The importance of system band broadening in modern size exclusion chromatography

In the last few years, highly efficient UHP-SEC columns packed with sub-3μm particles were commercialized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationary phases (150×4.6mm) was also modified compared to regular SEC columns (300×6 or 300×8mm). Because the analytes are excluded from the pores in SEC, the retention factors are very low, ranging from -1 <k < 0, resulting in very small column band variance. Therefore, the contribution of the system itself to peak variance can become significant under UHP-SEC conditions. 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 150×4.6mm, sub-3μm 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 (typicallyV_ec<10μL) 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-2μm 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.