Gradient HPLC of samples extracted from the green microalga Botryococcus braunii using highly efficient columns packed with 2.6 μm Kinetex-C18 core–shell particles

The analysis of the nonpolar extract of the cells of colonies of the green colonial microalgae Botryococcus braunii was performed by gradient HPLC. The growth of B. braunii was stressed by reducing its nitrogen nutrients by 90%, in order to enhance the production of nonpolar compounds. Highly efficient 4.6 mm × 100 mm columns packed with 2.6 μm Kinetex-C18 core–shell particles (Phenomenex, Torrance, CA, USA) were used. The gradient mobile phase was a mixture of acetonitrile and water (70–97%, v/v). Its initial and final compositions during the gradient elution were chosen so that the retention factors of the last eluted compound at the inlet and outlet of the column were 15 and 1, respectively. The highest peak capacity was obtained by optimizing several experimental parameters, including the injected sample volume, the flow rate, and the column length. The highest resolution was obtained by connecting one 4.6 mm × 150 mm and three 4.6 mm × 100 mm columns (total length 45 cm). The optimum flow rate was 1.5 mL/min, which provided the minimum plate height for the most retained compounds, the optimum inlet pressure was 930 bar and the injected volume 2 μL. The analysis time was then 14 min for a peak capacity of 121. The trends observed for the variation of the experimental peak capacity with the flow rate and the column length are in excellent agreement with theoretical predictions.