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The independent input variables were: composition of mobile phase, including its pH, and column temperature.
The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time.
The total volume of the consumed mobile phase including column conditioning in the SIC method was 4.0 mL.
This drawback causes the problem of solution leakage especially when applying high flow rates, adopting a mobile phase including a buffer or installing long monolithic columns.
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Different mobile phases, including acetonitrile with 0.05%, 0.1% aqueous formic acid, acetic acid, 5 mM and 10 mM ammonium formate solutions were tested.
The injected samples were eluted by mobile phases including: 90% of solvent A and 10% of solvent B [namely methanol (pre-dried over 3 Å molecular sieves) and 0.75% of acetic acid in distilled water respectively] at a flow rate of 1 mL/min for 16 min [41].
In this system two mobile phases including: (A) water and (B) acetonitril (CAN) were used.
The LC mobile phases included H2O containing 0.1‰ formic acid for solvent A, and methanol containing 0.1‰ formic acid for solvent B. As shown in Figure 1, a specially designed LC binary gradient program was used to separate the phytochemicals and the effluent was delivered at 0.3 mL/min throughout the gradient program.
To obtain sharp and symmetrical peaks, different mobile phase systems, including methanol water, acetonitrile water, acetonitrile water (containing 0.5% formic acid, v/v) and acetonitrile water (containing 0.1% phosphoric acid, v/v) were tested.
The new SV solved the problem of solution leakage especially when applying a high flow rate for propelling a mobile phase that includes a buffer.
For the separation of compounds 1– 5, columns types (Phenomenex Gemini C18, Waters SunFire C18, and OptimaPak C18 column), column temperatures (30, 35, and 40°C), and various mobile phases (acids including acetic acid and phosphoric acid and buffers such as SDS and ammonium acetate, and organic solvent with methanol and acetonitrile) were examined.
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