The importance of system band broadening in modern size exclusion chromatography

The goal of this study was to evaluate the loss of efficiency observed with three different instruments (regular HPLC, non-optimized UHPLC and fully optimized UHPLC) offering different system variances. It appears that the new 150 × 4.6 mm, sub-3 μm SEC columns cannot be employed on a regular HPLC instrument, since the efficiency loss was equal to 60-85%, when analyzing mAb sample. Optimized UHPLC systems having very low extra-column volumes (typically Vec < 10 μL) have therefore to be used to properly operate these columns. Due to the instrument contribution to band broadening, the apparent efficiency of SEC columns packed with sub-2 μm particles can indeed be hampered when using inappropriate system. Considering the extra-column band broadening contribution of current UHPLC instruments, a further decrease of SEC column dimension is therefore not desired.