Extracts were analyzed using an Agilent Technologies 1100 liquid chromatograph with a visible diode – array detector (DAD) equipped with Zorbax Eclipse XDB C8 column (150 X 4.6 mm I.D., dp = 5 μm) with gradient elution: A- water + 1% acetic acid; B – acetonitryle (0 min- 10% B; 0–10 min - 10-14% B; 10–25 min - 14-30% B; 25–35 min. – 30-35% B; 35–50 min. - 35-60% B; 50–57 min. 60% B). Injection volume for extracts and standards was 10 μL. The flow rate was 1 ml/min and column thermostat temp. 25 °C. The identification of the compounds was performed by comparing retention times and UV-DAD spectra (λ = 254, 280 and 325 nm) with those for standard solutions. Quantitative determination was performed on the basis of 3-fold determinations of the tested compounds. Determination of the content of the tested compounds in individual extracts was determined with the use of calibration curves and calculated mathematically. Calibration plot was prepared of five different concentrations (1, 0.75, 0.5, 0.25, 0.1 mg per 10 mL for all investigated coumarins, phenolic acids and flavonoids. In the HPLC-DAD analysis of compounds of examined extract calibration plots were highly linear – R2 > 0,9932 for phenolic acids R2 > 0,9966 for flavonoids and R2 > 0,9867 for coumarins.
Eclipse xdb c8 column
Manufactured by Agilent Technologies
Sourced in United States
The Eclipse XDB-C8 column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C8 stationary phase and provides reliable and reproducible chromatographic performance.
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Lab products found in correlation
Agilent 1100 series, by Agilent Technologies (3 mentions)
1100 series, by Agilent Technologies (2 mentions)
Luna c18 2 column, by Agilent Technologies (2 mentions)
1100 series hplc system, by Agilent Technologies (2 mentions)
Hplc 2d chemstation software, by Agilent Technologies (2 mentions)
Diode array detector, by Agilent Technologies (2 mentions)
Uflcxr triple tof 5600 lc ms system, by Agilent Technologies (2 mentions)
6460 triple quadruple mass spectrometer, by Agilent Technologies (1 mentions)
Vanquish ultra high performance liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Tlc plate, by Merck Group (1 mentions)
Avance 400 spectrometer, by Bruker (1 mentions)
1100 lc msd, by Agilent Technologies (1 mentions)
Lambda 900, by PerkinElmer (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Lc ms grade acetonitrile, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
1200 series, by Agilent Technologies (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
Squalene, by Merck Group (1 mentions)
37 protocols using eclipse xdb c8 column
1
HPLC-DAD Analysis of Phenolic Compounds
2
Quantification of Sphingolipids in Planthopper
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Total sphingolipids was extracted as described (Bielawski et al. 2006 (link)), and 0.3 g of third and fourth instar (mixed 1:1) nymphs were prepared for sphingolipids extraction. Total sphingolipids of two L. striatellus colonies were extracted in 50 ml iso-propanol: water: ethyl acetate (30:10:60) for 10 min. The organic phase containing total sphingolipids was dried under nitrogen and dissolved in 500 μl MeOH with 25 mmol/l ammonium formate. HPLC-MS/MS analysis was performed using agilent 6460 triple quadruple mass spectrometer coupled with agilent 1200 infinity LC modules. ZORBAX Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm particle size, water) was chosen for the separation of sphingolipid and the injection volume was 10 μl. The mobile phases were as follows: phase A (25 mmol/l ammonium formate) and phase B (acetonitrile). The follow rate was 0.3 ml/min at 30 °C with the following gradient elution conditions of phase B: 20–95%, 0–15 min; 95–100%, 15–30 min; 100%, 30–40 min. The ESI was carried out with gas temperature of 350 °C and the drying gas flow rate was 10 l/min, leading to nebulization pressure of 50 psi. The capillary voltage and fragmentor voltage was 3500 V and 100 V, respectively, and the collision energy was 20 V. Sphingolipid Mix I (Avanti Polar Lipids, Alabama, USA) was used as a standard.
3
Chromatographic Separation and Mass Spectrometry Analysis
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Chromatographic separation and MS detection were performed on samples using methods described in Reddy et al. [13 (link)]. Briefly, a Vanquish Ultra-High-Performance Liquid Chromatography system (Thermo Fisher Scientific, Bremen) was coupled to a QExactive Plus mass spectrometer (LC–MS) (Thermo Fisher, Waltham, MA, USA; Thermo, Bremen, Germany). The MS data were acquired with polarity switching between the positive and negative mode over a mass range of 70–1200 amu with resolution set at 35,000. Separation was achieved using an Agilent 150 × 2.1 mm, 3.5 µm Zorbax Eclipse XDB-C8 column with a linear gradient of 2% to 100% B over 11 min with mobile phase, A (0.1% formic acid in H2O) and B (0.1% formic acid in 55% isopropanol (IPA) in acetonitrile) at a flow rate of 0.5 mL/min. Injection volume was 3 µL.
