C18 analytical column

Manufactured by Waters Corporation

Sourced in United States

The C18 analytical column is a type of chromatography column used for the separation and analysis of a wide range of compounds. It is designed to facilitate the separation of molecules based on their hydrophobic interactions with the stationary phase, which is typically composed of silica particles coated with a C18 alkyl chain. The C18 column is widely used in various analytical techniques, such as high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC), to separate and identify a diverse range of molecules, including pharmaceuticals, environmental pollutants, and biochemical compounds.

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Lab products found in correlation

17 protocols using c18 analytical column

Eculizumab Hydrogen/Deuterium Exchange

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Hydrogen/deuterium exchange-MS (H/DX-MS) was initiated by dilution of eculizumab (SB12 and RP) in D₂O buffer for 10 s, 1 min, 10 min, 1 h and 4 h. After labeling, the solution was mixed with quenching buffer and injected into the Waters nano LC system. Loaded protein was digested on an immobilized pepsin column (Waters) and the digested peptide fragments was separated on an analytical C18 column (Waters). The analyte was then introduced to MS with MS^E mode. Mass spectra were analyzed using PLGS software (Waters) for peptide identification, and DynamX software (Waters) for deuterium uptake calculation and generation of the butterfly plot and uptake plots.

Kim H., Hong E., Lee J., Hong S., Kim J., Cho M., Kim Y, & Yoo T. (2023). Characterization for the Similarity Assessment between Proposed Biosimilar SB12 and Eculizumab Reference Product Using a State-of-the-Art Analytical Method. Biodrugs, 37(4), 569-581.

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Serum-Mediated Peptide Degradation Kinetics

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Diluted human serum (25%; H4522 human serum, Sigma) was incubated at 37 °C for 15 min and added to peptide stock in DMSO to ~100 μM final peptide concentration. The solution was incubated at 37 °C and 50-μL aliquots were withdrawn at various time points. This solution was mixed with 50 μL of 15% trichloroacetic acid (TCA) in MeOH and 50 μL of acetonitrile, and the mixture was stored at 4 °C overnight. Finally, the samples were centrifuged at 15,000 rpm for 10 min in a microcentrifuge equipped with Eppendorf FA-45-24-11 rotor, and the supernatant was analyzed by reversed-phase HPLC equipped with an analytical C18 column (Waters). The amount of peptide remaining at each time point was determined by integrating the area under the peptide peak in the resulting HPLC chromatogram (monitored at 214 nm) and comparing to the peptide amount at time zero.

Buyanova M., Cai S., Cooper J., Rhodes C., Salim H., Sahni A., Upadhyaya P., Yang R., Sarkar A., Li N., Wang Q.E, & Pei D. (2021). Discovery of a Bicyclic Peptidyl Pan-Ras Inhibitor. Journal of medicinal chemistry, 64(17), 13038-13053.

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Serum Amino Acid and Ghrelin Quantification

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The concentration of ghrelin in serum was determined according to the kit instructions (Jiancheng Bioengineering Institution, Nanjing, China). The determination of serum amino acid concentration was completed by Suzhou panomic Biomedical Technology Co., Ltd. (Suzhou, China). Briefly, 200 μL serum were transferred into a 2 ml EP tube, and then fully mixed with 400 μL 10% formic acid methanol-H₂O (1:1. v/v) solution, followed by centrifugation at 12,000 rpm at 4 °C for 5 min. Next, after mixing 10 μL centrifuged supernatant with 490 μL 10% formic acid methanol-H₂O (1:1. v/v) solution, we took 100 μL diluted samples and added 100 μL internal standard (13 C-labelled Phe, 100 ppb) into them. Finally, the mixed solution were filtered with the 0.22μM-pore-size membrane for subsequent detection by the liquid chromatography-tandem mass spectrometric (LC-MS) method. The LC-MS instrument consisted of a Waters ACQUITY UPLC system (Waters, Milford, Massachusetts, USA), equipped with an analytical C18 column (2.1 × 100 mm, 1.7 μm, Waters, Milford, MA, USA) and an AB 4000 triple quadrupole mass spectrometer.

Yan X., Zhang H., Lin A, & Su Y. (2022). Antagonization of Ghrelin Suppresses Muscle Protein Deposition by Altering Gut Microbiota and Serum Amino Acid Composition in a Pig Model. Biology, 11(6), 840.

