was performed using an Agilent 1100 series HPLC system (Agilent, Santa Clara, CA, USA), which was equipped with the Agilent 1200 series DAD detector and a reversed-phase C18 column (4.6 mm × 250 mm, 5 μm, Diamonsil plus). The data were captured and processed using Agilent ChemStation for LC 3D systems acquisition software. The mobile phase was a mixture of methanol, acetonitrile and water (pH = 3.0, 3:3:4, v/v) and eluted at a flow rate of 1.0 mL/min. Effluents were detected at 385 nm. This method has been validated for selectivity, linearity, limit of detection and quantification, accuracy, precision, as well as repeatability. The EE was determined using the following equation:

Jin W., Tan X., Wen J., Meng Y., Zhang Y., Li H., Jiang D., Song H, & Zheng W. (2019). A Novel Dantrolene Sodium-Loaded Mixed Micelle Containing a Small Amount of Cremophor EL: Characterization, Stability, Safety and Pharmacokinetics. Molecules, 24(4), 728.

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Isolation and Characterization of Algicidal Compounds

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Strain B1 was cultured in 2216E liquid medium (4 L) at 30°C with shaking at 160 rpm for 3 d. The bacterial culture was centrifuged at 4000 × g for 10 min to collect the supernatant, and then filtered through a 0.22 μm microporous membrane. The filtrate was evaporated to dryness by using a rotary evaporator. The residue was applied to the silica gel column chromatography (the silica particle size: 200–300 mesh) and eluted with six volume ratios of CHCl₃/CH₃OH (5:5, 4:6, 3:7, 2:8, 1:9 and 0:1). Fractions that exhibited algicidal activity were subjected to Sephadex G-15 (Pharmacia) column chromatography with ultrapure water as eluent. The active fraction was applied to a reversed-phase C₁₈ column (4.6 × 250 mm, Agilent) connected to an HPLC system and monitored at 254 nm using UV detector. The purified compounds from HPLC were identified by Q-TOF-MS and PeakView software (AB SCIEX TripleTOF 5600 + System with Accelerator TOF Analyzer and Electrospray Ionization source).

Yang Q., Chen L., Hu X., Zhao L., Yin P, & Li Q. (2015). Toxic Effect of a Marine Bacterium on Aquatic Organisms and Its Algicidal Substances against Phaeocystis globosa. PLoS ONE, 10(2), e0114933.

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