All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. The catalysts were synthesized following the procedure outlined by our group recently [41 (link),42 (link),43 (link),44 (link)]. Anhydrous toluene and THF were distilled from sodium-benzophenone. Dichloromethane and trimethylamine were distilled from calcium hydride. Flash column chromatography was performed using Tsingtao silica gel (60, particle size 0.040–0.063 mm). Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. 1H NMR (400 MHz and 600 MHz), 13C NMR (101 MHz and 151 MHz), and 19F NMR (565 MHz and 376 MHz) spectra were recorded on a Bruker AV III HD spectrometer (Romanshorn, Switzerland), and reported in terms of chemical shift relative to residual CDCl3 (δ 7.26 and δ 77.0 ppm, respectively). High-resolution mass spectra (HRMS) data were obtained by using Thermo Scientific™ Q Exactive™ Quadrupole-Orbitrap Mass Spectrometer (Midland, Canada). HPLC (High Performance Liquid Chromatography) analysis was conducted on Agilent 1260 instrument (Waldbronn, Germany) and Shimadzu LC-20A instrument (Kyoto, Japan) using chiral column described below in detail. Specific optical rotation was measured on a Rudolph-Autopol I (Hackettstown, NJ, USA).
Lc 20a instrument
Manufactured by Shimadzu
Sourced in Japan
The LC-20A is a high-performance liquid chromatography (HPLC) instrument manufactured by Shimadzu. It is designed for the separation, identification, and quantification of chemical compounds in various samples. The LC-20A provides reliable and accurate analytical results through its advanced technology and precise control of liquid flow and pressure.
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Lab products found in correlation
Sephadex lh 20, by GE Healthcare (2 mentions)
Q exactive quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Aviii hd spectrometer, by Bruker (1 mentions)
Agilent 5 tc c18 column, by Agilent Technologies (1 mentions)
Avance 400 nmr spectrometer, by Bruker (1 mentions)
Analytic c18 column, by Phenomenex (1 mentions)
Spd m20a photodiode array detector, by Shimadzu (1 mentions)
Silica gel, by Qingdao Marine Chemical (1 mentions)
Gf254 plates, by Qingdao Marine Chemical (1 mentions)
400 mr spectrometer, by Agilent Technologies (1 mentions)
Waters xselect csh, by Waters Corporation (1 mentions)
Vnmrs 600 spectrometer, by Agilent Technologies (1 mentions)
Dionex ultimate 3000 instrument, by Thermo Fisher Scientific (1 mentions)
Hplc grade, by Merck Group (1 mentions)
4 protocols using lc 20a instrument
1
Anhydrous Catalyst Synthesis and Characterization
2
Quantification of Lobetyolin and Saponins in Transgenic Hairy Roots
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Transgenic hairy roots sub-cultured for one month were used for determination of lobetyolin and total saponins.
To determine the concentration of lobetyolin, we ground the dried hairy roots into powder, followed by extracted three times with 10 mL methanol by sonication (50-150 W) in an ultrasonic bath (Kunshan Instrument Co., Ltd., China) for 30 min, 20 min, and 15 min, respectively. The extracts were put together and the methanol solution was evaporated, followed by dissolved with methanol to 5 mL volumetric ask. After ltration with 0.22 µm microporous membrane, the solution was used for HPLC analysis on a Shimadzu LC-20A instrument (Shimadzu, Japan) equipped with an Agilent 5 TC-C 18 column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of ultrapure water (A) and methanol (B) and the gradient condition was 0-5 min, 20-40% B; 5-10 min, 40-70% B; 10-12 min, 79-90% B; 12-25 min, 90% B. The separation was performed at 30 ℃, with the ow rate of 1.0 mL/min and UV detector wavelength at 220 nm.
The concentration of total saponins in transgenic hairy roots was determined as we described previously [39] .
