The polyphenolic compounds were quantified using high-performance liquid chromatography (HPLC) using a Shimadzu Prominence device (Shimadzu, Kyoto, Japan) equipped with LC-20AT binary pumps, CTO-20A thermostats, SIL-20A automatic dispensers, and SPD-20AV UV/Vis detectors. Different wavelengths of light were used to record chromatograms: 280 nm, 320 nm, and 360 nm. Separation was conducted using a Luna C-18 RP column, 5 μm, 250 × 4.6 mm (Phenomenex, Torrance, CA, USA), which was protected by a C18 pre-column, 4 × 30 mm (Phenomenex, Torrance, CA, USA). As the mobile phase, the following solvents were used: A (acetonitrile) and B (1% formic acid) at the flow rate of 1 mL/min and using the following linear gradient: 0–10 min, from 10 to 25% A, then 10–20 min to 60% A, then 20–30 min to 70% A. After the column has been equilibrated to initial conditions, 10% A in 10 min, there is an additional 5 min stabilization period. Filters with a pore size of 0.45 μm were used to filter all samples and solvents before analysis (Millipore, Bedford, MA, USA). The LC Solution Software (Shimadzu, Kyoto, Japan) was used to identify and quantify the peaks obtained.
Luna c 18 rp column
Manufactured by Phenomenex
Sourced in United States
The Luna C-18 RP column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. The column features a C-18 reversed-phase stationary phase, which is suitable for the separation of both polar and non-polar analytes. The column's dimensions and packing material are optimized for efficient chromatographic separations.
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Lab products found in correlation
C18 guard column, by Phenomenex (3 mentions)
Spd 20av, by Shimadzu (1 mentions)
Sil 20a, by Shimadzu (1 mentions)
C18 pre column, by Phenomenex (1 mentions)
Cto 20a, by Shimadzu (1 mentions)
Lc solution software, by Shimadzu (1 mentions)
Spd 20av uv vis detector, by Shimadzu (1 mentions)
Prominence hplc, by Shimadzu (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Alliance 2695 hplc system, by Waters Corporation (1 mentions)
7 protocols using luna c 18 rp column
1
Quantitative HPLC Analysis of Polyphenols
2
Synthesis of Withaperuvin M from Withaperuvin E
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Example 17
This example demonstrates a synthesis of withaperuvin M (6) from withaperuvin E.
To a stirred solution of triphenylphosphene (1.6 mg) and iodine (1.9 mg) in anhydrous dichloromethane (0.05 mL) at 0° C. was added a solution of withaperuvin E (3) (2.5 mg) in anhydrous dichloromethane (0.15 mL). Ice bath was removed and the reaction mixture was stirred at 25° C. After 1 hour, reaction mixture was diluted with dichloromethane (10 mL), washed with 10% aqueous Na2S2O3 solution (2×5 mL), brine (2×5 mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure to give crude product mixture. This mixture was separated by reversed phase HPLC on a Phenomenex, Luna, C18 RP column (250×10 mm) using methanol/water (72.5:27.5) as eluant to give withaperuvin M (6) (1.5 mg, 62% yield, tR=11.5 minutes) as a pale yellow solid; mp 176-178° C.; [α]25D+115 (c 0.8, CHCl3); 1H and 13C NMR data were consistent with those reported in the literature (Fang et al., Steroids (2012), 77, 36-44).
3
Chromatographic Analysis of Phenolic Compounds
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Samples were analysed by a chromatographic system Shimadzu Prominence (Shimadzu, Kyoto, Japan). Chromatograms were recorded using different wavelengths for individual compounds: 280 and 320 nm for phenolic acids and 360 nm for flavonoids. Separation was performed on a Luna C-18 RP column, 5 mm, 250 mm×4.6 mm with a C18 guard column, 4 mm×30 mm (both from Phenomenex, Torrance, CA, USA). Two mobile phases, A (acetonitrile) and B (1 % formic acid), were used at a flow rate of 1 mL/min with the following gradient profile: 0–10 min from 10 to 25 % B, 10–20 min linear rise up to 60 % B and from 20 to 30 min linear rise up to 70 % B, followed by 10 min reverse to initial 10 % B with additional 5 min of equilibration time.
4
Quantitative Analysis of Phenolic Compounds
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Total phenolic content (TPh) in the extracts was established using the Folin–Ciocalteau spectrophotometric method adapted to microscale [11 (link)]. The sample (15 μL) was mixed with distilled water (170 μL), 2 N Folin–Ciocalteu’s reagent (12 μL) and 20% Na2CO3 (30 μL) in a plate well. Absorbance was measured after 1 h (room temperature, dark conditions) at 750 nm, using distilled water as blank. Gallic acid (GAE) was used for the calibration curve.
Chromatographic analysis for identification and quantification of phenolic compounds were carried out as recommended by Tumbas Šaponjac et al. [11 (link)], using Shimadzu Prominence HPLC, connected to an SPD-20AV UV/VIS detector (Shimadzu, Kyoto, Japan), with Luna C-18 RP column, 5 lm, 250 mm × 4.6 mm with a C-18 guard column, 4 mm × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was applied using acetonitrile (A) and water acidified with 1% formic acid in d-water (B), at flow rates of 1 mL/min, at the following order: 10% to 25% A (0–10 min); 25% to 60% A (10–20 min); 60% to 70% A (20–30 min); 70% to 10% A (30–40 min); 10% A (5 min) (equilibration time). For hydroxybenzoic acids, chromatograms were recorded at 280 nm; for hydroxycinnamic acids at 320 nm; and for flavonoids at 360 nm. HPLC standards were dissolved in 50% methanol.
