Hypersil ods column

Manufactured by Agilent Technologies

Sourced in United States

The Hypersil ODS column is a reverse-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of chemical compounds. The column features a silica-based stationary phase with octadecylsilane (ODS) bonded ligands, which provide strong hydrophobic interactions for the retention of non-polar and moderately polar analytes. The Hypersil ODS column is widely used in various applications, including pharmaceutical, environmental, and food analysis.

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21 protocols using hypersil ods column

Separation of Pyridylamino-Labeled Glycans

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Separation of pyridylamino-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL; excitation at 320 nm and emission 400 nm). For standard RP-HPLC, a Hypersil ODS column (Agilent; a classical C18 column of the dimensions 250 × 4.6 mm) was used with 100 mM ammonium acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); a gradient of increasing buffer B (1% per minute) was programmed up to 30 minutes; a step up to 40% B for 5 minutes was followed by another step to 45% for 5 minutes followed by a return to starting conditions. A similar linear gradient (1% B per minute; 0.8 ml/min) for 45 minutes was used with a Kinetex™ 5μ XB-C18 column (250 × 4.6 mm; Phenomenex), which has iso-butyl side chains on fused core particles. For the Ascentis® Express 2.7 μ RP-Amide column (150 × 4.6 mm; Sigma-Aldrich) which has a fused core carrying alkyl amide chains as the bonded phase, a gradient of buffer B up to 35% over 34 minutes was applied at a flow rate of 0.8 ml/min as follows: 0-4 min, 0% B; 4-14 min, 0-5% B; 14-24 min, 5-15% B; 24-34 min, 15-35% B; 34-35 min, return to starting conditions. The recommended guard columns were used in all cases. The overall HPLC and MALDI-TOF MS results from two independently-grown nematode samples were similar, but only the data for one preparation are shown.

Yan S., Wilson I.B, & Paschinger K. (2015). Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus. Electrophoresis, 36(11-12), 1314-1329.

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Glycan Separation and Characterization by HPLC

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Separation of PA-labeled
glycans was carried out on a Shimadzu HPLC system equipped with a
fluorescence detector (RF 10 AXL; 320/400 nm). In the case of RP-HPLC,
a Hypersil ODS column (C18; Agilent) was used with 100 mM ammonium
acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); a gradient
of increasing buffer B (1% per minute) was programmed. The column
was calibrated daily in terms of glucose units (g.u.) with a pyridylaminated
partial dextran hydrolysate. For 2D-HPLC, either normal-phase HPLC
(Tosoh TSKgel Amide-80) with an inverse gradient of acetonitrile in
10 mM ammonium formate, pH 7, or combined hydrophobic-interaction
anionic-exchange HPLC (HIAX, Dionex IonPac AS11) with an inverse gradient
of acetonitrile in 800 mM ammonium acetate, pH 3, was applied as previously
described.^{4 (link),27 (link)}

Yan S., Jin C., Wilson I.B, & Paschinger K. (2015). Comparisons of Caenorhabditis Fucosyltransferase Mutants Reveal a Multiplicity of Isomeric N-Glycan Structures. Journal of Proteome Research, 14(12), 5291-5305.

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Separation and Identification of PA-Labeled Glycans

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Separation of PA-labelled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 20 AXS; excitation at 320 nm and emission at 400 nm). For RP-HPLC, a Hypersil ODS column (Agilent; a C18 column of the dimensions 250 × 4.6 mm) was used with 100 mM ammonium acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); after 5 minutes at 0% buffer B, a gradient of increasing buffer B (0.5% per minute) was programmed for 30 minutes. A pyridylaminated partial dextran hydrolysate was used for calibration in terms of glucose units; also commercial Man_5,9GlcNAc₂ (Takara) and previously-defined Man₈GlcNAc₂A and B isomers were also chromatographed to verify elution times on the shallower gradient employed in this study. Selected RP-HPLC fractions were then subject to hydrophilic-interaction/anion-exchange (HIAX) using an IonPac AS11 column (Dionex) as previously described ^{(15 (link))}. Buffer A was 0.8 M ammonium acetate, pH 3 and buffer B 80 % acetonitrile. The following gradient was applied at a flow rate of 1 ml/min: 0-5 min, 99 % B; 5-50 min, 90 % B; 50-65 min, 80 % B; 65-85 min, 75 % B. The HIAX column was calibrated using a mixture of oligomannosidic glycans (Man_3,6,7,9GlcNAc₂) derived from white beans.

