The conversion of d ,l -PL was estimated by HPLC on an ODS-3 column (4.6 × 250 mm, 5 μm, Shimadzu, Tokyo, Japan) at 210 nm with a mobile phase of 10% acetonitrile containing 0.02 mM KH2PO4 with a flow rate of 1.0 mL min−1. The retention times of d ,l -PL and d ,l -PA were 4.92 and 8.89 min respectively. The optical purity of d -PA was determined by HPLC on an MCI GEL CRS10W packed column (4.6 × 50 mm, 3 μm, Mitsubishi Kasei, Tokyo, Japan) at 254 nm with a mobile phase of 10% acetonitrile containing 1.8 mM CuSO4 with a flow rate of 0.8 mL min−1. The retention times of d -PA and l -PA were 2.82 and 3.75 min respectively.
Ods 3 column
Manufactured by Shimadzu
Sourced in Japan
The ODS-3 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. The column features a proprietary octadecylsilane (ODS) stationary phase that provides efficient and reproducible separations. The ODS-3 column is a versatile tool for various analytical applications.
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Lab products found in correlation
Hplc system, by Shimadzu (2 mentions)
Lc 20a system, by Shimadzu (1 mentions)
Prominence lc solution system, by Shimadzu (1 mentions)
Lc 20ap pump, by Shimadzu (1 mentions)
Lc 20ad pump, by Shimadzu (1 mentions)
J 810 circular dichroism spectrometer, by Jasco (1 mentions)
Agilent 6210 lc msd tof mass spectrometer, by Agilent Technologies (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Av 400 spectrometer, by Bruker (1 mentions)
P 1020 polarimeter, by Jasco (1 mentions)
V 550 uv vis spectrophotometer, by Jasco (1 mentions)
Jms t100cs, by JEOL (1 mentions)
Lsm 800 confocal laser scanning microscopy, by Zeiss (1 mentions)
Fp 6300 fluorescence spectrophotometer, by Jasco (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Agilent 7890b gas chromatograph, by Agilent Technologies (1 mentions)
Lcms it tof, by Shimadzu (1 mentions)
10 protocols using ods 3 column
1
HPLC Analysis of Pyridoxal Enantiomers
2
HPLC Analysis of Notopterol and Pterostilbene
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The concentrations of notopterol (nol) and pterostilbene (pte) in the test sample solution were determined by HPLC. The equipment utilized included the following: LC-2030C high-performance liquid chromatography system (Shimadzu), Inertsil ODS-3 column (4.6 mm × 250 mm, 5 µm), mobile phase: acetonitrile +0.5% glacial acetic acid (50:50), a flow rate of 1.0 mL/min, detection wavelength of 305 nm, injection volume of 10 μL, and column temperature of 35 °C. We utilized isocratic elution and Labsolutions software for HPLC to collect data and generate spectra.
3
LC-MS/MS Analysis of Ethyl Sulfate and Ethyl Glucuronide
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Chromatographic separation was performed with an Inertsil ODS-3 column (2.1 × 100 mm, 3 µm) through a LC-20A system (Shimadzu, Japan). The mobile phase was a mixture of solvent A (0.1% formic acid in ultrapure water) and solvent B (0.1% formic acid in acetonitrile). The chromatographic column was held at 35 °C and eluted for 14.0 min in total at a flow rate of 0.2 mL/min with a gradient of 5% B (0–2.0 min), 90% B (2.0–6.0 min), 90% B (6.0–8.0 min), 5% (8.0–8.5 min), and 5% (8.5–14.0 min), respectively. Mass spectrometry was performed on a mass spectrometer (TRAP 4000, Sciex, AB). All analytes were ionized by electrospray in negative mode, and tandem MS analysis was performed in multiple reaction monitoring (MRM) mode. The ionspray voltage was − 4000 V, and the temperature was 500 °C. The curtain gas, Gas1, and Gas2 were 40 psi, 50 psi, and 35 psi, respectively. Other specific MRM parameters for each analyte are shown as follows in Table 1 .
