Thin-layer chromatography was carried out on silica gel 60 GF254 (Merck) plates. Preparative HPLC (LC-20AR, Shimadzu, Kyoto, Japan) was conducted on a Shim-pack GIS C18 column (5 μm, 250 × 20 mm, Shimadzu). Column chromatography (CC) was performed on silica gel (200–300 mesh) and Sephadex LH-20. LC-HRMS spectra were obtained from an Agilent 1260 UPLC-DAD-6530 ESI Q-TOF MS (Agilent Technologies GmbH, Waldbronn, Germany). Optical rotation values were measured using a Jasco P-1020 polarimeter. NMR data were obtained using a Bruker Avance 500 MHz or Bruker Avance 300 MHz spectrometers (Bruker Biospin GmbH, Karlsruhe, Germany). Tetramethylsilane was used as an internal standard. The X-ray structures were solved by direct methods (SHELXL-97). The X-ray crystallographic data were collected on a Bruker SMART APEX-II CCD diffractometer using graphite monochromatic Cu Kα radiation.
Shim pack gis c18 column
Manufactured by Shimadzu
Sourced in Japan
The Shim-pack GIS C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a stationary phase of octadecylsilane (C18) bonded to silica particles, which provides efficient and reproducible chromatographic separations.
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Lab products found in correlation
Smart apex 2 ccd diffractometer, by Bruker (1 mentions)
Lc 20ar, by Shimadzu (1 mentions)
Avance 500 mhz, by Bruker (1 mentions)
Agilent 1260 uplc dad 6530 esi q tof ms, by Agilent Technologies (1 mentions)
P 1020 polarimeter, by Jasco (1 mentions)
Avance 300 mhz, by Bruker (1 mentions)
1260 infinity 2 system, by Agilent Technologies (1 mentions)
Hplc dad, by Shimadzu (1 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
8 protocols using shim pack gis c18 column
1
Isolation and Characterization of Natural Products
2
Purple Corn Anthocyanin Extraction and Purification
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Anthocyanin extraction was performed according to the reference method of Li et al., with slight modifications [25 (link)]. Purple corncob samples (1000 g) were soaked at 4 °C and extracted twice in methanol (4 L) containing 0.1% HCl for 12 h each time [25 (link)]. The extract was further purified on a column of AB-8 macroporous resin (C. Chen, Somavat, Singh, and Elvira, 2017). We used an LC-16P preparative-HPLC system combined with a Shimadzu Shim-pack GIS-C18 column (20 × 250 mm, 10 μm) [26 (link)] to separate anthocyanins. The mobile phase consisted of water/acetonitrile/formic acid (80/15/5, v/v/v). For isocratic elution, the flow rate was 10 mL/min, the column temperature was set to 35 °C, the UV detection was set to 520 nm, and 5 mL of sample solution (15.3 g/L total anthocyanins) was injected. An automated fraction collector was used for collecting the anthocyanin fraction.
3
Comparative HPLC Analysis of HSPE and HSCE
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To compare the chromatographic data of the water extracts from HSPE and HSCE, we performed HPLC using an Agilent 1260 Infinity II system. Separation was achieved using a Shim-pack GIS C18 column (5 μm, 4.6 × 250 mm) (Shimadzu, Kyoto, Japan) maintained at 30 °C. The mobile phase was a mixture of solvent A (water containing 0.1% trifluoroacetic acid) and solvent B (acetonitrile containing 0.1% trifluoroacetic acid), which were pumped under the following gradient conditions: 0→5 min, 0% B→0% B; 5→75 min, 0% B→75% B; 75→77 min, 75% B→95% B; 77→82 min, 95% B→95% B; 82→84 min, 95% B→0% B and 84→95 min 0% B→0% B. The flow rate was set at 1.0 mL/min, and the injection volume was 20 μL. Chromatographic data were collected using a diode array detector (DAD) within the wavelength range of 200–600 nm, and the results were extracted at three characteristic wavelengths (210, 255, and 305 nm).
4
HPLC Quantification of 5,6-Dehydrokawain
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The 5,6-dehydrokawain (chemical name 4-methoxy-6-[(E)-2-phenylethenyl]pyran-2-one, chemical formula C14H12O3, >98% purity) was purchased from abcam (Cambridge, UK). The 5,6-dehydrokawain was quantified using a high-performance liquid chromatography-diode array detector (HPLC-DAD; Shimadzu) equipped with a shim-pack GIS C18 column (250 × 4.6 mm, 5 μm ODS; Shimadzu), a quaternary pump, and an autosampler at 35 °C. The mobile phase eventually adopted for this study was acetonitrile/water (50/50), isocratic elution for 30 min and the flow rate was 1.0 mL/min. The sample injection volume was 10 μL. The detector was set to 343 nm. The peaks were identified by comparing their retention times and UV spectra with standards.
