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Reverse phase hplc system

Manufactured by Shimadzu
Sourced in Japan

The Shimadzu Reverse-phase HPLC system is a high-performance liquid chromatography instrument designed for the separation and analysis of a wide range of compounds. It utilizes a reverse-phase chromatographic stationary phase to separate analytes based on their hydrophobic interactions. The system is equipped with essential components such as a solvent delivery unit, an autosampler, a column oven, and a suitable detector for accurate quantification and identification of the separated compounds.

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10 protocols using reverse phase hplc system

1

Licofelone Modulates Prostaglandin Synthesis

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Frozen pancreatic tumor tissues from male mice fed 250 or 500 ppm of licofelone or control diet were homogenized using ice-cold homogenizing buffer. For COX-1 and COX-2 assays, 150 μL of reaction mixture containing 12 μmol/L [14C] arachidonic acid (AA; 420, 000 dpm), 1 mmol/L epinephrine, 1 mmol/L glutathione in 50 mmol/L phosphate buffer (pH 7.4) were incubated with 30 mg of lysate protein at 37°C for 20 minutes. The reactions were terminated by adding 40 μL of 0.2mol/L HCl. The COX-mediated metabolites of AA were extracted with ethyl acetate (3 × 0.5 mL). The combined extracts were evaporated to dryness under N2, dissolved in 1 mL of acetonitrile and 10 μL were injected into a reverse phase HPLC system (Shimadzu Scientific Instruments, USA) equipped with a Phenomenix C18 column (300 × 3.90 mm; pore size 10 μ). The [14C]-PGs, [14C]-TxB2 and [14C]-PGE2 were eluted with a gradient solvent system containing solvent A: Acetonitrile:Water:Acetic acid (35:65:0.1%) and solvent B: Acetonitrile:Water:acetic acid (65:30:0.1%). The eluted metabolites were monitored and quantified with an IN/US Systems β-RAM radio HPLC detector.
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2

Characterization of Novel Organic Compounds

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All the reagents and dried organic solvents were obtained from Sigma Aldrich (St. Louis, MO, USA.), TCI Chemicals (Tokyo, Japan), and Merck (Darmstadt, Germany). 1H and 13C NMR spectra were acquired with a Jeol NMR spectrophotometer USA at 400 and 100 MHz, respectively. Chemical shifts are shown in parts per million (ppm) proportional to tetramethylsilane (TMS). The coupling constants (J) are presented in hertz (Hz), and the splitting patterns are designated by using abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet), brs (broad singlet), and dd (double doublet). LC/MS data were recorded with an Agilent 6310 Ion trap LC/MS system and elemental analysis (C, H and N) was carried out on Elementar analysensysteme. Melting points were taken in open capillaries by using KSPII melting point apparatus (KRUSS, Germany). Purity of all target compounds were analysed by using Shimadzu reverse phase HPLC system (Kyoto, Japan) coupled with a photodiode array detector (PDA) and C-18 column. Acetonitrile and methanol (80 : 20) were used as a mobile phase, keeping flow rate 1 ml min−1. All target compounds displayed >97% purity.
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3

Validated HPLC Assay for Roflumilast

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Roflumilast was analyzed using a validated reverse-phase HPLC method modified from Belal et al. [19 (link)]. A reverse-phase HPLC system (Shimadzu, Kyoto, Japan) equipped with an LC-20AD solvent delivery unit, Prominence photodiode array (PDA) detector (Shimadzu SPD-M20A), degasser (Shimadzu DGU-20A5), and autosampler operated with Class-VP 7.4SP4 software was used. The mobile phase, ammonium acetate buffer (pH 6.3), acetonitrile, and methanol (30:35:35% v/v) was pumped at a 1.0 mL/min flow rate through a Synergi Fusion RP80A C18 column (4 µm, 150 × 4.6 mm; Phenomenex, Torrance, CA, USA) connected to a C18 security guard (Phenomenex Fusion RP, CA, USA; 4.0 × 3.0 mm). An aliquot (20 µL) of each sample was injected at ambient temperature and detected at a wavelength of 251 nm with a total run time of 8 min. The calibration curve (0.25–80 µg/mL) was linear (R2 = 0.9999) with the limit of detection (LOD) and limit of quantitation (LOQ) of 0.04 and 0.11 µg/mL, respectively. The repeatability and reproducibility of the separately prepared quality control samples (10, 50, and 100 µg/mL) were within the acceptable limit (%Bias: ≤15; coefficient variation, %CV: ≤15).
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4

Quantifying Curcumin Encapsulation in Nanoliposomes

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The preservation of curcumin incorporated in the nanoliposomes was determined by centrifugation of nanoliposomes at 10,000× g for 10 min and the supernatant was assayed by HPLC at 425 nm to follow curcumin and 280 nm to follow its degradation products, by dissolving in methanol. Curcumin concentration was determined via a reverse-phase HPLC system (Shimadzu, Kyoto, Japan), that is equipped with an auto-injector, a quaternary pump, a UV-Vis photodiode array detector, a Zorbex SB-C18 column, and the LabSolutions data software.
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5