4
Synthesis and Characterization of Novel Compounds
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Synthesis. All chemicals used were of reagent grade. Yields refer to purified products and are not optimized. Merck silica gel 60 (230−400 mesh) was used for column chromatography. Merck TLC plates and silica gel 60 F254 were used for TLC. NMR spectra were obtained with a Bruker 400 AVANCE spectrometer in the indicated solvents. Melting points were determined in open capillaries in a Gallenkamp apparatus and were uncorrected. The chemical shifts are referenced to the residual not deuterated solvent signal (CHD2OD: δ (1H) = 3.31 ppm, δ (13C) = 49.86 ppm). The values of the chemical shifts are expressed in ppm, and the coupling constants (J) in Hz. An Agilent 1100 LC/MSD operating with an electrospray source was used in mass spectrometry experiments. The purity of compounds 1b –h was assessed by RP-HPLC (Agilent 1100 series) and was found to be higher than 95% [40 (link)]. A Zorbax Eclipse XDB-C8 column (4.6 × 150 mm, 5 μm) was used in the HPLC analysis with methanol-H2O (0.1% formic acid) (80:20) as the mobile phase at a flow rate of 0.5 mL/min. UV detection was achieved at 280 nm. The absorption spectra were recorded with a PerkinElmer Lambda 900 in the indicated solvent. UV-B irradiations were conducted using a Multyrays chamber equipped with 2 GT15T8 Hg UV-B tubes (2 × 15 Watt) in continuous rotation.
5
Measuring Stapled Peptide Solubility
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Solubility was measured
by using an HPLC-UV method. Stapled peptides (∼2 mg) were added
to 1.5 mL Eppendorf tubes and either pH 7.4 phosphate buffer (100
μL) or double-distilled H2O (ddH2O) (100
μL) was added for dissolution with shaking for 24 h at 25 °C,
followed by centrifugation of the mixture at 10000 rpm for 15 min.
The saturated supernatant solution was filtered through a 0.45 μm
filter membrane and then transferred to other vials for analysis by
HPLC with UV detection. Each sample was assayed in triplicate. For
quantification, analytical RP-HPLC was used with a Zorbax Eclipse
XDB-C8 column (4.6 mm × 150 mm, 5 μm). Solvent A, 0.1%
TFA in H2O; Solvent B, 0.1% TFA in 70% CH3CN/H2O; flow rate, 1 mL/min; gradient, 5–100% solvent B
in solvent A over 25 min. The aqueous concentration was determined
by comparison of the peak area of the saturated solution with a standard
curve plotted for the peak area versus known concentrations, which
was prepared by solutions of test compound in PBS or ddH2O at 20, 10, 5, 2.5, 0.5, and 0.05 mg/mL.
by using an HPLC-UV method. Stapled peptides (∼2 mg) were added
to 1.5 mL Eppendorf tubes and either pH 7.4 phosphate buffer (100
μL) or double-distilled H2O (ddH2O) (100
μL) was added for dissolution with shaking for 24 h at 25 °C,
followed by centrifugation of the mixture at 10000 rpm for 15 min.
The saturated supernatant solution was filtered through a 0.45 μm
filter membrane and then transferred to other vials for analysis by
HPLC with UV detection. Each sample was assayed in triplicate. For
quantification, analytical RP-HPLC was used with a Zorbax Eclipse
XDB-C8 column (4.6 mm × 150 mm, 5 μm). Solvent A, 0.1%
TFA in H2O; Solvent B, 0.1% TFA in 70% CH3CN/H2O; flow rate, 1 mL/min; gradient, 5–100% solvent B
in solvent A over 25 min. The aqueous concentration was determined
by comparison of the peak area of the saturated solution with a standard
curve plotted for the peak area versus known concentrations, which
was prepared by solutions of test compound in PBS or ddH2O at 20, 10, 5, 2.5, 0.5, and 0.05 mg/mL.
6
UPLC-MS/MS Analysis of Metabolites
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Liquid chromatography was performed with ExionL CAD UPLC equipped with a TripleTOF 5600 MS system. The samples were separated by a ZORBAX Eclipse XDB-C8 column (4.6 × 150 mm, 5 μm) at 30 °C, the injection volume was 15.0 μL. Mobile phase A: 0.1% formic acid; mobile phase B: 95% acetonitrile. Samples elution was performed at a flow rate of 0.2 mL/min. Gradient elution procedure is as follows: 0 - 2.5 min, 2% B; 2.5 - 4.0 min, 2% - 50% B; 4.0 - 6.0 min, 50% B; 6.0 - 6.1 min, 50% - 5% B; 6.1 min - 9.9 min, 5% B; 9.9 min - 10.0 min, 2% B. Samples were detected by electrospray ion source in positive ion mode. Samples were scanned with following parameters: DP = 60 V, CE = 10 eV, 50 - 250 Da, curtain gas (CUR) = 30 psi, atomizing gas (GS1) = 50 psi, heating gas (GS2) = 50 psi, ion spray voltage (ISVF) = 5500 V, ion source temperature = 500 °C.
