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The independent input variables were: composition of mobile phase, including its pH, and column temperature.
The optimum composition of mobile phase for the separation of the eight isoflavones was obtained.
For the method, robustness was studied by changing the composition of mobile phase, flow rate, and detection wavelength.
Hence, it can be concluded that the composition of mobile phase (M3) remains unaltered for several hours, and therefore, it is most suitable for chromatographic analysis.
Robustness of the proposed method was determined at single concentration level (100 μg/mL) in three different ways, i.e., by changing the composition of mobile phase, making deliberate change in the flow rate, and changing the detecting wavelength.
Table 1 The composition details of mobile phases Code Composition of mobile phase M1-M5 10, 5, 2, 1 and 0.1% (w/v) aqueous 1-methyl-imidazolium chloride respectively M6-M10 10, 5, 2, 1 and 0.1% (w/v) aqueous 1,2,3-trimethylimidazolium-methyl sulphate, respectively M11-M15 10, 5, 2, 1 and 0.1% (w/v) aqueous 1-ethyl 3-methyl-imidazolium tetrafluoroborate, respectively.
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Different compositions of mobile phase and resulting Rf values of standards are summarized in (see Additional file 1: Table S1).
No obvious effects on the chromatographic parameters were observed in all of the deliberately varied chromatographic conditions (different flow rates, compositions of mobile phase and buffer pH).
Robustness of the method was achieved by introducing small changes in the compositions of mobile phase and detection wavelength.
Some data of composition optimization of mobile phases were shown in Table 1, in which a Diamonsil C18 column was used.
The method was performed with small variations such as a composition of the mobile phase (±2% of IPA), a change to the mobile phase flow rate (±0.2 mL/min), and increasing the temperature from normal (±5 °C).
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