Early work demonstrated excellent potential for analytical applications and this has come to fruition. Silica-based reverse-phase monoliths introduced by Tanaka in 1996 are now offered commercially by Merck (www.merck.de) and Phenomenex (www.phenomenex.com).
Compressed gel-based analytical ion exchange monoliths (UNO™) are provided by Bio-Rad Laboratories (www.bio-rad.com). Dionex (www.dionex.com) sells rigid porous polymer-based monolithic columns (SwiftPro®) in various formats for reversed-phase and ion-exchange chromatography. A complete line of ion exchange, reversed phase, hydrophobic interaction, and affinity monoliths (CIM®) are available from BIA Separations (www.biaseparations.com).
The performance of commercial analytical monoliths already equals the best of their particle-based counterparts, and the technology endows capabilities that have yet to be exploited. Capillary monoliths presented at the meeting demonstrated efficiencies in excess of 100,000 plates per meter and complete separations in less than 10 seconds. These columns also minimize consumption of expensive organic solvents, minimize sample volume, then compound overall savings by minimizing used solvent disposal costs.
The relationship of monoliths to other contemporary separation media is illustrated in the Figure. Professor Alois Jungbauer, department of biotechnology, University of Natural Resources and Applied Life Sciences (Vienna), emphasized the most prominent feature of monoliths—they exploit convective mass transport through large channels (1–5 µm) while porous particles must rely on diffusion through relatively narrow pores (600–1000 Å).
Diffusion is slow and increasingly so for larger solutes (Table). This is the reason why both capacity and resolution decline with increasing flow rate and solute size on traditional chromatography media.
Ales Podgornik, Ph.D., head of R&D for BIA Separations, likens convective flow to a river: the mass of the objects flowing down the river doesn’t matter. A tree trunk or a cork flow equally with the current, however fast it might be. Access to the river banks—the binding surface—is unrestricted, so capacity and resolution are both independent of flow rate, regardless of solute size.
Although the kinetics of binding and elution are independent of solute size, there is an important difference with respect to absolute capacity. Prof. Jungbauer and Prof. Shuichi Yamamoto, of Yamaguchi University (Japan), both emphasized that in contrast to porous particles, binding capacity on monoliths increases with the size of the molecule. This suggests that the number of chemical binding sites may be the limiting factor and highlights a valuable application feature for large proteins, plasmids, and viruses.
Another important distinction from microparticles is that the channels in monoliths are highly interconnected. This creates high surface accessibility and uniform frontal migration throughout the support. The combination of convective flow with high interconnectivity allows rapid separations to be carried out with extremely short beds.
CIM disks sold by BIA Separations for analytical applications and preparative method scouting have a bed height of 3 mm. Scale-up products have bed heights ranging up to 46 mm. Interconnected channels and short beds translate into low back pressure, allowing high throughput without requiring costly high pressure pumps.
One other distinction is that up to 40% of packed particle beds are occupied by wasted space: the void volume between the particles. This represents not only lost capacity but also lost resolution because turbulent mixing in the void erodes the separation achieved by the surface chemistry.
Monoliths have no void volume. The entire bed, less the volume occupied by the support matrix, is functional. In this respect, monoliths have an advantage over membrane chromatography as well. Mass transport on membranes is convective as it is with monoliths, but flow aberrations between layers and dead volumes within the housings contribute to turbulent mixing and sacrifice some of the resolution achieved by the surface chemistry.
Finally, monoliths do not require packing. Packing, process development, validation, and labor are eliminated. Differences in packing skills among operators become a non-issue. Scale-up scale-down variations in packing quality become a non-issue. Channeling from inadvertent introduction of air and the subsequent need to repack a column are eliminated.