Hykollari A., Eckmair B., Voglmeir J., Vanbeselaere J., Razzazi-Fazeli E., Wilson I.B, & Paschinger K. (2015). More Than Just Oligomannose: An N-glycomic Comparison of Penicillium Species. Molecular & Cellular Proteomics : MCP, 15(1), 73-92.

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Quantitative Analysis of Plumbagin in PI Extract

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The PI extract was analyzed for the plumbagin content using high-performance liquid chromatography (HPLC) which was carried out on a Hypersil ODS column (Agilent Technologies, CA, USA, particle size 5 μm, 250 × 4 mm) coupled with an Agilent 1260 Infinity system and a UV detector (Agilent Technologies), before being eluted by an isocratic linear solvent system of acetonitrile and water (50:50 by volume) with a flow rate of 1 mL/min. The chromatogram was monitored at a wavelength of 410 nm and analyzed by the ChemStation software (Agilent Technologies). Identification and quantification of plumbagin were performed on the basis of retention time and peak area of the authentic standard of plumbagin (LKT Laboratories).

Sukkasem N., Chatuphonprasert W., Tatiya-aphiradee N, & Jarukamjorn K. (2016). Imbalance of the antioxidative system by plumbagin and Plumbago indica L. extract induces hepatotoxicity in mice. Journal of Intercultural Ethnopharmacology, 5(2), 137-145.

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Amino Acid Quantification by RP-HPLC

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Amino acids were determined by using reverse-phase high-performance liquid chromatography (RP-HPLC) via pre-column derivatization with O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-Cl). The 17 amino acid standards for quantification included glutamic acid, proline, aspartic acid, glycine, alanine, arginine, histidine, serine, tyrosine, cysteine, valine, leucine, isoleucine, phenylalanine, threonine, lysine, and methionine.
An Agilent 1100 HPLC (NYSE: A; Agilent Technologies Inc., Palo Alto, CA, USA) and an Agilent Hypersil ODS column (5 μm, 4.0 × 250 mm) were applied for RP-HPLC analysis. A 27.6 mM sodium acetate–triethylamine–tetrahydrofuran (500:0.11:2.5, v/v/v, pH = 7.2) was used as solvent A, and an 80.9 mM sodium acetate–ethanol–acetonitrile (1:2:2, v/v/v, pH = 7.2) was used as solvent B for mobile phases at a flow rate of 1 ml/min. The elution gradient employed was as follows: 0–17 min, 8–50% B; 17–20 min, 50–100% B; 20–24 min, 100–0% B. Amino acids were detected by ultraviolet (UV) detector at 338 nm, whereas proline was detected at 262 nm.

Guan Q., Gong T., Lu Z.M., Geng Y., Duan W., Ren Y.L., Zhang X.J., Chai L.J., Shi J.S, & Xu Z.H. (2021). Hepatoprotective Effect of Cereal Vinegar Sediment in Acute Liver Injury Mice and Its Influence on Gut Microbiota. Frontiers in Nutrition, 8, 798273.