5
Hepatoprotective Herb Analysis by HPLC-MS/MS
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The decoction of the herb with the strongest hepatoprotective effect (S. officinalis L.) was analyzed by reverse-phase HPLC using a Shim-pack GIS C18 column (5 µm, 4.6 mm × 250 mm; Shimadzu) maintained at 30 °C and a gradient elution (solvent A: H2O containing 0.1% (v/v) formic acid; solvent B, methanol containing 0.1% (v/v) formic acid). The phytochemical compounds were separated using a 70-min linear gradient from 20% to 70% solvent B at a flow rate of 1.0 mL/min and detected at 280 nm. The injection volumes were 20 µL (sample without dilution) for HPLC analysis and 50 µL (sample with 10-fold dilution) for HPLC-MS/MS analysis.
The MS analysis was performed by using an Ion Trap Mass Spectrometer (ITMS; Thermo Scientific, United States) equipped with an ESI under the following conditions: ESI source temperature, 320 °C; source voltages, 4.0 kV for positive mode and 3.5 kV for negative mode; capillary voltages, 24.0 V for positive polarity, and -12.0 V for negative polarity; desolvation gas, nitrogen; full scan mass range, m/z 50–1500. Tandem mass spectrometry analyses were performed using nitrogen as the collision gas with a collision energy of 35 eV. The MS/MS spectrum was obtained to the tenth most intense ion from ITMS.
The MS analysis was performed by using an Ion Trap Mass Spectrometer (ITMS; Thermo Scientific, United States) equipped with an ESI under the following conditions: ESI source temperature, 320 °C; source voltages, 4.0 kV for positive mode and 3.5 kV for negative mode; capillary voltages, 24.0 V for positive polarity, and -12.0 V for negative polarity; desolvation gas, nitrogen; full scan mass range, m/z 50–1500. Tandem mass spectrometry analyses were performed using nitrogen as the collision gas with a collision energy of 35 eV. The MS/MS spectrum was obtained to the tenth most intense ion from ITMS.
6
Quantitative HPLC Analysis of Bioactive Compounds
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High-performance liquid chromatography (HPLC) analysis of EAHDT, ampelopsin, and quercetin was performed using an UltiMate™ 3000 UHPLC (Thermo Fisher Scientific, Waltham, MA, USA) and a Shim-pack GIS C18 column (5 μm, 4.6 mm × 250 mm, Shimadzu Co., Kyoto, Japan). The binary mobile phase consisted of solvent A (water with 0.1% TFA) and solvent B (100% acetonitrile). The mobile phase flow rate and oven temperature were maintained at 1.0 mL/min and 50 °C, respectively. The linear gradient elution conditions were set at 5% B to 90% B (0–28 min), 90% B (28–30 min), 90% B to 10% B (30–33 min), and 10% B (33–35 min). Chromatograms were obtained at a wavelength of 270 nm.
7
Quantifying Riboflavin in Fermented Soymilk
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The concentration of riboflavin was determined by high-performance liquid chromatography (HPLC) coupled with a photodiode array (PDA) detector (Shimadzu, Kyoto, Japan) based on a previous study [19 (link)]. A Shim-pack GIS C18 column (4.6 × 250 mm, 5 μm) (Shimadzu, Kyoto, Japan) was used to separate riboflavin in the culture media or the LAB-fermented soymilk. The sample was passed through a 0.22 μm pore size membrane filter. The mobile phase consisting of methanol/water/formic acid (70:29.25:0.75, v/v/v) was used to elute riboflavin. All solvents used in the mobile phase were HPLC-grade and degassed with ultrasound for 30 min. The flow speed was 0.8 mL/min, the injection volume was 20 μL, and the detection wavelength was set at 267 nm. A standard curve was prepared based on a riboflavin standard (concentration range 0.2–5 mg/L, R2 = 0.9996). By comparing the retention time and peak area of the standard sample, the content of the sample was determined.
8
Purification and Characterization of Peptides
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Crude peptides were dissolved in 1:2:2 DMSO:ACN:H2O and then purified using an acetonitrile:water gradient on a reverse-phase preparative HPLC column (Shim-pack GIS C18 column, 20 × 250 mm, 10 μm, Shimadzu). Purified peptides were analyzed for purity using the Genesis peak extraction program (ThermoFisher) using high resolution mass spectrometry. All LC–MS spectra and purities can be found in the Supporting Information (Supplementary Figures S3-S9 ).
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