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The importance of system band broadening in modern size exclusion chromatography

Published in Journal of Pharmaceutical and Biomedical Analysis. 2017, vol. 135, p. 50-60
Abstract In the last few years, highly efficient UHP-SEC columns packed with sub–3 m particles were commercial-ized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationaryphases (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 itselfto 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. Itappears that the new 150 × 4.6 mm, sub–3 m SEC columns cannot be employed on a regular HPLCinstrument, since the efficiency loss was equal to 60–85%, when analyzing mAb sample. OptimizedUHPLC systems having very low extra-column volumes (typically Vec< 10 L) have therefore to be usedto properly operate these columns. Due to the instrument contribution to band broadening, the appar-ent efficiency of SEC columns packed with sub–2 m particles can indeed be hampered when usinginappropriate system. Considering the extra-column band broadening contribution of current UHPLCinstruments, a further decrease of SEC column dimension is therefore not desired.
Keywords Size exclusion chromatographyKinetic efficiencyExtra-column band broadeningAggregatesMonoclonal antibodies
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GOYON, Alexandre, GUILLARME, Davy, FEKETE, Szabolcs. The importance of system band broadening in modern size exclusion chromatography. In: Journal of Pharmaceutical and Biomedical Analysis, 2017, vol. 135, p. 50-60. https://archive-ouverte.unige.ch/unige:91132

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Deposited on : 2017-01-17

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