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Phytochemical Profiling of D. alatus Leaves

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The leaf extract of D. alatus was analyzed by a reversed-phase HPLC system using a Hypersil ODS column (Agilent Technologies Inc., Santa Clara, CA, USA, 4 × 250 mm, 5 µm). Methanol (Solvent A) and 0.2% formic acid in aqueous solution (solvent B) were used as a mobile phase, with gradient elution (30–60% solvent A, cycle time 65 min.) with the flow rate of 1.2 mL/min. Standards were detected at 254, 275, and 370 nm, as previously described [42 (link),43 (link)]. The amount of gallic acid, caffeic acid, ferulic acid, luteolin-7-O-glucoside, rutin, and kaempferol-3-glucoside present in the extract was then determined by comparison with standard curves (1–6 µg/mL). The analytical method was validated for specificity by the absence of undesired peaks in HPLC chromatograms and accuracy by the percentage recovery of all standards in 5 replicates. Precision (%RSD) was validated for within-day and between-day determinations (n = 5. Linearity was validated by using linear regression analysis to calculate the coefficient of determination (r2) of standard curve (1–6 µg/mL, n = 5). Limit of detection (LOD) and limit of quantitation (LOQ) were also determined, and signal to noise ratios were calculated (n = 5).

Daodee S., Monthakantirat O., Ruengwinitwong K., Gatenakorn K., Maneenet J., Khamphukdee C., Sekeroglu N., Chulikhit Y, & Kijjoa A. (2019). Effects of the Ethanol Extract of Dipterocarpus alatus Leaf on the Unpredictable Chronic Mild Stress-Induced Depression in ICR Mice and Its Possible Mechanism of Action. Molecules, 24(18), 3396.

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Glycan Separation and Analysis via HPLC

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Separation of PA-labelled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL). First the glycans were fractionated by NP-HPLC using a Tosoh Amide-80 column (4.6 × 250 mm); dried samples were taken up in 50 μl of a 25:75 mixture of buffer A (10 mM ammonium formate, pH 7) and buffer B (95% acetonitrile) prior to injection and the following gradient was applied: 0-5 mins, 75% B; 5-15 mins, 75-65% B; 15-40 mins, 65% B; 40-55 mins, 65-57% B; followed by a return to the starting conditions. Selected fractions were then subject to RP-HPLC on a Hypersil ODS column (5 μm, 4 × 250 mm; Agilent), whereby buffer C was 0.1 M ammonium acetate, pH 4, and buffer D was 30% (v/v) methanol. Gradients of increasing methanol (1% buffer D per minute) were applied. Fluorescence was recorded at 320 nm (excitation) and 400 nm (emission). The columns were calibrated daily in terms of glucose units (g.u.) with a pyridylaminated partial dextran hydrolysate. Various modifications of N-glycans have different effects on retention time as compared to the ‘parent’ structure: α1,3-fucose and α1,6-fucose resulting in either 2 g.u. earlier or 3 g.u. later RP-HPLC elution; bisecting galactose in 3 g.u. earlier elution on RP-HPLC; intersecting galactose in 1 g.u. earlier elution on RP-HPLC; methylation in 0.3 g.u. earlier on NP-HPLC, but later elution on RP-HPLC.

Yan S (.闫.石.)., Wang H (.王.彗.捷.)., Schachter H., Jin C (.金.春.生.)., Wilson I.B, & Paschinger K. (2018). Ablation of N-acetylglucosaminyltransferases in Caenorhabditis induces expression of unusual intersected and bisected N-glycans. Biochimica et biophysica acta. General subjects, 1862(10), 2191-2203.

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Quantifying Free Amino Acids in Simulated Intestinal Fluid

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Here, 1 mL simulated intestinal fluid after 2 h digestion was mixed with an equal volume of 10% TCA and let stand for 1 h to remove large proteins. The mixture was centrifuged at 15000 rpm for 30 min. Then, the composition and content of free amino acids were determined by reversed-phase high performance liquid chromatography (Agilent 1260). Amino acid separation was achieved using Agilent Hypersil ODS column (5 μm，4.6 × 100 mm) and a binary gradient system. Solvent A was sodium acetate, triethylamine, and tetrahydroquantine (500/0.11/2.5, v/v/v), at pH 7.2, and Solvent B contained sodium diacetate and methanol-acetonitrile (1/2/2, v/v/v) at pH 7.2. The detection wavelength of UV detector (VWD) was 338 nm, and amino acid content was determined by the external standard method basing 17 amino acid standards.