7
Quantitative Fungal Metabolite Extraction
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U. virens strains were cultured on PSA plates with cellophane at 28°C for 28 days. The mycelia (1 g) were collected and immersed in 50 mL ethyl acetate for 12 h. The extracts were condensed to dry, and were then dissolved in acetonitrile (ACN, 1 mL) followed by filtration with a 0.22‐μm filter (Lu et al., 2015 (link)). The extracts were quantified by high‐performance liquid chromatography (HPLC) on a ZORBAX Eclipse XDB‐C8 column (4.6 × 150 mm, 5 μm) with UV detector (wavelength 290 nm) using an HPLC system (LC‐20A; SHIMADZU). The extracts were eluted with ACN using the following gradient: 0–2 min, 55% ACN; 2–9 min, 55%–65% ACN; 9–10 min, 65% ACN; 10–15 min, 65%–70% ACN; 15–18 min, 70% ACN; 18–20 min, 70%–100% ACN; 20–28 min, 100% ACN; 28–30 min, 100%–70% ACN; 30–35 min, 70% ACN; 35–36 min, 70%–55% ACN; 35–36 min, 55% ACN at a flow rate of 1 mL/min. These assays were repeated at least three times.
8
HPLC-DAD-MS Analysis of Compounds
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HPLC-DAD-MS analyses were performed on a HPLC Dyonex Ultimate 3000 equipped with a diode array UV detector and mass spectrometer TSQ Quantum Access Max with an electronspray ionization source. Samples 0.5 mL in size were used as sources for the automated injection. LC-MS grade acetonitrile was purchased from Sigma-Aldrich in the highest available purity and was used without any further purification. Ultrapure water (resistivity 18.2 MΩ/cm at 25 °C) was produced in our laboratory by means of a Millipore Milli-Q system. The chromatographic separation was performed on a reverse phase Zorbax Eclipse XDB-C8 column 4.6 × 150 mm, 5 µm, at flow rate of 0.5 mL/min; linear gradient HCOOH 1% aqueous solution/ACN from 95:5 to 20:80. Mass data acquisition was performed in negative ionization (ESI-) and full-scan mode in the range of m/z 240–1200.
9
HPLC-based Indole Quantification
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To determine the indole content in the plant samples, the same eluates as for ITC analysis were used. The HPLC-DAD-FLD system (Agilent Technologies 1200 Series) equipped with a Zorbax Eclipse XDB-C8 column was employed for the analysis of indolic compounds as described previously [54] . A linear gradient of acetonitrile in water changing from 10 to 100% within 30 min, the mobile phase flow 1 mL/min and the injection volume of 20 µL were applied. Monitoring of florescence of indolic analytes was carried out at 280/360 nm (Ex/Em).
10
Quantification of Isothiocyanates in Plants
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The content of ITC was determined by a modified method developed by Zhang et al. [53] with modifications [54] . Lyophilized plant material (0.2 g) was mixed with 5 mL of 0.01M sodium phosphate buffer (pH 7.4) and incubated for 3 h at 37 °C to achieve complete myrosinase catalyzed conversion of parent aliphatic and aromatic GLs into degradation products. The samples were concentrated on Bakerbond SPE C18 500 mg cartridges (J. T. Baker, Center Valley, PA). Concentrated ITC extracts (0.1 mL of the eluate) were submitted to cyclocondensation reaction in a mixture containing 0.5 mL of 2-propanol, 0.5 mL of 0.1M potassium phosphate buffer (pH 8.5), and 0.1 mL of 60 mM 1,2-benzenedithiol dissolved in 2-propanol. After 1 h at 65 °C, the product of reaction, 1,3-benzenedithiol-2-thione, was analyzed by reverse phase HPLC using an Agilent 1200 system coupled with a DAD detector. The reaction mixture (30 µL) was injected onto a 150 mm, 4.6 mm, 3.5 mm Zorbax Eclipse XDBC8 column. The mobile phase used was 4.8% [v/v] formic acid in water (A) and methanol (B) with a flow rate of 1 mL/min. The gradient changed as follows: 60% B to 100% B within 12 min, then 100% B for 3 min. Chromatograms were traced at 365 nm.
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