To determine the concentration of lobetyolin, we ground the dried hairy roots into powder, followed by extracted three times with 10 mL methanol by sonication (50-150 W) in an ultrasonic bath (Kunshan Instrument Co., Ltd., China) for 30 min, 20 min, and 15 min, respectively. The extracts were put together and the methanol solution was evaporated, followed by dissolved with methanol to 5 mL volumetric ask. After ltration with 0.22 µm microporous membrane, the solution was used for HPLC analysis on a Shimadzu LC-20A instrument (Shimadzu, Japan) equipped with an Agilent 5 TC-C 18 column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of ultrapure water (A) and methanol (B) and the gradient condition was 0-5 min, 20-40% B; 5-10 min, 40-70% B; 10-12 min, 79-90% B; 12-25 min, 90% B. The separation was performed at 30 ℃, with the ow rate of 1.0 mL/min and UV detector wavelength at 220 nm.
The concentration of total saponins in transgenic hairy roots was determined as we described previously [39] .
3
Analytical Techniques for Natural Product Characterization
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High resolution electrospray ionization mass spectrometry (HRESIMS) spectra were recorded on an LC1260-Q-TOF/MS 6520 machine (Agilent Technologies, CA, USA). 1 H, 13 C, and 2D NMR spectra were measured on an Avance 400 NMR spectrometer (Bruker BioSpin, Zurich, Switzerland) . Chemical shifts were expressed in δ (ppm) referenced to the solvent residual peaks (δ H 2.05/δ C 29.84, δ H 3.31/δ C 49.0 for acetone-d 6 and CD 3 OD, respectively), and coupling constants (J) in hertz. Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden), and silica gel Qingdao Marine Chemical Inc., Qingdao, China) were used for column chromatography. Semi-preparative HPLC separation was carried out on a Lumtech K-501 pump (Lumiere Tech. Ltd., Beijing, China) with a K-2501 UV detector using a Luna-C18 c umn (250 mm × 10 mm i. ., 5 μm, Ph n m n x Inc., T anc , CA, USA). High performance liquid chromatography (HPLC) analysis was performed using a Shimadzu LC-20A instrument with a SPD-M20A photodiode array detector (Shimadzu Corp., Tokyo, Japan) and an analytic C 18 column (250 mm × 4.6 mm i.d., 5 µm; Phenomenex Inc., Torrance, California, USA). The precoated silica gel GF-254 plates (Qingdao Marine Chemical Inc., China) on glass were used for analytical thin layer chromatography (TLC). Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H 2 SO 4 in 95% ethanol followed by heating.
4
Characterization of Bioactive Compounds
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The NMR spectra were recorded on a Varian VNMRS 600 spectrometer (Varian, Palo Alto, USA, 600 MHz for 1 H) and Varian 400-MR spectrometer (Varian, Palo Alto, USA, 400 MHz for 1 H) with TMS as an internal reference. The carbon signal of solvent (CD 3 OD, 49.00 ppm) was used as a reference for 13 C NMR shifts. ESI-MS spectra were measured using a Bio-TOF Q spectrometer (QStar Elite Hybrid LC/MS/MS). Analytical HPLC was carried on a Dionex UltiMate 3000 instrument (Thermo, Waltham, MA, USA) with UV detection using Waters XSELECT TM CSH TM on C 18 (4.6 × 250 mm, 5 µm) column. Semi-preparative HPLC was conducted on a Shimadzu LC-20A instrument (Shimadzu Corporation, Kyoto, Japan) with UV detection using a Waters XSELECT TM CSH TM on C 18 (10 × 250 mm, 5 µm, Waters Co., Milford, MA, USA) column. Silica gel (200-300 mesh, Jiangyou silica gel Development Co. Ltd., Yantai, China), ODS (Merck, Germany) and Sephadex LH-20 (GE, Sweden) were used for column chromatography. Solvents were analytical grade (Baishi Chemical Co. Ltd., Tianjin, China) for column chromatography and HPLC grade (Merck, Germany) for HPLC analysis. B16 melanoma cells line were acquired from the Chinese Academy of Sciences (Beijing, China) .
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