title: The importance of system band broadening in modern size exclusion chromatography.
abstract: 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 (typicallyVec<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.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet, 1, 1211 Geneva 4, Switzerland.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Goyon
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet, 1, 1211 Geneva 4, Switzerland.
authors.forename: Davy
authors.initials: D
authors.lastname: Guillarme
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet, 1, 1211 Geneva 4, Switzerland. Electronic address: szabolcs.fekete@unige.ch.
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Fekete
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D002850:Chromatography, Gel / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards
chemical_list: D000911:Antibodies, Monoclonal
keywords: Aggregates; Extra-column band broadening; Kinetic efficiency; Monoclonal antibodies; Size exclusion chromatography