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Active RP fractions were further fractionated by normal-phase (NP) LC into eight fractions with increasing polarity.
The separated fractions were further fractionated in the 2nd dimension by SDS-PAGE.
Active fractions were further fractionated and assayed.
These fractions were further fractionated on Diaion HP-20 absorbent resin column (2.5 × 20 cm).
The oligosaccharides extracted from the initial hydrolysate (8.5 g) by charcoal fractionation were further fractionated by SEC chromatography.
The water-soluble fractions of IYD extracts were further fractionated by ultrafiltration to characterize the nature of the viability-enhancing compounds present.
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The methylene chloride extract (Fraction C) was further fractionated into four fractions (C1 C4) of different Rf values using preparative thin layer chromatography (TLC) plates (0.2 mm, POLYGRAM® SIL G/UV254, Macherey-Nagel GmbH & Co, Duren, Germany) and a solvent system: acetone:ethyl acetate = 1 4 (v/v).
More specifically, the initial aqueous fraction was further fractionated in aqueous and methanolic fractions.
The water soluble fraction was further fractionated into chloroform, ethyl acetate and n-butanol soluble fraction.
The candidate fraction (fraction 4) was further fractionated by the ODS column (NX-ODS-9-120A 20 mm i.d. × 250 mm; NX-ODS-9-120A 20with NX-ODS-9-120A 20nt commri.dng MeOH/acetonitrile/acetic acid/H2O (4:3:1:92).
When the CDEb fraction was further fractionated by quick column silica gel 60 chromatography, the seven obtained fractions (CDEb1-CDEb7) all provided a lower free radical scavenging activity than the starting CDEb material at each concentration, with the EC50 values of CDEb1-CDEb7 being much higher (> 100 μg/ml) than that for CDEb (7.47 ± 0.12 μg/ml).
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