Zorbax sb c18 column

Manufactured by Agilent Technologies

Sourced in United States, Germany, Japan, China, France

The Zorbax SB-C18 column is a 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 n-octadecylsilane (C18) ligands, which provides reversible phase chromatography for the separation of both polar and non-polar analytes.

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558 protocols using zorbax sb c18 column

Intracellular Ascorbate and Glutathione Analysis

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The intracellular D-erythroascorbic acid content was measured by employing known techniques [28 (link), 29 (link)]. Samples were applied to an analytical HPLC system (Waters) that was equipped with a Waters 460 electrochemical detector. The resulting extracts were separated with ZORBAX SB-C18 columns (Agilent, 250 mm × 4.6 mm) and eluted with 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min.
Cellular GSH was labeled with monobromobimane (mBBr) and measured as described previously [30 (link)]. The mBBr-derivatized thiol compounds were analyzed by HPLC with a Hewlett-Packard 1050 series fluorescence detector with ZORBAX SB-C18 columns (Agilent, 250 mm × 4.6 mm). Samples were eluted with 0.1% trifluoroacetic acid at a flow rate of 0.7 ml/min. The mobile phase consisted of 15% methanol and 85% trifluoroacetic acid (0.1%) at a wavelength at 370 nm.

Kang S.O, & Kwak M.K. (2020). Methylglyoxal-Scavenging Enzyme Activities Trigger Erythroascorbate Peroxidase and Cytochrome c Peroxidase in Glutathione-Depleted Candida albicans. Journal of Microbiology and Biotechnology, 31(1), 79-91.

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Analytical Techniques for Compound Characterization

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Optical rotations were measured in MeOH with Horiba SEPA-300 (Horiba, Kyoto, Japan) and JASCO P-1020 polarimeters (Jasco, Tokyo, Japan). 1D and 2D NMR spectra were taken on Avance III HD 600, and Avance III HD 800 (Bruker, Karlsruhe, Germany) instruments, using TMS as the internal standard. Mass spectrometry was performed on an API QSTAR TOF spectrometer (Waters, Manchester, America) equipped with an ESI source in the positive-ion mode. Column chromatography was performed with silica gel (100–200 or 200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and reverse-phase C₁₈ silica gel (40–63 μm, Merck, Darmstadt, Germany). Precoated TLC sheets of silica gel 60 GF₂₅₄ (Qingdao Haiyang Chemical Plant, Qingdao, China) were used, and compounds were visualized either by UV light (254 nm) or by spraying heated silica gel plates with 10% H₂SO₄ in EtOH. A Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column (Shimadzu, Kyoto, Japan) was used for preparative HPLC. An Agilent 1100 liquid chromatograph (Agilent, Walter Bloem, America) equipped with a Zorbax SB-C₁₈ column (4.6 mm × 250 mm, 5 μm) was used for HPLC analysis, and a semi-preparative Zorbax SB-C₁₈ column (9.4 mm × 250 mm, 5 μm, Agilent, Walter Bloem, America) was used for sample preparation.

Li Y., Lou S., Yang R., Zhang L., Zou Q., Shang S., Gao L, & Wang W. (2023). Cytotoxic sesquiterpene aryl esters from Armillaria gallica 012m. Chinese Herbal Medicines, 15(2), 343-346.

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UPLC-MS/MS Analysis of Reaction Products

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The analyses of reaction products and determination of conversion rate were performed on an Agilent 1260 Infinity UPLC system (Agilent Technologies, Germany)^{17 (link)}. The analyses were performed on an Agilent ZORBAX SB-C18 column (3.0 × 100 mm, 1.8 μm). The flow rate was 0.3 ml/min, the column temperature was set at 40 °C, and the injection volume was 10 μl. The chromatogram was monitored at 280 nm. The mobile phases comprised solvents A (ultrapure water) and B (methanol). The elution program was as follows: 0–30 min, 5–100% B; 30–40 min, 100% B^{18 (link)}.
UPLC-MS/MS was analysed on a maXis LC-ESI-QTOF-MS system (Bruker, Germany) equipped with an Agilent ZORBAX SB-C18 column (3.0 × 100 mm, 1.8 μm). The elution program was the same as UPLC analysis. Ionization of the analytes was achieved by using electron spray ionization interface in negative mode. The collision voltage was 10 eV. Mass scan was set in the range of m/z 50–1000. The daughter ions were monitored at a collision voltage range of 28–42 eV^{19 (link)}.

Tao Z., Liu J., Jiang Y., Gong L, & Yang B. (2017). Synthesis of prenylated flavonols and their potents as estrogen receptor modulator. Scientific Reports, 7, 12445.

