Fast separation of large biomolecules using short monolithic columns

Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control.