A New Segmented Gradient HPLC-DAD and Spectra Index Plots for Measuring Caffeine and Chlorogenic Acid in Dark and...

A rapid and sensitive segmented gradient elution high-performance liquid chromatographic methodology with a diode array detector (SGE-HPLC-DAD) has been developed for quantifying important bioactive compounds, chlorogenic acid (ChGA) and caffeine (CAFF), in different coffee samples. A Luna cyano-column (5 μm, 25 × 0.46 cm) conditioned at ambient temperature (22 ± 1 °C) and a gradient elution system consisting of solvents A (1% trifluoroacetic acid in water) and B (acetonitrile) were employed. The elution process used a segmented gradient program, starting with a gradual increase in solvent B from 5% to 8% over the first 4 min. This was followed by a rapid ramp-up from 8% to 100% over 1 min (4-5 min) and then an isocratic elution at 100% B lasting 5-7 min. Thereafter, there was a linear decrease from 100% to 5% B over the next minute 7-8 min and finally an isocratic elution by a mixture of A and B (95:5, v/v) until 11 min. The flow rate was maintained at 1.5 mL/min with effluent detection at 254 nm. The SGE-HPLC-DAD method revealed linear relationships between peak areas and concentrations, with CAFF ranging from 0.4 to 1000 μg/mL and ChGA ranging from 0.6 to 1000 μg/mL. The recovery percentages for CAFF and ChGA were estimated to be between 100.97% and 101.33%, respectively. The relationship between ChGA and CAFF contents in dark and green coffees commercially available in the Egyptian market that were brewed using stainless-steel and glass cookware materials on direct heat was also investigated.