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Quantifying Amphotericin B in Liposomes

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The AmB content in the liposome formulations was determined by high-performance liquid chromatography (HPLC) [25 (link)]. Liposomes were ruptured by MeOH and CHCl₃ (1:1) and the solution was then diluted 10-fold with MeOH. The liposomes were filtered through polytetrafluoroethylene (PTFE) syringe filters with 0.45-μm pore size Whatman^® syringe filters. The chromatographic analyses were performed on a HPLC Agilent 1200 Infinity Series HPLC system (Agilent Technologies, Waldbronn, Germany), using a C₁₈ analytical column (Waters; 250 × 4.6 mm, 5 µm). The mobile phase consisted of (A) MeOH containing 0.1% v/v trifluoroaceticacid and (B) water containing 0.1% v/v trifluoroaceticacid, with a gradient flow of 60–84% (A) over 9 min. The flow rate was 1 mL/min. (Model 1260 Quat Pump VL), sample injection volumes were 20 µL (Model 1260 ALS), and the detection wavelength was 390 nm.

Lim C.B., Abuzar S.M., Karn P.R., Cho W., Park H.J., Cho C.W, & Hwang S.J. (2019). Preparation, Characterization, and In Vivo Pharmacokinetic Study of the Supercritical Fluid-Processed Liposomal Amphotericin B. Pharmaceutics, 11(11), 589.

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Carotenoid Extraction and HPLC Analysis

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Pigment extraction and HPLC analysis followed the reported method [64 (link)]. Cells were harvested after 12 h cultivation and centrifuged at 10,000 rpm for 5 min. Subsequently, the pellets were resuspended in 1 mL of acetone and incubated at 55 °C for 15 min in the dark to extract carotenoids. The supernatant was then collected at 10,000 rpm for 10 min. The accurate β-carotene yield of each strain was determined and analyzed using the HPLC (Shimadzu A20 system, Shimadzu, Kyoto, Japan) configured with a C18 analytical column (5 μm, 4.6 mm × 250 mm, Waters, Wexford, Ireland). Methanol and isopropanol (8:2, V:V) were chosen as the mobile phase at a flow rate of 1 mL/min at 35 °C. A β-carotene standard sample (Cat. No. C4582, Sigma, St. Louis, MI, USA) was used for carotenoid identification and quantitation with the absorbance signal recorded at 450 nm. Results represented the means ± S.D. of three independent experiments.

Shi S., Chang Y., Yu J., Chen H., Wang Q, & Bi Y. (2023). Identification and Functional Analysis of Two Novel Genes—Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)—from Aurantiochytrium limacinum Significantly Enhance De Novo β-Carotene Biosynthesis in Escherichia coli. Marine Drugs, 21(4), 249.

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Quantification of Tryptophan Metabolites

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Serum KYNA, KYN and TRP levels were determined by the ultra-high-pressure liquid
chromatography (UHPLC) method with fluorescence detection (KYNA) and ultraviolet
(UV) detection (KYN, TRP) using Waters Acquity UHPLC system and Waters C18
analytical column, according to Zhao and colleagues²² in modification. The mobile phase contained 20 mmol/l sodium acetate,
3 mmol/l zinc acetate and 7% acetonitrile, at a flow rate of 0.1 ml/min.
Quantification of TRP and its metabolites was performed by a UV variable
wavelength detector (KYN at 365 nm; TRP at 250 nm) and by a fluorescence
detector (KYNA-344 nm excitation and 398 nm emission). The 3-OH-KYN levels were
determined fluorometrically, using an electrochemical detector (potential of
working electrode: +0.20 V; Coulochem III, ESA) as described before. The HPLC
column (HR-80; 3 µm; C₁₈ reverse-phase column; ESA) was perfused at
0.6 ml/min using a mobile phase consisting of 2% acetonitrile, 0.9%
triethylamine, 0.59% phosphoric acid, 0.27 mM sodium ethylenediaminetetraacetic
acid and 8.9 mM heptane sulfonic acid.

Dudzińska E., Szymona K., Kloc R., Gil-Kulik P., Kocki T., Świstowska M., Bogucki J., Kocki J, & Urbanska E.M. (2019). Increased expression of kynurenine aminotransferases mRNA in lymphocytes of patients with inflammatory bowel disease. Therapeutic Advances in Gastroenterology, 12, 1756284819881304.

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UHPLC for Kynurenine Pathway Metabolites

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Serum KYNA, KYN and TRP levels were determined by the ultra-high pressure liquid chromatography (UHPLC) method (Waters Acquity UHPLC system; Waters C18 analytical column), as described before [40 (link)]. Briefly, the mobile phase containing 20 mM sodium acetate, 3 mM zinc acetate and 7% acetonitrile was run with the flow rate of 0.1 mL/min. Quantification of TRP and its metabolites was performed by a UV variable wavelength detector (KYN at 365 nm; TRP at 250 nm) and by a fluorescence detector (KYNA-344 nm excitation and 398 nm emission). 3-OH-KYN levels were determined fluorometrically with an electrochemical detector (potential of working electrode: + 0.20 V; Coulochem III, ESA), as described before [28 (link)]. The HPLC column (HR-80; 3µm; C18 reverse-phase column; ESA) was perfused at 0.6 mL/min using a mobile phase consisting of 2% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM sodium EDTA and 8.9 mM heptane sulfonic acid.

