The HRESIMS analyses were performed on a Waters Xevo G2-XS QTof mass spectrometer (Waters Corp., Milford Massachusetts, America). NMR spectra were recorded on an Ascend 600 MHz instrument (Bruker-Biospin, Billerica, MA, America). The analytical experiments were performed on a Shimadzu LC-20AT HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a Shimadzu InertSustain C18 column (4.6 I.D. × 250 mm, 5 μm, S/N 6LR98081). A Hanbon NP700 semipreparative HPLC (Hanbon Sci. & Tech., Jiangsu, China) equipped with a Shimadzu InertSustain C18 column (10 I.D. × 250 mm, 5 μm, S/N 7ER43006) was used for purifying compounds. Biological assays were monitored on a BioTek ELx808 microplate spectrophotometer (BioTek Instruments, Inc., Winooski, America), a BD FACSCalibur flow cytometry (BD Biosciences, Franklin Lakes, America) and an Olympus BX-51 Fluorescence Microscopy (Olympus Corporation, Tokyo, Japan).
Inertsustain c18 column
Manufactured by Shimadzu
Sourced in Japan
The InertSustain C18 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 spherical silica-based stationary phase with a C18 modification, providing excellent compatibility with a variety of mobile phases and analytes. The column's inert nature and sustained performance make it a reliable choice for various analytical applications.
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Lab products found in correlation
Lc 30, by Shimadzu (2 mentions)
Lc 20a hplc system, by Shimadzu (2 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Xevo g2 xs qtof mass spectrometer, by Waters Corporation (1 mentions)
Bx51 fluorescence microscopy, by Olympus (1 mentions)
Lc 20at hplc system, by Shimadzu (1 mentions)
Elx808 microplate spectrophotometer, by Agilent Technologies (1 mentions)
Ascend 600 mhz instrument, by Bruker (1 mentions)
Lc 2030c, by Shimadzu (1 mentions)
Lc 20a, by Shimadzu (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
11 protocols using inertsustain c18 column
1
Multi-Instrumental Analytical and Biological Assays
2
HPLC-MS Analysis of Ethanolic Leaf Extract
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Agilant 1290uplc liquid chromatography equipped with Agilant mass spectrometry (MS) qtof6550 was used to detect and analyze the component of EAF. A Shimadzu InertSustain C18 column (100 × 2.1 mm, 2 μm) was used for HPLC (column temperature: 35 °C, flow rate: 0.3 mL/min). Scanning mode, data-independent analysis (100–1500 m/z), sheath gas temperature 500 °C, and sheath gas flow rate 12 L/min were used in mass MS conditions. The HPLC analysis was performed by step-gradient method, and the mobile phase with solvent A (acetonitrile) and solvent B (water) was as follows: 10% of eluent A at 0 min, 25% at 15 min, 40% at 30 min, and 55% at 45 min. The HPLC-MS chromatogram was preprocessed and compared with the TCM database, and the data came from the analytical results of TCM database.
3
Chinese Medicine Extraction and Analysis
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A total of 2.6 g of the Chinese medicine granule mixture was weighed, 10 ml of hot water added and brewed for 20 min. Next, the mixture was sonicated for 5 min followed by a 10min centrifugation at 12000 r/min. The supernatant was filtered through a 0.45 um membrane, and the filtrate was injected into a hybrid quadrupole time-of-flight liquid chromatography mass spectrometer, an ultra-high performance liquid chromatography (UPLC, Shimadzu LC-30) connected to a mass spectrometer (MS, SCIEX 5600+) with a Shimadzu InertSustain C18 column (100 × 2.1 mm, 2 µm). The column temperature was set at 35°C and 1 ml/min flow rate. The mobile phase consisted of acetonitrile (A) and 0.1% HCOOH-H2O (B).
Mass spectrometric detection was performed using electrospray ionization (ESI) in the positive and negative ion modes.
Mass spectrometric detection was performed using electrospray ionization (ESI) in the positive and negative ion modes.
