HPLC analysis was performed to determine the content of metabolites in the methanol extracts from biomass (2 h, at the solvent boiling point of 64.7 °C) and in hydrolysates (2 M HCl, 30 min, at the solvent boiling point of 100 °C) obtained from the agitated shoot cultures. RP-HPLC analysis was carried out as previously described [41 (link)] on Merck-Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher® RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Analysis was carried out at 25 °C. The mobile phase used for the analysis consisted of methanol (A), and methanol:0.5% acetic acid (1:4, v/v) (B). The flow rate was 1 mL/min, and the gradient was as follows: 100% B for 0–20 min; 100–80% B for 20–35 min; 80–60% B for 35–55 min; 60–0% B for 55–70 min; 0% B for 70–75 min; 0–100% B for 75–80 min; and 100% B for 80–90 min. Quantification was done by measuring the peak area with reference to a standard curve derived from five concentrations (0.03125–0.5 mg/mL). Exemplary chromatograms showing the content of the analyzed compounds in an extract from R. graveolens cultures and chromatograms of the standards are included in the Supplementary Files (Figures S1–S4) .
L 2455
Manufactured by Merck Group
Sourced in Japan
The L-2455 is a laboratory equipment product designed for general scientific applications. It serves as a tool for performing various laboratory procedures and experiments. The core function of the L-2455 is to provide a controlled and reliable environment for carrying out research and analysis tasks. Detailed specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
L 2130, by Merck Group (3 mentions)
Purospher rp 18e 250 4 mm 5 mm column, by Merck Group (2 mentions)
Lachrom elite, by Hitachi (2 mentions)
L 2200, by Merck Group (2 mentions)
Chlorogenic, by Merck Group (1 mentions)
Isoquercetin, by Merck Group (1 mentions)
Rosmarinic, by Merck Group (1 mentions)
Neochlorogenic, by Merck Group (1 mentions)
Quercitrin, by Merck Group (1 mentions)
Millex gp, by Merck Group (1 mentions)
Hplc dad, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Elitelachrom, by Merck Group (1 mentions)
5 protocols using l 2455
1
Quantitative HPLC Analysis of Metabolites
2
HPLC Analysis of Phenolic Compounds
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Lyophilised and powdered samples (about 250 mg) were extracted with 2 mL of methanol using sonication (10 min at 25 ± 2 °C) and centrifuged for 10 min at 15,000 rpm. The supernatant was filtered through 0.22 μm filter (Millex®GP, Millipore, Merck, Darmstadt, Germany). RP-HPLC analyses was carried out according to Simlat et al.34 (link) using a Merck–Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher®RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Quantification was carried out at λ = 254 nm for phenolic acids and λ = 370 nm for flavonoids. Identification was performed through a comparison of the retention times of the peaks with authentic reference compounds and co-chromatography with standards. Quantification consisted of measurement of the peak area with reference to the standard curve derived from five concentrations (0.03125–0.5 mg ml−1). Commercially available standards of the phenolic acids: chlorogenic, neochlorogenic (Sigma–Aldrich, St Louis, MO, USA), isochlorogenic A (ChromaDex, Irvine, CA, USA), and rosmarinic (Sigma–Aldrich, St Louis, MO, USA), as well as the flavonoids isoquercetin, and quercitrin (Sigma–Aldrich, St Louis, MO, USA), were used to generate the calibration curves. The analysis was repeated three times.
3
Quantitative HPLC Analysis of Chlorogenic Acids
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The instrumental analysis of CQAs was performed using HPLC-DAD, Merck Hitachi Elite La Chromatograph (Tokyo, Japan) equipped with a quaternary system of pumping (L-2130) and L-2455 UV/vis spectrophotometry diode array detector. Separation was achieved using LiChroCART RP-18 endcapped (250 × 4 mm, 5 μm) column, attached to a guard column (4 × 4 mm, 5 μm) of the same kind.
Quantitative analysis of chlorogenic acids was performed based on the method described previously by Tfouni et al. [15 (link)] with slight modifications. The mobile phase was constituted eluent A: 10 mM citric acid aqueous solution (pH of 2.4) and eluent B: acetonitrile. The gradient was programmed as follows: from 0 to 30 min 8% of B, 30 to 35 min increase to 80% of B, 35 to 40 min 80% of B, 40 to 45 min decrease to 8% of B, and 45 to 50 min 8% of B. Injected volume was 10 μL and the flow rate of analysis was 1 mL/min. Detection of CQAs was carried out at 325 nm. Identification of the target compounds was confirmed by retention time and spectrum comparison with standard solutions.
