To resolve coelution phenomenon of acetone and acrolein in the HPLC‐based analysis of carbonyls, we attempted to find the optimal conditions for their separation. For the purpose of this study, the collection of carbonyl Compounds is made by DNPH‐coated cartridges. Quantification of carbonyls is then initiated by the formation of hydrazone derivatives that are then separated by HPLC and detected by UV‐VIS spectroscopic detector (at 360 nm). To the course of this experiment, we examined the influences of the three major experimental variables such as 5 temperature (25, 30, 40, 50 and 60℃), 2 flow rate (1.0 and 1.2 mL min‐1), and variable relative composition of mobile phase (among acetonitrile, water, and tetrahydrofuran). According to our experiments, the optimal condition for separation was found at flow rate of 1.2 mL min‐1 and temperature of 32℃ with the mobile phase composition of acetonitrile:water:tetrahydrofuran = 34 : 52.8 : 13.2.