The method described by Gu et al. (2016) [87 (link)] was used to detect the levels of 5-HT, 5-HIAA and QA in the LSSC of control, diabetic, and diabetic + 8-OH-DPAT-treated rats. Briefly, on the day of the experiment, stock solutions (10 µg/mL) of 5-HT, 5-HIAA and QA were prepared in 0.2 M perchloric acid solution and stored on ice in the dark. The neuronal tissues dissected from rats were weighed and homogenized in ice-cold 0.2 M perchloric acid solution (10 µL/mg tissue). After centrifugation at 12,000× g at 4 °C for 20 min, supernatants were collected for chromatographic separation. HPLC Equipment Waters 2535 Quaternary Gradient Module with a 15 uL fixed injection loop was used. The PDA detector used was a Waters 2998 Photodiode array controlled by Empower Software 3 for data acquisition and analysis. Chromatographic separation was performed using XBridge C18 5 um, 4.6 × 100 mm column (Waters XBridge, Wexford, Ireland). An amount of 5 mM perchloric acid solution containing 5% acetonitrile was used as a mobile phase, and the separation temperature was set at 12 °C with a flow rate adjusted to 2 mL/min [87 (link)].
Waters 2535 quaternary gradient module
Manufactured by Waters Corporation
Sourced in United States
The Waters 2535 Quaternary Gradient Module is a laboratory equipment designed to precisely control and deliver multiple solvents for use in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) systems. It features four independent solvent channels and can create accurate, reproducible gradient elution profiles to facilitate effective separation and analysis of complex samples.
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2 protocols using waters 2535 quaternary gradient module
1
HPLC Detection of Neurotransmitters in Diabetic Rats
2
Isolation and Purification of Compound 10
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For structural elucidations, compound 10 was isolated and purified from a crude H. rhamnoides leaf extract. 9.46 grams of the extract was dissolved in 40 ml of water, centrifuged and the supernatant was applied onto a column (Chromaflex, 320 × 55 mm; Kimble-Chase Kontes, Vineland, NJ, USA) packed with Sephadex LH-20 gel equilibrated in water. Fractionation was performed with 10-50% aqueous methanol and 20-80% aqueous acetone with compound 10 eluting using 40-50% methanol. Methanol was evaporated from the main fractions containing compound 10 followed by their lyophilization, yielding 407 mg of fractions with compound 10. The purification was completed with reversed-phase highperformance liquid chromatograph (consisting of a Waters 2535 Quaternary Gradient Module, Waters 2998 Photodiode Array Detector, and a Waters Fraction Collector III; Waters Corporation, Milford, USA) equipped with a Gemini 10µ C18 110 Å (150 × 21.2 mm i.d., 10 µm, Phenomenex, Torrance, CA, USA) column using a flow rate of 8 ml min -1 and a gradient elution with acetonitrile and 0.1% aqueous formic acid as eluents. The total yield of purified compound 10 was 9.6 mg.
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