4
HPLC Analysis of Saururus Rhizome Components
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Twenty batches of SR were quantitative content analyzed by HPLC on an HPLC system (Shimadzu LC-2030C, Japan) with a UV detector using an InertSustain C18 column (250 mm × 4.6 mm, 5 µm, Shimadzu, USA). Column oven temperature was maintained at 40 °C. Sample compartment temperature was 4 °C. 254 nm was choosen for UV detection. Flow rate of 0.8 mL/min and a gradient elution of mobile phase (A: 0.1% aqueous formic acid in water, B: methanol) were used. The gradient was started at 5% B, then rised to 100% B at 60 min. and returns to the initial conditions after 10 min.
Standard curve method was used to quantify nine contents (5-HMF, VA, FA, PH, PHA, VN, PA, PCA, IA). Bivariate correlation analysis between anti-thrombotic activity and the main components in SR was conducted by SPSS 19.0 software, which was represented by Spearman correlation coefficient (R) and significance (P).
Standard curve method was used to quantify nine contents (5-HMF, VA, FA, PH, PHA, VN, PA, PCA, IA). Bivariate correlation analysis between anti-thrombotic activity and the main components in SR was conducted by SPSS 19.0 software, which was represented by Spearman correlation coefficient (R) and significance (P).
5
HPLC Separation of Compounds
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The samples were determined by an LC-20A HPLC system (SHIMADZU, Kyoto, Japan). The separation was achieved on an InertSustain C18 column (4.6 mm × 250 mm, 5 µm, SHIMADZU, Kyoto, Japan) with the flow rate keeping at 1.0 mL/min, the column temperature maintaining at 40 °C and mobile phase consisting of methanol:water (25:75, v/v) with 0.4% phosphoric acid in water. Injection volume for each run was 50 μL, and detection wavelength was 254 nm.
6
Measurement of IsPETase and Fast-PETase Activity
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The enzyme activity of IsPETase was measured as previously described7 (link). Briefly, 200 µL of the diluted enzyme sample was mixed with 3 mg of amorphous PET (percentage crystallinity of 25.7%) in 50 mM glycine-NaOH buffer (pH 9.0) to a final volume of 1 mL, followed by incubating at 30 °C for 6 h. The reaction was terminated by heating at 85 °C for 10 min, and the mixture was centrifuged at 14000 g for 1 min, followed by analysis with HPLC. The activity of Fast-PETase was analyzed with the same method except the buffer was changed to 100 mM KH2PO4 buffer (pH 8.0). TPA and MHET released from the PET depolymerization was quantified with HPLC. A Prominence Ultra Fast Liquid Chromatograph (Shimadzu) equipped with an GL Sciences InertSustain C18 column (4.6 × 250 mm, 5 µm). The mobile phase was methanol with 20 mM phosphate buffer (pH 2.5) at a flow rate of 0.8 mL/min. The elution condition was 0–25 min with 25–85% (v/v) methanol linear gradient. The effluent was monitored at a wavelength of 240 nm. A standard curve was plot using TPA and MHET. All of the experiments were carried out in triplicate. One unit of enzyme (U) was defined as the amount of enzyme causing the release of 1 µg of TPA and MHET at 30 °C for 1 h.
7
HPLC Analysis of Phenolic Compounds
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All samples taken from the cultures were centrifuged at 12,000 rpm for 2 min and the supernatants were filtered through a 0.22 μm polytetrafluorethylene (PTFE) filter and analyzed by HPLC (Shimadzu LC-20A, Kyoto, Japan) with a reverse phase Shimadzu InertSustain C18 column (5 μm, 4.6 mm × 250 mm) and a Shimadzu SPD-M20A photodiode array detector. The mobile phase used was a gradient of solvent A (H2O containing 1.3% acetic acid) and solvent B (100% acetonitrile) applied as following time procedure: 0–20 min, 10–100% B linear; 20–20.5 min, 100%–10% linear; 20.5–30 min, 10% B isocratic. The flow rate was set at 1.0 mL·min−1, and the injection volume was 10 μL. The column was maintained at 35 °C, and the eluted compounds were monitored at 309 nm for p-coumaric acid and at 274 nm for trans-cinnamic acid respectively. The concentration of trans-cinnamic acid and p-coumaric acid were quantified by fitting the peak area with a standard curve (R2 > 0.999) of the corresponding standard.
