The method described by Cristea et al. [21 (link)] was used to determine the content of individual polyphenols. Agilent 1100 Series HPLC was employed and the gradient was optimized using trifluoroacetic acid (TFA) as an eluent with acidification of 1% CH3OH (A channel) and 50% CH3OH (B channel) acidified to 2.15 pH with TFA. The column system was composed of a pre-column SecurityGuard ULTRA Cartridges HPLC C18 for 4.6 mm ID coupled to Kinetex 5 μm C18 100 Å 250 × 4.6 mm columns manufactured by Phenomenex at 35 °C. The injection volume was 20 μL and the run time 90 min. The phases were A—H2O:CH3OH (99:1) and B—H2O:CH3OH (50:50), with a flow of 1.5 mL/min and detection was carried out at 256, 280, 324, and 365 nm. The gradient of elution was as follows: 100% (A) for 10 min; 82% (A) and 18% (B) for the next 10 min; 70% (A) and 30% (B) for 10 min; 65% (A) and 35% (B) for 6 min; 40% (A) and 60% (B) for 15 min; 20% (A) and 80% (B) for 5 min; 100% (B) for 15 min and 100% (A) for 10 min. The content of specific polyphenols was determined by comparison of retention times and peaks with the ones from the chromatogram of a synthetic mix containing the standards listed in Table 5 .
Kinetex 5 μm c18 100
Manufactured by Phenomenex
Sourced in United States
The Kinetex 5 μm C18 100 Å 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 a pore size of 100 Angstroms and particle size of 5 micrometers, providing efficient chromatographic separation.
Automatically generated - may contain errors
8 protocols using kinetex 5 μm c18 100
1
HPLC-Based Polyphenol Profiling Protocol
2
Characterization of Organic Compounds by NMR and MS
3
Quantification of Furfural in Xylan Samples
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The quantification of furfural in each sample was carried out by HPLC-DAD (Shimadzu, Kyoto, Japan, model PROMINENCE). HPLC analyses were performed with an analytical C18 reverse-phase column (250 mm length × 4.60 mm internal diameter), Kinetex 5 μm C18 100 Å, from Phenomenex, Torrance, CA, USA. The mobile phase consisted of 20% of methanol and 80% of ultra-pure water. The separation was performed in isocratic mode, at a flow rate of 0.8 mL.min−1 and an injection volume of 10 μL. Furfural detection was carried out at 268 nm with a diode array detector (DAD). Each sample was analyzed at least in duplicate. The column oven and the autosampler were operated at a controlled temperature of 35 °C. Calibration curves were established with pure furfural dissolved in water. Furfural yield is expressed in moles of furfural in relation to the number of moles of xylose present in the xylan sample used.
4
Curcumin Solubility in Water and DES
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The determination of the solubility of curcumin in water and in the aqueous solutions of Bet:Lev followed a previously reported procedure [33 (link)]. Briefly, the solubility was determined by saturation of 2.0 g of pure water or of each DES aqueous solution (0–90% (w/w) of DES) with curcumin at both room (25 °C) and human body (37 °C) temperatures. A measured aliquot of each saturated solution was diluted to a well-defined final total v/v, carefully filtered with a 0.45 μm syringe filter to remove any solid and subsequently quantified by high-performance liquid chromatography with diode-array detection (HPLC-DAD) (Shimadzu, model PROMINENCE, Kyoto, Japan) to determine the curcumin solubility. HPLC analyses were performed with an analytical C18 reversed-phase column (250 × 4.60 mm2), Kinetex 5 μm C18 100 Å, from Phenomenex conducted in isocratic mode under a flow rate of 1 mL min−1 and operated at 35 °C. The mobile phase contained 40% (v/v) of methanol, 15% (v/v) of acetonitrile and 45% (v/v) of ultra-pure water with 0.3% (v/v) of ortho-phosphoric acid. Samples were analyzed at 377 nm in duplicates and using an injection volume of 10 μL.
