Theoretical performance of multiple size-exclusion chromatography columns connected in series

The fundamental relationships are derived for the retention, peak width, and peak capacity of non-retained polymers eluting from multiple standard size-exclusion chromatography (SEC) columns connected in series. The standard SEC columns may have different dimensions and are packed with particles having distinct average particle diameters (APDs) and average mesopore sizes (AMSs). The performances (peak capacity, local resolution power, and sensitivity) of three standard SEC columns connected in series (called a tri-SEC column) packed with bridged-ethylene-hybrid (BEH) fully porous particles (FPPs) having three different APDs (1.7, 2.5, and 3.5 μm) and AMSs (200, 450, and 900 Å, respectively) are calculated as a function of the applied flow rate and size of polystyrene standards. Irrespective of the APD and AMS, the present investigation assumes isomorphological materials relative to the mesopore space of the three different BEH particles. The advantage of a 15 cm long tri-SEC column over a single reference SEC column (APD=3.5 μm, AMS=900 Å), which generates the same back pressure and separation window as those of the tri-SEC column, is expected at flow rates larger than the optimum flow rate generating the maximum peak capacity. The calculations predict a significant relative increase of the peak capacity (from +25% to +85%), resolution of small molecules (from +75% to +225%), and of the detection limit of intermediate size (from +15% to +70%) and largest polymers (from +25 to +110%). This is explained by 1) the exclusion of the largest polymers from the internal volume of the particles having the smallest mesopores (restricted access media) and 2) the minimum dispersion along the columns packed with the smallest particle sizes in the tri-SEC column. The main benefit of multi-SEC columns is to easily adjust the desired pore size distribution by properly selecting the lengths of each individual SEC column. The user can then control the pore size distribution for any specific separation problem. A potential application is theoretically demonstrated for the fast purification of monoclonal antibodies from metabolites, host cell proteins, aggregated forms, and from virus-like particles.