(640b) A Comprehensive Rate Model for Predicting the Adsorption and Elution of Lispro Insulin in Reversed Phase Chromatography with Gradient and Stepwise Elution
A comprehensive rate model was developed and verified for lispro insulin in reversed phase chromatography. The mobile phase consisted of a buffer and various fractions of acetonitrile. The system was operated at 15°C. More than 95% of adsorbed insulin was eluted under the conditions tested. A modified reversed-phase modulator model was developed to correlate the nonlinear adsorption isotherm of insulin over a wide range of acetonitrile concentration. An efficient method for estimating the isotherm parameters was developed. The linear isotherm parameters were estimated using isocratic and linear gradient pulse elution. Frontals at three organic fractions were used to estimate the nonlinear isotherm parameters. The isotherm parameters were verified using rate model simulations and the data from frontal chromatography and high-loading gradient and stepwise elution. At a low organic fraction, the slow approach to saturation in the breakthrough curve was apparently due to surface diffusion. Tailing at high elution velocities could be attributed to viscous fingering. The pore diffusion model gave close predictions of the chromatograms of insulin at high loading in linear gradient and stepwise elution. For C8 columns with10 micron particles and a linear velocity less than 10 cm/min, the peak shape of lispro insulin in linear gradient or stepwise elution was controlled by self-sharpening and gradient focusing effects.