8
Characterization of Organic Compounds
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The reagents and solvents were purchased from commercial sources and used without further purification. 1H NMR and 13C NMR spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) with DMSO-d6 or CDCl3 as the solvent. Chemical shifts are given in ppm throughout. Coupling constants (J) are expressed in hertz (Hz). NMR chemical shifts (δ) are reported in parts per million (ppm) units. The high-resolution mass spectra were performed on a UPLC G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). TLC analysis was performed on GF254 silica gel plates (Qingdao Haiyang Chemical, China) under ZF-20D UV light (254 nm) (Yuhua instrument, Gongyi, China) or I2 in silica gel for monitoring all reactions. Flash column chromatography was performed on silica gel (300–400 mesh). Melting point was measured (uncorrected) on X-6 melting point apparatus (Beijing Tech, China). Purity of all compounds was determined by LC-20AT prominence high-performance liquid chromatography (HPLC) (SHIMADZU, Kyoto, Japan) using an InertSustain C18 column (150 mm × 4.6 mm, 5 μm) with the 65% solvent A (MeOH) and 35% solvent B (H2O) as eluents. The flow rate was set as 0.8 mL/min and the signals were monitored by a SPD-20A prominence UV/VIS detector at 254 nm. The purity of all the final compounds was determined by HPLC to be >97%.
9
Quantitative HPLC Analysis of Str
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The samples were determined by an LC-20A HPLC system (SHIMADZU, Tokyo, Japan), which was equipped with an LC20A pump, an SPD-M20A diode array detector, a CTO-10AS column oven and an SIL-20A automated sampler. The separation was achieved on an InertSustain C18 column (4.6 mm × 250 mm, 5 µm, SHIMADZU, Tokyo, Japan) with the flow rate kept at 1.0 mL/min, the column temperature maintained at 40 °C and the mobile phase consisting of methanol: water solution (25:75, v/v) with 1.0% formic acid and 0.3% triethylamine in water. The run duration was fifteen minutes, and the retention time of the Str was 10.0 min. The injection volume for each run was 50 μL, and the detection wavelength was 254 nm. The linear equation of the Str was Y = 114,005X − 2340.1 (r2 = 0.999) at concentrations ranging from 0.0428 μg/mL to 10.7 μg/mL. The LOQ and LOD of the Str were 0.0428 μg/mL and 0.00856 μg/mL, respectively.
10
JWYY Herbal Formula Composition Analysis
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30 g JWYY formula (Table 1 ) was prepared by mixing the powder of CH (20.85 g), BD (20.85 g), BT(20.85 g), PP(20.85 g), CWT(20.85 g), SK(6.25 g), GL(6.25 g), and pearl powder (6.25 g). Briefly, CH, BD, BT, PP, CWT, and GL were boiled and concentrated, and the SK and pearl powder were added. To validate the components in the formula, a connected system of LC-30 (Shimadzu)-hybrid quadruple time-of-flight mass spectrometer (TOF-MS) with electrospray ionization source (ESI) was used. InertSustain C18 column (Shimadzu, 100 × 2.1 mm, 2 μm) was used to perform chromatographic separation with a flow rate of 0.3 ml/min at 35°C. Mobile phase system was composed of equate A (acetonitrile) and equate B (0.1% HCOOH-H2O): 4 min (A: 5% : B: 95%), 8 min (A: 20% : B: 80%), 2 min (A: 15% : B: 75%), 2 min (A: 46% : B: 54%), 3 min (A: 100% : B: 0%), and 1 min (A: 5% : B: 95%). Data were acquired in information-dependent acquisition (IDA) with high sensitivity mode, collision energy was 35 ± 15 eV, and declustering potential was ±60 V (Supplementary Table 1 ) (Supplementary Figure 1 ).
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