The feature ion pair and mass spectrum data of each analyte.
| Analyte | Qualitative ions (m/z) | Quantitative ions (m/z) | DP (V) | CE (V) |
|---|---|---|---|---|
| EtS | 125.0/80.0 | 125.0/97.0 | 46 | 45 |
| 125.0/97.0 | 21 | |||
| EtG | 221.1/75.0 | 221.1/75.0 | 63 | 22 |
| 221.1/85.0 | 23 | |||
| EtG-D5 | 226.1/75.0 | 226.1/75.0 | 63 | 23 |
| 226.1/85.0 | 26 | |||
| EtS-D5 | 130.0/80.0 | 130.0/80.0 | 46 | 46 |
| 130.0/97.9 | 25 |
4
Polyphenol Dynamics in Root Growth
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Samples from five growth stages were analyzed using HPLC to investigate changes in polyphenol content and composition during the growth stages of the root system.
The total polyphenol content and composition were measured using HPLC and were used to separate and determine individual polyphenols in the sample. Freeze-dried root powder (0.02 g) was dissolved in 5 mL of 80% (v/v) methanol and passed through a 0.20 μm membrane filter (Sartorius, Göttingen, Germany) for analysis.
The extracts were analyzed by reverse-phase HPLC using the Prominence LC solution system with an ODS-3 column (Shimadzu). The chromatographic conditions were as follows: solvent A, 100% ethanol; solvent B, 20 mM KH2PO4 (pH 2.4); column temperature, 40 °C; detection at 320 nm; and flow rate, 1.0 mL·min−1. The binary gradient was as follows: 85–68% B (0–12 min), 68% B (12–15 min), 50–55% B (15–20 min), and 85% B (20–29 min). Retention times and spectra were compared with pure standards of chlorogenic acid, caffeic acid, 3,4-dicaffeoylquinic, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. The results are expressed as mg·100 g−1 FW. The measurements were replicated three times.
The total polyphenol content and composition were measured using HPLC and were used to separate and determine individual polyphenols in the sample. Freeze-dried root powder (0.02 g) was dissolved in 5 mL of 80% (v/v) methanol and passed through a 0.20 μm membrane filter (Sartorius, Göttingen, Germany) for analysis.
The extracts were analyzed by reverse-phase HPLC using the Prominence LC solution system with an ODS-3 column (Shimadzu). The chromatographic conditions were as follows: solvent A, 100% ethanol; solvent B, 20 mM KH2PO4 (pH 2.4); column temperature, 40 °C; detection at 320 nm; and flow rate, 1.0 mL·min−1. The binary gradient was as follows: 85–68% B (0–12 min), 68% B (12–15 min), 50–55% B (15–20 min), and 85% B (20–29 min). Retention times and spectra were compared with pure standards of chlorogenic acid, caffeic acid, 3,4-dicaffeoylquinic, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. The results are expressed as mg·100 g−1 FW. The measurements were replicated three times.
5
Spectroscopic Characterization of Compounds
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Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were recorded on a JASCO V-550 UV/VIS spectrophotometer (JASCO Corporation). A JASCO FT/IR-480 plus FT-IR spectrometer (JASCO Corporation) was used for scanning the IR spectra with KBr pellets. 1H, 13C, and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker Corporation, Rheinstetten, Germany). HR-ESI-MS data were conducted on an Agilent 6210 LC/MSD TOF mass spectrometer. CD spectra were obtained using a JASCO J-810 circular dichroism spectrometer. For column chromatography, silica gel (300–400 mesh; Qingdao Marine Chemical Group Corporation, Qingdao, China) and Sephadex LH-20 (Pharmacia, Pittsburgh, PA, USA) were used. TLC analyses were carried out using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, Yantai, China). Analytic high-performance liquid chromatography (HPLC) was performed on a SHIMADZU chromatography (SHIMADZU Corporation, Kyoto, Japan) equipped with an LC-20AD pump and an SPD-M20A diode-array detector (DAD) with an Inertsil ODS-3 column (4.6 mm × 250 mm, 5 μm). Preparative HPLC was carried out on a SHIMADZU instrument equipped with an LC-20AP pump and an SPD-20A (SHIMADZU Corporation) detector with a YMC-Pack ODS-A column (20 mm × 250 mm, 5 μm).
