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Modifications in mobile phase concentration, mobile phase volume, mobile phase saturation time, and temperature were examined to check the robustness.
Variations made in mobile phase composition, mobile phase volume, saturation time, and temperature showed less %RSD and SD for FA and vanillin by spotting 150 ng/spot (n = 3) (Table 4).
By employing modified silica gel column chromatography to obtain the squalene-rich fraction, the mobile phase volume and elution time required as fractions of those needed in classical silica gel column chromatography are 1/73 and 1/18, respectively.
To obtain the FASEs-rich fraction, the corresponding mobile phase volume and elution time are 1/221 and 1/23, respectively of those needed in classical silica gel column chromatography.
In order to check the robustness, following parameters were deliberately changed at three different concentration levels (300, 500 and 800 ng of each standard), scanning wavelength (λmax ± 2nm), mobile phase volume (15 ± 2 mL), and time variation (30 minutes) before chromatographic process, were studied and effects on the results were examined.
The standard deviation of % yield of three standard levels 300, 500 and 800 was estimated for each parameter, mean R.S.D. % was 1.16 (TP), 1.78 (TPP), 1.32 (TI) and 1.18 (TD) for varying mobile phase volume, 2.12 (TP), 1.37 (TPP), 1.15 (TI) and 2.10 (TD) for the effect of varying wavelength, and 2.21 (TP), 1.96 (TPP), 2.27 (TI) and 1.85 (TD) for varying time from chromatography to scanning.
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However, methylene chloride-acetone mobile phase in volume composition 1 : 2 and methylene chloride-methanol in volume composition 5 : 1 were applied for the separation of ecdysones.
Parameters including flow rate, mobile phase composition (volume fraction of ACN in water), temperature, response time, sampling frequency, and injection volume for quantitative analysis of OTA in red wines were investigated in detail.
Then, 4.0 ml of this solution was transferred to a second 25-ml volumetric flask and diluted with mobile phase to volume.
In the intervals of 5, 10, 20, 30, 60, 120 min, 2.0 ml aliquot samples was taken and transferred to a 25-ml volumetric flask and then diluted with mobile phase to volume and filtered through 0.45 μm syringe filter.
The optimization of the mobile phase composition (volume fractions of MeOH, ACN, and THF) for a mixture of nine benzodiazepines is achieved using a classical mixture design (Fig. 2).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com