Accuracy and precision of adsorption isotherm parameters measured by dynamic HPLC methods

The fluctuations of the column temperature, the composition and the flow rate of the mobile phase affect the accuracy and precision of the adsorption isotherm parameters measured by dynamic HPLC methods. Experimental data were acquired by frontal analysis (FA) for phenol in equilibrium between C18-bonded Symmetry and a methanol:water mixture (20:80, v/v), at 303 K and a flow rate of 1 mL/min. The fluctuations of the experimental parameters were 0.1 K for the temperature, 0.1% for the mobile phase composition and 0.001 mL/min for the flow rate. The best isotherm model was shown to be the tri-Langmuir isotherm. Random errors were calculated and shown to agree with experimental results. Overloaded band profiles of phenol were acquired at low (sample size, 100 μL, concentration 3 g/L) and high (same sample size, concentration 60 g/L) loadings, at seven temperatures (298, 300, 302, 303, 304, 306, and 308 K), for seven mobile phase compositions (methanol 16, 18, 19, 20, 21, 22, and 24%), and with seven mobile phase flow rates (0.95, 0.97, 0.99, 1.00, 1.01, 1.03, and 1.05 mL/min), always keeping two experimental parameters at the values selected for the FA runs. Assuming that the isotherm model stays the same, the inverse method (IM) was used to derive the isotherm parameters in each case. Temperature affects the equilibrium constants according to Van’t Hoff law. A temperature change of 1 K around 303 K causes a relative variation of 1.5% of the high-energy adsorption constant b3 and of 0.6% of the saturation capacity q3. The isotherm parameters are very sensitive to the mobile phase composition, especially the highest energy mode. Both adsorption constants b2 and b3 follow the linear strength solvent model (LSSM). A methanol volume fraction change of 1% causes a relative decrease of 3.2 and 5.0% of b2 and b3, respectively and a 2% decrease of the saturation capacity q3. Finally, flow rate changes affect only the saturation capacities. A flow rate change of 1% causes a 2% change in the saturation capacity parameters.