Chromatographic analysis for identification and quantification of phenolic compounds were carried out as recommended by Tumbas Šaponjac et al. [11 (link)], using Shimadzu Prominence HPLC, connected to an SPD-20AV UV/VIS detector (Shimadzu, Kyoto, Japan), with Luna C-18 RP column, 5 lm, 250 mm × 4.6 mm with a C-18 guard column, 4 mm × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was applied using acetonitrile (A) and water acidified with 1% formic acid in d-water (B), at flow rates of 1 mL/min, at the following order: 10% to 25% A (0–10 min); 25% to 60% A (10–20 min); 60% to 70% A (20–30 min); 70% to 10% A (30–40 min); 10% A (5 min) (equilibration time). For hydroxybenzoic acids, chromatograms were recorded at 280 nm; for hydroxycinnamic acids at 320 nm; and for flavonoids at 360 nm. HPLC standards were dissolved in 50% methanol.
5
HPLC Analysis of Carnosic Acid
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CA determination was performed by HPLC analysis using a Waters Alliance 2695 HPLC system (Milford, MA, USA) equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatic column compartment, a model 2996 diode array detector (DAD), and Empower software (Waters Corporation, Milford, MD, USA) for data collection. The chromatographic separation was performed by using a Luna C 18 RP column (250 × 4.6 mm, 5 µm, Phenomenex, Torrance, CA, USA).
The mobile phase consisted of 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The following gradient system was used: 0 min, 90% A and 10% B; 30 min, 50% A and 50% B; 35 min, 0% A and 100% B. Analyses were finished at 50 min. The system was equilibrated between runs for 10 min using the start mobile phase composition. The flow was pumped at 1 mL/min and the injection volume was 10 µL. Diode array detection was set between 200 and 600 nm, and absorbance was recorded at 325 nm. The CA concentration was identified by matching the retention time and the spectral characteristics against those of the standards.
The mobile phase consisted of 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The following gradient system was used: 0 min, 90% A and 10% B; 30 min, 50% A and 50% B; 35 min, 0% A and 100% B. Analyses were finished at 50 min. The system was equilibrated between runs for 10 min using the start mobile phase composition. The flow was pumped at 1 mL/min and the injection volume was 10 µL. Diode array detection was set between 200 and 600 nm, and absorbance was recorded at 325 nm. The CA concentration was identified by matching the retention time and the spectral characteristics against those of the standards.
6
HPLC Analysis of Synergistic Mixtures
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The most synergistic mixtures were dissolved in ACN, filtered by a membrane filter and subjected to HPLC analysis in a Hewlett Packard 1050 (Palo Alto, CA), coupled to a DAD detector (HP/Agilent series 1050 DAD), with a quaternary pump and autosampler (HP/Agilent series 1050)
and Luna C18-RP column (Phenomenex) of 25 cm x 4.6 mm, 5 µm of particle size. The isocratic solvent phase was composed of ultra-pure water supplemented with formic acid 0.1% (40%) and HPLC-grade ACN (60%). The flow rate was 0.5 ml/min and the injection volume, 5 µl. Peaks in the extracts, monitored at λ = 254 nm, were assigned based on the Rt of reference compounds.
and Luna C18-RP column (Phenomenex) of 25 cm x 4.6 mm, 5 µm of particle size. The isocratic solvent phase was composed of ultra-pure water supplemented with formic acid 0.1% (40%) and HPLC-grade ACN (60%). The flow rate was 0.5 ml/min and the injection volume, 5 µl. Peaks in the extracts, monitored at λ = 254 nm, were assigned based on the Rt of reference compounds.
7
Quantification of Phenolic Compounds in FDSP
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Total phenolic content (TPC) TPC of methanolic FDSP extracts were evaluated using the Folin-Ciocalteu assay as described by Singleton et al. [43] . The results are expressed in mg gallic acid equivalent (GAE) in mg/100 g FDSP dm.
Total flavonoids content (TFC) TFC of methanolic FDSP extracts were estimated using a method by Zhishen et al. [44] and the results were expressed as rutin equivalents (RE) in mg/100 g FDSP dm.
Individual phenolic compounds HPLC separation and quantification of phenolic compounds in FDSP extracts was performed according to Aborus et al. [17] . Chromatograms were recorded using different wavelengths for individual phenolic compounds. Phenolic compounds were separated using Luna C-18 RP column, 5 µm, 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) with a C18 guard column, 4 × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was performed using acetonitrile (A) and 1% formic acid (B) mobile phases, at flow rates of 1 ml/min, following the gradient profile: 0 -10 min from 10% to 25% A; 10 -20 min linear rise up to 60% A; and from 20 min to 30 min linear rise up to 70% A, followed by 10 min reverse to initial 10% A with 5 min of equilibration time.
Total flavonoids content (TFC) TFC of methanolic FDSP extracts were estimated using a method by Zhishen et al. [44] and the results were expressed as rutin equivalents (RE) in mg/100 g FDSP dm.
Individual phenolic compounds HPLC separation and quantification of phenolic compounds in FDSP extracts was performed according to Aborus et al. [17] . Chromatograms were recorded using different wavelengths for individual phenolic compounds. Phenolic compounds were separated using Luna C-18 RP column, 5 µm, 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) with a C18 guard column, 4 × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was performed using acetonitrile (A) and 1% formic acid (B) mobile phases, at flow rates of 1 ml/min, following the gradient profile: 0 -10 min from 10% to 25% A; 10 -20 min linear rise up to 60% A; and from 20 min to 30 min linear rise up to 70% A, followed by 10 min reverse to initial 10% A with 5 min of equilibration time.
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