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Preparative and Analytical RP-HPLC Purification of Peptides

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Crude peptides were purified by reverse phase-HPLC. The peptides were dissolved in 99:1 or 95:5 A:B (A: water + 0.1% TFA; B: acetonitrile + 0.1% TFA) and 6 M guanidine hydrochloride was added depending on the solubility of the peptide. For preparative RP-HPLC purification, we used an Agilent Zorbax SB C₁₈ column (21.2 × 250 mm, 7 μm) at a flow rate of 10 mL min⁻¹ at 1–41% or 5–45% B over 80 min. For semi-preparative RP-HPLC purification, we used an Agilent Zorbax SB C₁₈ column (9.4 × 250 mm, 5 μm) at a flow rate of 5 mL/min over the same gradient. UV absorbance was monitored at 214 nm. Purity of the fractions was analyzed by MALDI or LC-MS. HPLC fractions from preparative or semi-preparative HPLC were spotted with MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) in 50% A: 50% B and checked for the correct molecular masses. The analytical RP-HPLC Agilent C₁₈ Zorbax SB column (2.1 × 150 mm, 5 μm) was used to confirm the purity of fractions at a flow rate of 0.5 mL/min over a linear gradient of 1–51% B over 12 min. Analytical HPLC UV absorbance traces were measured at 214 nm.

Rabideau A.E., Liao X., Akçay G, & Pentelute B.L. (2015). Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen. Scientific Reports, 5, 11944.

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HPLC Analysis of Compound Fractions from Black Garlic

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The HPLC analysis was performed with an Agilent model 1100. Five fractions were separated by reverse-phase HPLC on an Agilent Zorbax SB-C18 column (4.6 mm × 250 mm) equilibrated in pure water solvent A and methanol solvent B. Elution was performed with a linear gradient by increasing the concentration of solvent B, as followed for the ethyl acetate extracts from black garlic: 0–30 minutes, 5–100%; 30–45 minutes, 100%; 45–55 minutes, 100–5%. F3 was detected by HPLC with the solution of methanol:water (1:9, v/v) for 45 minutes; F4 was detected by HPLC with the solution of methanol:water (2:8, v/v) for 55 minutes. The temperature of the column was maintained at 25°C. The flow rate was 0.8 mL/min, and the wavelength was set at 254 nm for UV detection [24 ].
The semiprep-HPLC was performed with analysis/circular semipreparative system of SHIMADZU LC-6AD system on an Agilent Zorbax SB-C18 column (9.4 mm × 250 mm); F3 was detected by semiprep-HPLC with the solution of methanol:-water (1:9, v/v) for 45 minutes. F4 was detected by semiprep-HPLC with the solution of methanol:water (2:8, v/v) for 55 minutes at a flow rate of 2 mL/min, and the wavelength was set at 254 nm for UV detection. The compounds we purified from F3 and F4 were detected by HPLC with the solution of methanol:water (2:8, v/v) for 55 minutes at a flow rate of 0.8 mL/min.

Lu X., Li N., Qiao X., Qiu Z, & Liu P. (2016). Composition analysis and antioxidant properties of black garlic extract. Journal of Food and Drug Analysis, 25(2), 340-349.

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Phytochemical Analysis of Compounds

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A Jasco digital polarimeter (DIP-370, purchased from JASCO Corporation, Tokyo, Japan) was employed to examine optical rotations. NMR spectra were monitored by a Bruker AV 600 MHz spectrometer using an internal standard (tetramethylsilane) (Bruker BioSpin Group, Germany). An API-QSTAR Pulsar (Applied Biosystem Corporation, Canada) was hired to achieve HR-ESI–MS and ESI–MS. A Shimadzu UV-2401 spectrometer (Beckman, Brea, USA) was implemented to obtain the UV spectra. Column chromatography with various gels was conducted, including 75 μM of ODS-C₁₈ (YMC Co., Ltd., Japan), 75–150 μM of MCI gel (GHP20P, Mitsubishi Chemical Corporation, Tokyo, Japan), 43–63 mm of LiChroprep RP-18 (Merck), 80–100 & 200–300 mesh of silica gels (Qingdao Marine Chemical Co., Ltd., China), and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). An Agilent 1260 liquid chromatography system (Agilent, USA) was implemented for semipreparative and analytical HPLC analysis on a semipreparative Zorbax SB-C₁₈ column (5 μm, 250 × 9.4 mm, 3 ml/min) and an analytical Zorbax SB-C₁₈ column (5 μm, 250 × 4.6 mm, 1 ml/min), respectively. TLC was run for monitoring collected fractions on silica gel GF₂₅₄ plates (Qingdao Marine Chemical Co., Ltd., China). The visualization of spots on the plates were conducted by using an ultraviolet lamp at the wavelength of 254 nm or by heating with H₂SO₄-EtOH (5%).

Feng S.Y., Jiang N., Yang J.Y., Yang L.Y., Du J.C., Chen X.Q., Liu D., Li R.T, & Zhong J.D. (2024). Antiviral and anti-inflammatory activities of chemical constituents from twigs of Mosla chinensis Maxim. Natural Products and Bioprospecting, 14(1), 26.