Quantitative analysis of chlorogenic acids was performed based on the method described previously by Tfouni et al. [15 (link)] with slight modifications. The mobile phase was constituted eluent A: 10 mM citric acid aqueous solution (pH of 2.4) and eluent B: acetonitrile. The gradient was programmed as follows: from 0 to 30 min 8% of B, 30 to 35 min increase to 80% of B, 35 to 40 min 80% of B, 40 to 45 min decrease to 8% of B, and 45 to 50 min 8% of B. Injected volume was 10 μL and the flow rate of analysis was 1 mL/min. Detection of CQAs was carried out at 325 nm. Identification of the target compounds was confirmed by retention time and spectrum comparison with standard solutions.
4
HPLC-MS Analysis of Organic Compounds
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All chemical solvents and standards were of analytical grade. Acetonitrile was delivered from Merck (Darmstadt, Germany) and formic acid from POCh (Katowice, Poland). HPLC ELITE LaChrom VWR (Merck) instrumentation consisted of the following components: a pump series L-2130 in gradient mode, an autosampler series L-2200 with a 100 μL injection loop, Column Oven series L-2350, and the diode array detector (DAD) series L-2455. The chromatographic separation was performed in the gradient mode with a Discovery RP 18 column (250 × 4.6 mm I.D., 5 μm particle size) from Sigma-Aldrich (Supelco).
The mobile stage consisted of the phase A which was 0.1% v/v formic acid (HCOOH) in water and the phase B which was Acetonitrile (0.050% TFA in Acetonitrile/water 80 : 20 v/v). The flow rate increased from 0.6 mL/min to 1.2 mL/min for 11minutes and then decreased to 0.6 mL/min. The gradient was programmed as follows: 6% B for 11 min, then increased to 14% B for 3 min, and then decreased to 6% B. Temperature of the column was equal to 15°C. The applied gradient was linear from 0 to 55% in 11 min, at a flow rate of 1.2 mL/min.
The DAD was set at a wavelength of 274 nm. Mass spectra were collected every 3 ms in the positive ion mode. MS spray voltage was 4.50 kV and the capillary temperature was 250°C.
The mobile stage consisted of the phase A which was 0.1% v/v formic acid (HCOOH) in water and the phase B which was Acetonitrile (0.050% TFA in Acetonitrile/water 80 : 20 v/v). The flow rate increased from 0.6 mL/min to 1.2 mL/min for 11minutes and then decreased to 0.6 mL/min. The gradient was programmed as follows: 6% B for 11 min, then increased to 14% B for 3 min, and then decreased to 6% B. Temperature of the column was equal to 15°C. The applied gradient was linear from 0 to 55% in 11 min, at a flow rate of 1.2 mL/min.
The DAD was set at a wavelength of 274 nm. Mass spectra were collected every 3 ms in the positive ion mode. MS spray voltage was 4.50 kV and the capillary temperature was 250°C.
5
HPLC Analysis of Diterpene Compounds
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HPLC analysis was performed in a Merck Hitachi Elite LaChrom (Tokyo, Japan) system equipped with a quaternary pump (L-2130), an L-2200 autosampler and a L-2455 UV/vis spectrophotometry diode array detector. Separation was achieved using a Purospher STAR LichroCART RP 18 end-capped (250 × 4 mm, 5 μm) column attached to a guard column (4 × 4 mm, 5 μm) of the same kind. The detection wavelengths were 225 nm for cafestol and 290 nm for kahweol. EZChrom Elite 3.1.6 software was used for data acquisition and analysis.
Before chromatographic analysis, the dried extracts were made up to 2.5 mL (ECs) or 10 mL (R&G Arabica coffee) with acetonitrile. Twenty microliters of the reconstituted samples were injected after filtration (0.45 µm polytetrafluoroethylene membranes (PTFE), VWR, USA). Mobile phase was acetonitrile/water (55/45, v/v) with an isocratic flow rate of 0.8 mL/min [27] (link). Target compounds were identified by comparing spectra and retention times of reference standard solutions. Quantitative analysis was performed using external standard calibration curves by plotting the peak area vs. the corresponding concentrations.
Before chromatographic analysis, the dried extracts were made up to 2.5 mL (ECs) or 10 mL (R&G Arabica coffee) with acetonitrile. Twenty microliters of the reconstituted samples were injected after filtration (0.45 µm polytetrafluoroethylene membranes (PTFE), VWR, USA). Mobile phase was acetonitrile/water (55/45, v/v) with an isocratic flow rate of 0.8 mL/min [27] (link). Target compounds were identified by comparing spectra and retention times of reference standard solutions. Quantitative analysis was performed using external standard calibration curves by plotting the peak area vs. the corresponding concentrations.
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