Dudzińska E., Szymona K., Kloc R., Kocki T., Gil-Kulik P., Bogucki J., Kocki J., Paduch R, & Urbańska E.M. (2021). Fractalkine, sICAM-1 and Kynurenine Pathway in Restrictive Anorexia Nervosa–Exploratory Study. Nutrients, 13(2), 339.

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Quantitative Proteomic Analysis of GBM Cells

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GBM cell lines were grown as tumourspheres for three days. Cells were lysed in buffer containing 2% SDS, 100 mM Tris pH 7.6, protease and phosphatase inhibitors followed by precipitation with acetone. The protein precipitate was resuspended in 8 M urea, followed by reduction with DTT, alkylation with iodoacetamide and digested with trypsin using a standard protocol (https://www.nature.com/articles/ni.3693). Peptides were desalted and separated on a C18 analytical column (Waters) interfaced to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). All raw files were searched against human proteome sequences from UniProt using the based label-free quantification algorithm in MaxQuant (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159666/) (protein and peptide FDR = 0.01). Proteins of interest were extracted manually from the total list.

Stringer B.W., Day B.W., D’Souza R.C., Jamieson P.R., Ensbey K.S., Bruce Z.C., Lim Y.C., Goasdoué K., Offenhäuser C., Akgül S., Allan S., Robertson T., Lucas P., Tollesson G., Campbell S., Winter C., Do H., Dobrovic A., Inglis P.L., Jeffree R.L., Johns T.G, & Boyd A.W. (2019). A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma. Scientific Reports, 9, 4902.

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Quantitative Analysis of Kynurenic Acid

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The content of kynurenic acid in samples from in vitro and enzymatic experiments was performed with the ultra-high-pressure liquid chromatography (UHPLC) method with fluorescence detection using Waters Acquity UHPLC system and Waters C18 analytical column, as previously described [30 (link)]. The mobile phase (20 mmol/L sodium acetate, 3 mmol/L zinc acetate and 7% acetonitrile) was run at a flow rate of 0.1 mL/min. The content of kynurenic acid in studied samples was carried out by a fluorescence detector with excitation at 344 nm and emission at 398 nm. The assay sensitivity reached 150 fmol of kynurenic acid/injection (signal:noise ratio = 5). During each assay, the calibration curve was calculated from external standards (0.2 pmol up to 1 pmol of kynurenic acid). The linearity of the calibration curve was not less than r² > 0.999.

Bednarz K., Kozieł K, & Urbańska E.M. (2024). Novel Activity of Oral Hypoglycemic Agents Linked with Decreased Formation of Tryptophan Metabolite, Kynurenic Acid. Life, 14(1), 127.

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Quantifying SN-38 Encapsulation in Nanoparticles

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A Waters HPLC system equipped with a diode array UV detector was used to analyze the SN-38 that encapsulated in NPM. The mobile phase consisted of a mixture of water and acetonitrile (50:50, v/v%). C18 analytical column (150 mm 4.6 mm, 5 μm, Waters) was employed to separate the samples, and the flow speed was set to 1 mL/min, the whole eluting procedure lasted for 10 min. SN-38 was detected at 360 nm and quantitatively determined by an external calibration curve. The calibration curve was obtained using a series of drug/DMSO standard solutions with different concentrations. The SN-NPM were diluted with DMSO (SN-NPM solution/DMSO=1:9, v/v) to dissociate nanoparticles. The encapsulation efficiency (EE%) and loading content of SN-38 in NPM were calculated by using the following equations: EE%= [(SN-38 added - Free “unencapsulated SN-38”)/SN-38 added] *100, while the drug content (%) = [encapsulated SN-38/weight of SN-NPM] * 100.
The mean diameter and polydispersity of micelles were measured by dynamic light scattering (DLS) instruments (Microtrac), and data was analyzed by Microtrac FLEX Software 10.5.3. The concentration was kept at 1.0 mg/mL for DLS measurements. All measurements were performed at 25 °C. The morphology of the micelles was observed by TEM (Philips CM-120).

Yang X., Xue X., Luo Y., Lin T.Y., Zhang H., Lac D., Xiao K., He Y., Jia B., Lam K.S, & Li Y. (2017). Sub-100 nm, Long Tumor Retention SN-38-Loaded Photonic Micelles for Tri-modal Cancer Therapy. Journal of controlled release : official journal of the Controlled Release Society, 261, 297-306.

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