The objective of this research is to investigate large-scale "buffer-focusing" chromatgraphy in which a precisely controlled, internal pH gradient traveling much more slowly than the fluid velocity is used to simultaneously concentrate and purify dilute mixtures of biomolecules. The internal pH gradient is characterized by a low salt concentration, a high buffer capacity, and is produced using multicomponent mixtures of common amphoteric and non-amphoteric buffer compounds of defined chemical composition. Each of the buffer compounds interacts with the column by adsorption or through acid-base reactions. Reliable methods for producing internal pH gradients (e.g., criteria for selecting buffer compounds, type of sorbent, starting and final pH, column dimensions, and particle size) are to be developed using numerical simulations and ancillary experiments. The use of buffer-focusing chromatography to separate model mixtures of proteins is also to be investigated theoretically and experimentally. The results from this work can be used to evaluate buffer- focusing chromatography as an efficient, gently (i.e. non- denaturing) alternative to conventional large-scale methods for concentrating and purifying dilute mixtures of biomolecules.
