In this work the retention factor was calculated according to equation (1). (1) k = ( t R − t 0 ) / t 0where t₀ is the hold-up time, t_R is the retention time, and k is retention factor. The resolution (R) is calculated using the eq. (2). (2) R = 2 ( t R 2 − t R 1 ) / ( w 1 + w 2 )where t_R1 and t_R2 are the retention times of the first and second peak, respectively, and w₁ and w₂ are the peak widths at the baselines of the first and second peaks, respectively. (3) Related   erro = ( 1 − k a / k a v e ) × 100 %where k_a is the retention factor for each target compound and k_ave is the average retention factor for each target compound with several injections.

The acidity or basicity of the mobile phase can have a significant influence on separation. Acetic acid and triethylamine, as two traditional mobile phase additives in reverse phase HPLC, were employed to establish acidic or alkaline conditions, respectively. Chlorogenic acid and caffeic acid are organic acids which contain hydroxyl and carboxyl groups. Thus, acetic acid had more efficiency in increasing the resolution of these two compounds than triethylamine (Table 1). Furthermore, the interactions between target compounds and the stationary phase were reduced with increasing concentrations of acetic acid in the mobile phase. In this case, the resolution of the four target compounds was decreased (Figure 3). Moreover it should be noted that high concentrations of acid in the mobile phase may be harmful for the HPLC system. Therefore, a slightly acidic mobile phase was found to be the most suitable for the separation of the four compounds (Figure 4). The presence of a shoulder on the chromatographic peaks can be explained by the difference between the sample and the mobile phase solvents.

Next, the effect of inorganic salts was evaluated. When Na₂HPO₄ was added, the mobile phase showed basicity and the target compounds could not be separated (Table 1). The retention factors of the four target compounds increased with increasing concentrations of NaH₂PO₄ until 0.02 mol/L, after, which the retention factors decreased. This result reveals that at a low concentration of NaH₂PO₄ in mobile phase, ion exchange interaction between cations and anions play a major role in aqueous solution. With this interaction between target compounds and the surface of the stationary phase, the retention time was increased. When more NaH₂PO₄ was added, the acidity of the mobile phase was increased because of the high concentration of hydrogen ion. At this point, the interaction between target compounds and the stationary phase was decreased and the retention times of target compounds were reduced. The resolution of the four compounds via NaH₂PO₄ as the additive is shown in Figure 5. The figure shows that 0.02 mol/L was the optimum concentration (Figure 6).

As mobile phase additives, organic solvents have animal toxicity; additionally, inorganic buffers are potentially harmful for the HPLC system and the reverse-phase column. In this case, ionic liquid, which has organic cations and anions, was used.

Standards of chlorogenic acid, caffeic acid, scoparone, and rutin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol, triethylamine, Na₂HPO₄, NaH₂PO₄, and acetic acid were obtained from the Duksan Pure Chemical Co., LTD (Ansan, Korea). Ionic liquids were offered by the Chem Tech Research Incorparation (Hwaseong, Korea). All the other reagents used in the experiment were HPLC or analytical grade. Double distilled water was filtered with a vacuum pump (Division of Millipore, Waters, U.S.A.) and filter (HA-0.45, Division of Millipore, Waters, U.S.A.) before use.

Acetic acid, triethylamine, inorganic salt and ionic liquids were added into the methanol/water (40:60, v/v) solution directly. The molar concentrations were adjusted from 0.01 mol/L to 0.03 mol/L. All the mobile phase solvents were mixed completely for further experiments. Stock solutions of chlorogenic acid, caffeic acid, scoparone, and rutin were dissolved in methanol to make a final concentration of 25.0 μg/mL. It should be emphasized that the working solutions were re-prepared every 3 days so as to avoid potential errors arising from decomposition.

Chromatography was performed with a Waters 600s multisolvent delivery system, a Waters 616 liquid chromatography, and a Waters 2487 variable wavelength, dual-channel UV detector (Waters Associates, Milford, MA, USA). A six-port Rheodyne injector (20-μL sample loop) was also used. Data processing was performed with Millennium 3.2 software resident in an HP Vectra 500PC. Compounds were separated on a 150 mm × 4.6 mm, 5-μm particle, OptimaPak C₁₈ column (RStech, Daejeon, Korea). The flow rate was set at 0.5 mL/min. The wavelength was fixed at 325 nm. An injection volume of 10 μL was applied throughout the experiments. All procedures were carried out at ambient temperature.

Individual compounds and mixtures of standard compounds were injected 5 times to confirm the retention times and scientific replications.