process. Also, increasing the flow rate from 1 mL/min to
1.5 mL/min showed a similar decrease in the retention time. Sufficient flow rate of 1.3 mL/min was chosen to avoid overlap between peaks and the loss of its acceptable resolu- tion values (Fig. 4).
3.1.4. Ratios of the mobile phase components
We examined the effect of a gradual increase in acetonitrile from 10% to 25%, which led to a decrease in the total time required for the separation to 9 min (Fig. 5).
In order to improve the shapes of the peaks and to improve the separation between Bromazepam and Midazolam, we improved the previous experimental conditions and started to change the mobile phase pH using an ammonia solution (35%, w/w).