5
HPLC-DAD Quantification of 5-HMF
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5-HMF quantification was carried out by HPLC-DAD (Shimadzu, model PROMINENCE). HPLC analyses were performed with an analytical C18 reverse-phase column (250 mm length × 4.60 mm diameter), Kinetex 5 μm C18 100 Å, from Phenomenex. The column oven and the autosampler operated at a controlled temperature of 35 °C and the mobile phase consisted of 20% of methanol and 80% of ultra-pure water. The separation was performed in isocratic mode, at a flow rate of 0.6 mL.min−1 and an injection volume of 10 μL. 5-HMF detection was carried out at 272 nm with a diode array detector (DAD). Calibration curves were established with pure 5-HMF aqueous standards. Each sample was analyzed at least in duplicate.
6
Quantification of Rosmarinic and Carnosic Acids
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The quantification of rosmarinic acid and carnosic acid was made using a HPLC-DAD (Shimadzu, model PROMINENCE). The analyses were performed with an analytical C18 reversed-phase column (250 × 4.60 mm), Kinetex 5 μm C18 100 Å, from Phenomenex.
The separation was conducted in a gradient system of 0.1% of acetic acid-methanol (phase A) and 0.1% of acetic acid-ultra-pure water, at least in duplicate, according to Wojeicchowski and co-workers [30] (link). For this, the following conditions were applied: 0 min of phase A; 7 min of phase A; 11 min 80% of phase A; 23 min 80 % of phase A; 24 min 90 % of phase A; 28 min 40 % of phase A; 40 min 40 % of phase. Rosmarinic acid and carnosic acid displayed a retention time of 14 and 32.2 min, and DAD was set at 330 nm and 280 nm, respectively. The amount of biomolecules extracted were expressed in mg of extracted compound per g dry weight of rosemary leaves (mg/g).
The separation was conducted in a gradient system of 0.1% of acetic acid-methanol (phase A) and 0.1% of acetic acid-ultra-pure water, at least in duplicate, according to Wojeicchowski and co-workers [30] (link). For this, the following conditions were applied: 0 min of phase A; 7 min of phase A; 11 min 80% of phase A; 23 min 80 % of phase A; 24 min 90 % of phase A; 28 min 40 % of phase A; 40 min 40 % of phase. Rosmarinic acid and carnosic acid displayed a retention time of 14 and 32.2 min, and DAD was set at 330 nm and 280 nm, respectively. The amount of biomolecules extracted were expressed in mg of extracted compound per g dry weight of rosemary leaves (mg/g).
7
E. coli Metabolite Profiling
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5 μl of E. coli cultures (3 × 5 ml in LB medium) were inoculated into 5 ml LB broth in the presence of a sub-inhibitory range of SMX and incubated at 37 °C and 250 r.p.m for time-course analysis. Untreated E. coli cultures were used as controls (no drug). At three time-points (12 h, 24 h, and 48 h), metabolites from E. coli cultures were extracted as follows: whole cultures were centrifuged (2,000 × g, 20 min, 4 °C) and the supernatants were extracted with n-butanol (1 × 6 ml). The n-butanol-soluble layer was dried by a Genevac HT-4X system for 6 h. The dried samples were re-suspended in 100 μl of methanol and the insoluble materials were removed centrifugation for 5 min at 21,000 × g at 4 °C. The clarified samples were analyzed by high-resolution ESI-Q-TOF-MS (Injection volume: 2 μl; Column: Phenomenex Kinetex C18 (100Å) 5 μm (250 × 4.6 mm) column; Flow rate: 0.7 ml min−1; Mobile phase composition: 10 to 100% acetonitrile in water containing 0.1% formic acid for 30 min). Relative metabolite production was achieved by comparing extracted ion count (EIC) chromatograms with an error window of 10 ppm. The time-course experiment was performed in three biological replicates.
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E. coli Metabolite Profiling
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