Quantification of Acetate in Bacterial Cultures

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For quantification of acetate in the culture, a part of the culture was taken and analyzed using a reverse-phase HPLC system (Shimadzu, Kyoto, Japan) equipped with L-column2 ODS (4.6 × 250 mm) (CERI, Tokyo, Japan) and a UV detector as described previously46. Direct total cell counts in the enrichment cultures, RW, PS, and WC were determined by epifluorescence microscopy with SYBR GREEN II staining47. To monitor cell growth in a pure culture of strain R4, real-time qPCR was conducted by using a universal primer set for the bacterial 16S rRNA genes, 357f and 517r (supplementary Table S2). The qPCR was performed with a LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Indianapolis, IN) and the LightCycler Nano system (Roche Applied Science, Indianapolis, IN) as described previously45. As the standard, the purified 16S rRNA gene amplicon from genomic DNA of strain R4 was used.
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6

HPLC Fingerprinting of YK Samples

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The fingerprints of YK samples were monitored on a Shimadzu reverse-phase HPLC system (C18 column - 250 mm, 4.6 mm) with SCl-10AVp system controller and SPD-10AVvp UV–vis detector. The mobile phase (acetonitrile and water) was degassed and filtered through 0.2 μm membrane filter before pumping into the HPLC system. A linear gradient of acetonitrile from 5% to 95% over 55 min at a flow rate of 1 ml/min was maintained and the samples were monitored at 220 and 280 nm using Photo Diode Array (PDA) detector. The YK samples (20 μg in 200 μl of glass distilled water) were used for injection.
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7

Synthesis of Nucleoside Analogues and RNA Conjugates

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Reactions were carried out under argon atmosphere and performed using freshly distilled solvents. Dimethyl formamide (DMF) and MeOH were dried over calcium hydride. Phosphoramidite nucleosides, tetrazole and solvents for dinucleotides synthesis were purchased from Eurogentec. Progress of the reactions was monitored by thin layer chromatography (TLC). TLC: precoated silica gel thin layer sheets 60 F254 (Merck, Darmstadt, Germany) and detection by charring with 10% H2SO4 in ethanol followed by heating.
Preparative HPLC were performed using a Reverse-phase HPLC system (Shimadzu, Marne-la-Vallée, France) with a reverse phase C-18 NUCLEOSIL column (250 mm × 21.2 mm, 5 μm) using a solvent system consisting of A: 50 mM aqueous NH4OAc pH 4.5 and B: MeCN (linear gradient from 0% B to 63% B in 30 min) at a flow rate of 15 ml/min and UV detection at 254 nm.
The NMR characterizations for all synthesized compounds are presented in the Supplementary data and the NMR spectra are provided in Supplementary Figures S1–S29. The reverse-phase HPLC chromatograms of the final compounds (A*A, GA*A, GA* and the 13mer RNA–SAM conjugate) are shown in Supplementary Figures S30–S36.
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8

Reverse Phase HPLC Purification

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All the compounds were purified using a reverse phase HPLC system (Shimadzu, Japan) using the phenomenex C18 column (dimension 250×10mm) by linear gradient elution technique using a dual solvent system (water and methanol, 0-80% CH3OH in 40 min).
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9

Quantification of Curcumin Encapsulation in Nanoliposomes

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The percentage of drug incorporated was determined by centrifuging the drug-loaded nanoliposomes at 9000× g for 15 min to separate the unloaded curcumin crystals from the liposome. After centrifugation, an aliquot of liposomal dispersion was dissolved in 2 mL of methanol and the concentration of curcumin was assayed by HPLC at 425 nm after filtration through a membrane filter (0.2 µm) (Minisart® RC15 Syringe Filters, Sartorius, Germany). The concentration of curcumin was determined by a reverse-phase HPLC system (Shimadzu, Kyoto, Japan) equipped with a quaternary pump (LC-20AD), an auto-injector (SIL-20AC HT), a UV-Vis photodiode array detector (UV-Vis PDA, SPD-M20A, Shimadzu, Marne-la-Vallée, France), a Zorbex SB-C18 column (5 µm, 4.6 mm × 250 mm) and Labsolution data software (Shimadzu, Marne-la-Vallée, France). Suspension was analyzed in isocratic mode using methanol (v/v, 5%), acetic acid 2% (v/v, 30%) and acetonitrile (v/v, 65%) at a flow rate of 0.5 mL·min−1. Aliquot (20 µL) was injected onto an AlltimaTM [HP C18, 5 µm (250 × 4.6 mm i.d.) column (GRACE, Deerfield, IL, USA)] at 25 °C. Detection of curcumin was performed at 425 nm after 8 min and 49 s. The experiments were performed in triplicate.
The encapsulation efficiency (EE) was calculated as: EE(%)=Initial drug (g)Free drug(g)Initial drug×100.
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10

Fmoc-based Peptide Synthesis and Purification

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Peptides (Table 1) are synthesized in Solid phase Peptide synthesizer (Aapptec Endeavor 90) using the principles of Fmoc chemistry. Fmoc-protected amino acids are sequentially coupled followed by fmoc deprotection using 20% piperidine solution for 60 and 40 min, respectively. DIPEA and PyBOP are used as activator base and activator respectively. After washing in DMF, peptide-attached resin is cleaved by standard resin cleavage cocktail solution containing 92.5% TFA, 2.5% milliQ water, 2.5% TIS, and 2.5% phenol. The cleaved filtrates are precipitated using diethyl ether solvent and purified using reverse phase HPLC system (SHIMADZU, Japan) with Phenomenix C18 column. Peptide masses are determined by MALDI-TOF mass spectrometry (Bruker). (Supplementary information Section 5).
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