Zhang J., Zou Y., Yan B., Zhang N., Zhao J., Zhang H., Chen W, & Fan D. (2023). Microwave treatment on structure and digestibility characteristics of Spirulina platensis protein. Current Research in Food Science, 7, 100581.

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Quantification of Plasma Keto Acids by HPLC

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Concentrations of the plasma α-keto acids α-ketoisocaproate, α-ketoisovalerate and α-keto-β-methylvalerate were determined by HPLC after derivatization with o-phenylendiamine using α-ketocaproic acid as the internal standard [25 (link)]. Forty μL of plasma was mixed with 4 μL of 500 μM internal standard and 80 μL of 1 M HClO₄. After a 10 min incubation at 4°C and centrifugation (10 min, 22,000 g, 4°C), 50 μL of the supernatant was mixed with 50 μL 25 mM o-phenylendiamine in 2 M HCl and incubated for 30 min at 50°C. After cooling to room temperature the samples were centrifuged again (5 min, 22,000 g, 4°C) and the supernatants were analyzed by HPLC with an Agilent 1100 with a Hypersil ODS column (250 mm x 4 mm, 5 μm, Agilent) at 30°C and fluorescence detection (350 nm/410 nm). The α-keto acid derivates were eluted by a gradient of methanol and water with a flow rate of 0.8 mL/min (0 min 32.5%, 5 min 32.5%, 10 min 41.5%, 12 min 55%, 20 min 88.5%, and 32 min 100% methanol). The injection volume was 10 μL.
Because Leu is a ketogenic amino acid, we hypothesized that a high Leu diet would increase plasma concentrations of ketone bodies. The plasma concentration of 3-hydroxybutyrate was analyzed with a test kit (Autokit 3-HB test system; Wako Chemicals GmbH, Neuss, Germany).

Wessels A.G., Kluge H., Hirche F., Kiowski A., Schutkowski A., Corrent E., Bartelt J., König B, & Stangl G.I. (2016). High Leucine Diets Stimulate Cerebral Branched-Chain Amino Acid Degradation and Modify Serotonin and Ketone Body Concentrations in a Pig Model. PLoS ONE, 11(3), e0150376.

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Quantitative HPLC Analysis of Phytochemical Markers

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A reversed-phase HPLC system with a UV detector (Agilent Technologies Inc., Santa Clara, CA, USA) and a Hypersil ODS column (4 × 250 mm, 5 μm) was used for analyzing the YPJ extract and the gallic acid, myricetin, quercetin, luteolin, genistein, and coumestrol standards. Ultrapure water (solvent A) and 80% methanol containing 0.25% acetic acid (solvent B) was used as the mobile phase by gradient elution for 50 min at a flow rate of 2.5 mL/min and with detection at 254, 280, 340, and 370 nm.
Calibration curves were prepared from 1, 2, 4, 6.8, and 10 μg/mL dilutions of 1 mg/mL stock solutions of the gallic acid, myricetin, quercetin, luteolin, genistein, and coumestrol standards diluted with methanol. The analytical HPLC methods were validated following ICH guidelines. The linearity was validated by using linear regression analysis to calculate the coefficient of determination (R²) of the standard calibration curve (1–10 μg/mL, n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) were also determined, and the signal level of the substance reached at least 3 and 10 times the signal-to-noise ratios (n = 10). The accuracy was measured by the percentage recovery of three standard concentrations in 5 replicates. Precision (%RSD) was validated for within-day and between-day determinations (n = 5).

Daodee S., Monthakantirat O., Tantipongpiradet A., Maneenet J., Chotritthirong Y., Boonyarat C., Khamphukdee C., Kwankhao P., Pitiporn S., Awale S., Matsumoto K, & Chulikhit Y. (2022). Effect of Yakae-Prajamduen-Jamod Traditional Thai Remedy on Cognitive Impairment in an Ovariectomized Mouse Model and Its Mechanism of Action. Molecules, 27(13), 4310.

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