6
Quantitative Monosaccharide Analysis via HPLC
7
HPLC Analysis of β-Alanine and Glutathione Adducts
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For measurement of intact β-alanine or methylglyoxal-conjugated β-alanine, a Shimadzu HPLC system equipped with a Triart Diol-HILIC column (250 × 4.6 mm, YMC) was used. A flow rate of 1 ml/min was used with an 80:20 mix of acetonitrile:10 mM ammonium acetate as the mobile phase. Signals were detected at 210 nm at 40 °C and the retention times of β-alanine and methylglyoxal-conjugated β-alanine were defined as ~ 13 min and 14.5 min, respectively. For measurement of intact GSH or methylglyoxal-conjugated GSH, a Shimadzu HPLC system equipped with an Inertsil ODS-3 column (250 × 4.6 mm, YMC) was used. A flow rate of 1 ml/min was used with a 5:95 mix of acetonitrile:20 mM H3PO4 + 10 mM NaClO as the mobile phase. Signals were detected at 200 and 210 nm at 40 °C and the retention times of GSH and methylglyoxal-conjugated GSH defined as ~4.5 min and 5.5 min, respectively.
8
Spectroscopic Characterization of Fluorescent Probes
9
Optical Purity Analysis of D,L-PL and D,L-PA
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The conversion of d ,l -PL was analyzed by HPLC on an ODS-3 column (4.6 × 250 mm, 5 μm, Shimadzu, Tokyo, Japan) at 210 nm with a mobile phase of 10% acetonitrile containing 0.02 mM KH2PO4 with a flow rate of 1.0 mL min−1. The retention times of d ,l -PA and d ,l -PL were 4.93 and 8.98 min, respectively. The optical purity of D-PA was determined by HPLC on an MCI GEL CRS10W packed column (4.6 × 50 mm, 3 μm, Mitsubishi Kasei, Tokyo, Japan) at 254 nm with a mobile phase of 10% acetonitrile containing 1.8 mM CuSO4 with a flow rate of 0.8 mL min−1. The retention times of d -PA and l -PA were 5.4 and 7.5 min, respectively. Chiral GC analysis of d -PL and l -PL was performed on Agilent 7890B gas chromatograph equipped with a flame ionization detector (FID) using CYCLODEX-B column with nitrogen as the carrier gas (80 °C for 10 min, 10 °C min to 130 °C, and 130 °C for 10 min, total 25 min). The retention times of l -PL and d -PL were 17.8 and 18.3 min, respectively.
10
High-Resolution LC-MS Analysis of Antimicrobial Compounds
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High resolution LC-MS analysis was done to elucidate the compounds present in the samples with detectable MIC and MBC. LC-MS-IT-TOF, Shimadzu, Tokyo was fitted with an ODS-3 column (150 mm 9 1.5 mm i.d., 5-lm particle size). The solvent system comprised water with 0.1% formic acid as solvent A and acetonitrile with 0.1% formic acid as solvent B. The analysis was carried out at room temperature with a flow rate of 0.2 mL/min. The gradient flow of the mobile phase was set as: 0-10 min, 5-30% B; 10-15 min, 30-40% B; 15-30 min, 40% B; 30-60 min, 40-70% B; 60-70 min, 70-100% B; 70-80 min, 100% B; 80-85 min, 100-5% B; 85-95 min, 5% B. The LC chromatograms were recorded at the wavelengths of 280 and 254 nm.
For MS, electron spray ionization (ESI) source was used in positive and negative ionization mode with m/z values of 100-1000 for MS and 50-700 for MS/MS. A probe voltage of ±4.5 kV, nebulizer gas flow of 1.5 L/min, curved desolvation line (CDL) temperature of 200 °C, and heat block temperature of 200 °C were used. The peaks obtained were partially identified on the basis of retention time, m/z values, and comparison with standard compounds.
For MS, electron spray ionization (ESI) source was used in positive and negative ionization mode with m/z values of 100-1000 for MS and 50-700 for MS/MS. A probe voltage of ±4.5 kV, nebulizer gas flow of 1.5 L/min, curved desolvation line (CDL) temperature of 200 °C, and heat block temperature of 200 °C were used. The peaks obtained were partially identified on the basis of retention time, m/z values, and comparison with standard compounds.
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