The purposes of this study are to develop a process to analyze fatty acids in recombinant animal cells using reversed-phase chromatography and simulate liquid chromatography processes using the general rate model.
A method was developed for the separation and analysis of various fatty acids in recombinant animal cells. The cell sample was saponified with 0.5M NaOH in 96% ethanol and then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C18 RP-HPLC column to separate and identify fatty acids using a gradient ACN-H 2O mobile phase. The fatty acids were monitored by measuring ultraviolet light absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolves these fatty acids and can be used either analytically or preparatively. The fatty acids separation and analysis process developed in this work can be adapted to quantitatively analyze fatty acids from others sources.
The general rate model was used to simulate liquid chromatography processes. Two kinds of liquid chromatography processes were studied: size exclusion chromatography and reverse-phase chromatography. The effects of various parameters were analyzed using the mathematical model. Methods to estimate the model parameters and the procedures using the general rate model to simulate and predict the results of these two types of chromatography process were developed. For SEC, the particle tortuosity was used as an adjustable parameter while, for RP-HPLC, the eluite-modulator relationship parameter α was used as an adjustable parameter. The values of these two parameters were obtained by matching the model-predicted results and experimental results using trial and error method. The validity of these two simulation procedures was verified by the comparison between the model predictions and the experimental results for larger SEC and RP-HPLC columns. The simulation and scale-up strategies developed in this work can be used to facilitate the development and optimization of liquid chromatographic processes.