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HPLC-MS Analysis of MCLR

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The method used in the analysis was previously described by Weihua Song [16 (link)]. MCLR was analyzed using an HPLC (Agilent 1200) with photodiode array detection under the following conditions: the column used was ZORBAX SB-C18 column (5 μm, 4.6 mm×250 mm, Agilent, USA) and the mobile phase consisted of 40% ACN and 60% water, both containing 0.05% trifluoroacetic acid (TFA). The liquid chromatography-mass spectrometry (LC-MS) system used in this study consisted of a Dionex Ultimate 3000 HPLC Pump, a Dionex Ultimate 3000 auto-sampler, and a Bruker micrOTOF II Mass Spectrometer with an electrospray ionization source. The HPLC column used was ZORBAX SB-C18 column (5 μm, 4.6 mm×250 mm, Agilent, USA). Moreover, the injection volume of the treated sample was 80 μL, and the mobile phase used was water and ACN, both containing 0.05% TFA. Gradient elution was performed based on the method described by Liu et al. [18 (link)]. Mass spectral data were also obtained in the positive ion mode by full scanning from m/z 400 to 1200.

Liu Y., Ren J., Wang X, & Fan Z. (2016). Mechanism and Reaction Pathways for Microcystin-LR Degradation through UV/H2O2 Treatment. PLoS ONE, 11(6), e0156236.

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Organic Acids Detection in Bacterial Cultures

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Organic acids (OA) produced by bacterial strains were detected as described previously with some modifications [36] . HX2 and derivative strains were cultivated in NBRIP medium at 28°C for 7 days. The culture was centrifuged at 12,000 g for 5 min and filtrated through a 0.22 µm filter (Pall Corporation, USA). Twenty microliters of filtrates were injected to high-performance liquid chromatography (HPLC) (Waters 2998, USA) equipped with ZORBAX SB-C₁₈ columns (4.6×250 mm, 5 µm; Agilent Technologies, USA). The chromatogram class (NH₄)₂HPO₄ (0.5%, w/v) with a pH of 2.81 was used as mobile phase at flow rate of 0.4 ml min⁻¹. UV absorption was routinely monitored at a wavelength of 214 nm. Five types of OA served as standards for all samples; gluconic acid (GA), lactic acid (LA), citric acid (CA), succinic acid (SA) and propionic acid (PA).

Li L., Jiao Z., Hale L., Wu W, & Guo Y. (2014). Disruption of Gene pqqA or pqqB Reduces Plant Growth Promotion Activity and Biocontrol of Crown Gall Disease by Rahnella aquatilis HX2. PLoS ONE, 9(12), e115010.

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Fluorescent Peptide Conjugation and Characterization

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Lys-WT and SNARF-1 were dissolved in DMF (dimethylformamide, Sigma), and incubated at a ratio of 2:1 in 60% DMF (dimethylformamide), 30% 0.1 M PBS pH 9.0 and 10% pH 9.5 0.1 M sodium bicarbonate buffer for a final pH of 9.0. SNARF-1 was converted to its fluorescent form after conjugation by raising the conjugation solution’s volume by 50% with methanol and raising the solution pH to 14 with 2 M potassium hydroxide for 1 hour at room temperature. Then, pH was adjusted to pH 7.0 by adding 30% HCl. The reaction progress was monitored by reverse phase (Zorbax SB-C18 columns, 9.4 × 250 mm 5 μm, Agilent Technology) high-performance liquid chromatography (HPLC) using a gradient of 25−75% acetonitrile and water containing 0.05% of trifluoroacetic acid. The concentration of each labeled peptide in buffer was determined by SNARF-1 absorption at 548 nm, ε₅₄₈=27,000 M⁻¹ cm⁻¹. The purity and characterization of the construct were performed by analytical HPLC and surface-enhanced laser desorption/ionization–TOF mass spectrometry.

Wei D., Engelman D.M., Reshetnyak Y.K, & Andreev O.A. (2019). Mapping pH at Cancer Cell Surfaces. Molecular imaging and biology, 21(6), 1020-1025.

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Quantification of Gibberellic Acid 4 in Fruits

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GA₄ extracted from mature fruit skin and “Red Zaosu” and “Zaosu” explants was quantified as previously described^{29 (link),30 (link)}. Three biological replicates were assessed. Samples of 200 mg were ground in 1.5 mL of extraction buffer (20% methanol, 79% isopropanol, and 1% acetic acid) at 4 °C. The supernatant was filtered through a 0.22-μm syringe filter prior to HPLC analysis. LC-MS^{2 (link)} analysis was performed using an HPLC system (Agilent 1290) coupled to a SCIEX 6500 Qtrap (AB Sciex). Samples were injected onto ZORBAX SB-C18 columns (2.1 × 150 mm, 3.5 μm; Agilent). GA₄ was screened and quantified using the multiple reaction monitoring model with a transition of 331.0 > 213.0.

Zhai R., Wang Z., Yang C., Lin-Wang K., Espley R., Liu J., Li X., Wu Z., Li P., Guan Q., Ma F, & Xu L. (2019). PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit. Horticulture Research, 6, 137.

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