The importance of system band broadening in modern size exclusion chromatography

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In the last few years, highly efficient UHP-SEC columns packed with sub–3 μm particles were commercialized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationary phases (150 × 4.6 mm) was also modified compared to regular SEC columns (300 × 6 or 300 × 8 mm). 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 × 4.6 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.

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