Analytical hplc

Manufactured by Shimadzu

Sourced in Japan

Analytical HPLC is a high-performance liquid chromatography system used for the separation, identification, and quantification of compounds in complex mixtures. It employs a stationary phase and a mobile phase to separate analytes based on their physical and chemical properties.

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5 protocols using analytical hplc

Detailed Analytical Methods for Novel Compounds

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DMEM, FBS, L-glutamine, penicillin-streptomycin and SYBR Gold (molecular biology grade) were procured from Invitrogen. Caspase-3 assay kit was procured from Thermo Scientific. All single stranded oligonucleotide sequences (Table S1) and the primers for RT-PCR (Table S2) were purchased from Integrated DNA technologies (IDT). DMSO, MTT, Hoechst 33258 and DAPI were obtained from Sigma Aldrich (Merck). All other chemicals were of analytical reagent grade and used without further purification unless otherwise stated. Ultrapure water (double-distilled) obtained from Milli-Q Gradient ultrapure water system (Millipore) and was used in all experiments. ¹H and ¹³C NMR spectra were recorded on Bruker AV-400 MHz spectrometer with chemical shifts reported as parts per million (ppm) (in DMSO-d6, tetramethylsilane as an internal standard) at 20 °C. UV-vis absorption and emission spectra were measured in quartz cuvettes of 1 cm path length. HRMS were obtained on Agilent Technologies 6538 UHD Accurate-Mass Q-TOF LC/MS spectrometer. HPLC traces were obtained from Shimadzu analytical HPLC.

Suseela Y.V., Satha P., Murugan N.A, & Govindaraju T. (2020). Recognition of G-quadruplex topology through hybrid binding with implications in cancer theranostics. Theranostics, 10(23), 10394-10414.

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Quantifying Peptide Release from Hydrogels

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HA-SH and HA-ASE solutions were mixed and aliquoted into 12-mm cell culture inserts. Five minutes later, RGDSP-MI and AG73-MI (100 μL, 0.5 mM each) was added on top of the gel mixture and incubated for 30 min. The supernatant was aspirated and the hydrogel was washed with PBS (100 μL) three times. The supernatant and the wash solutions were pooled and lyophilized for the respective hydrogel. The dry product was dissolved in 100 μL deionized water and ran on the Shimadzu analytical HPLC by monitoring peptide elution at 220 nm. Pure peptides of known concentrations were analyzed similarly, and standard curves were constructed based on peak integration at each concentration. Peptide concentration in the combined wash solutions was determined using the standard curve.

Ravikrishnan A., Fowler E.W., Stuffer A.J, & Jia X. (2021). Hydrogel-Supported, Engineered Model of Vocal Fold Epithelium. ACS biomaterials science & engineering, 7(9), 4305-4317.

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HPLC Analysis of Alkaloid Extract

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The identification of DA was done according to Fico and Tomé (1998 ). The alkaloid extract was dissolved in acetonitrile and the analysis was performed using Shimadzu Analytical HPLC (Shimadzu, Japan) equipped with RP-C 18 and with an autosampler and UV-Vis detector column. MilliQ: CH₃CN (3:7) (pH 8 with diethylamine) was used as eluent in isocratic conditions. The alkaloid extract was monitored at 230 nm.

Bouguezza Y., Khettal B., Tir L, & Boudrioua S. (2015). Damascenine induced hepatotoxicity and nephrotoxicity in mice and in vitro assessed human erythrocyte toxicity. Interdisciplinary Toxicology, 8(3), 118-124.

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HPLC Analysis of Vitamin K1 in Formulations

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A High-Performance Liquid Chromatography (HPLC)-based analysis method, based on USP recommended assay, was used to quantify vitamin K1 in the formulations, and the extracts from receiving phase used for in vitro and ex-vivo experiments. Vitamin K1 was extracted using hexanes:1-penthanol 199:1 mixture. Internal standard (cholesteryl benzoate; 10 μg/mL) was added to the sample and the mixture of sample and extraction mixture were vortexed, and centrifuged at 12,000 g for 5 minutes. The hexanes:1-pentanol portion was then collected for analysis. The HPLC system consisted of an Ascentis Si 5um L3 (25cm x 4.6mm) column connected to a Prominance-i Shimadzu Analytical HPLC. Then 15 μL of extraction was injected onto the column and eluted at a flow rate of 1.0mL/min room temperature under isocratic conditions with hexanes:1-pentanol as the mobile phase. Vitamin k1 (retention time ≈ 8.2 minutes) and internal standard (retention time ≈ 3 minutes) were analyzed at 254nm. Standard curves were created in concentration range of 10 ng/mL—10 μg/mL, based on the ratio of the area under the curves (AUC; Vitamin K1 AUC / Internal Standard AUC) vs. concentration. A sample peak for the internal standard and different vitamin K1 concentrations is presented in S1 Fig. See the S1 File for the validation of the analytical method.

Nabiee R., Dubois B., Green L., Sharma A., Wong S.F, & Montazeri Aliabadi H. (2018). In vitro and ex-vivo evaluation of topical formulations designed to minimize transdermal absorption of Vitamin K1. PLoS ONE, 13(10), e0204531.

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Synthesis and Purification of Fluorescent Peptide Aptamers

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All the peptide sequences were synthesised according to the principles of solid-phase peptide synthesis (SPPS). HPLC grade fluorenyl-methyloxy-carbonyl chloride (Fmoc-Cl) protected amino acids and Wang resins were supplied by Novabiochem®. Analytical grade dimethyl-formamide (DMF), dichloromethane (DCM), and acetonitrile were supplied by Fisher Scientific. Analytical grade piperidine, tri-isopropyl-silane, tri-fluoro-acetic acid (TFA), ninhydrin, 5(6)-carboxy-fluorescein dye (5-FAM) and other reagents used in the synthesis of peptides were supplied by Sigma Aldrich. The different sequences that were synthesized and labelled with 5-FAM are presented in Table 1 together with their isoelectric points that were calculated using the PepCalc online calculator [35] .
The purification of the sequences was performed using a preparative high performance liquid chromatographer (Dionex, USA) with a stationary phase of C18-bonded silica and a mobile phase gradient of 0.1 vol% TFA in water and 0.1 vol % TFA in acetonitrile. The peptide aptamer sequences were analysed under the same mobile and stationary phase of the purification technique in an analytical HPLC Shimadzu. The sequence compositions were verified by electrospray mass spectrometry on a Micromass LCT (Waters, UK).

Rodriguez G.M., Bowen J., Grossin D., Ben-Nissan B, & Stamboulis A. (2017). Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides. Colloids and surfaces. B, Biointerfaces, 160.

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