SPONGY With micrometer-sized channels winding through a mass of fused particles, one-piece porous monoliths offer key advantages relative to traditional column chromatography media.
MONOLITHIC CHROMATOGRAPHY Nontraditional COLUMN MATERIALS improve separations of biomixtures MITCH JACOBY, C&EN CHICAGO
THEY SAY YOU CANT teach an old dog new tricks, but sometimes you really can. Case in point: For more than a century, chromatography practitioners have been separating the components of chemical mixtures by using columns packed with various types of particulate matter. Recent ly, however, some chemists have turned to an alternative type of column packing: onepiece porous solids known as monoliths. These nontraditional column materials recently have been commercialized and are being touted by manufacturers and users for the enhanced speed and thoroughness with which they can separate complex mix tures of biological molecules. "Packed-column chromatography is a mature technology," Frantisek Svec says, "but it's not flawless." Svec, who is a re search group leader at Lawrence Berkeley National Laboratory's Molecular Foundry,
explains that despite years of advances in separations media, packed columns have intrinsic characteristics that inevitably lead to limitations in chromatography. For example, interstitial voids—empty spaces between the tiny particles—take up space in the column but do not aid separa tion. The fraction of wasted space can be significant. According to Svec, in an ideally packed column with equal-sized spherical particles, some 30% of the column volume is lost to voids. "In reality, the percentage is even larger," he says. During the course of a chromatogra phy run, as the mobile phase (the fluid that transports the analytes) is pumped through the column, the fluid flows freely through the voids but meets resistance as it permeates the interior of porous pack ing materials. When a sample solution is injected into the column, the effects of WWW.CEN-0NLINE.ORG
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diffusion, which are linked to differences between analyte concentrations in the void spaces and the interiors of the particles, cause the analyte molecules to be trans ferred back and forth between the two regions. This process limits the column's ability to thoroughly separate analytes with similar properties and can lead to slow separations, especially for large molecules such as proteins and synthetic polymers, which tend to move sluggishly due to their small diffusion coefficients. One way chromatographers have tried to sidestep the problem is by reducing the size of the particles, which in turn shortens the distances that molecules travel as they diffuse in and out of the pores. But there's a tradeoff. Smaller particles mean smaller interstitial voids, and a reduction in the size of the voids lowers a column's perme ability. Low permeability means that fluids
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merely trickle through a column unless they're pushed with high pressure. Using high pressure, however, presents technical challenges and can destroy some types of columns, so it isn't al ways an option. That's where monolithic pack ing materials come in. Using chemical methods to polymerize liquid precursors into a continu ous porous mass of coalesced particles, scientists can control two sets of parameters simulta neously. They can optimize the nature of the material, porosity, and other properties that affect separations in monoliths, and they can independently control the size of the channels and open spaces that dictate the materials' permeability and give them their spongelike structures. To date, several monoliths based on organic and inorganic polymers have been prepared and shown to be efficient in
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[email protected] T A K E YOUR P I C K Chromatography monoliths are one-piece porous solids made of fused micrometer-sized glob ules of silica (top) or an organic poly mer (bottom) that can be synthesized directly inside a chromatography tube (visible in lower left corner).
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samples are extremely complex," Pohl says. To aid in separating mixtures with thousands of compo nents, as encountered, for example, in proteomics research, manufacturers of HPLC equipment have moved toward columns with diameters of a couple of micrometers or less. With traditional pack ing materials, however, ever greater operating Svec pressures are needed to overcome the resulting low column permeability. But the extreme pressures (up to 1,000 bar) require special ized and expensive equipment. With monoliths, such separation can
on the surface of the growing monolith and on the inside walls of quartz tubes. As such, the functional groups are readily joined via polymerization reactions, thereby firmly at taching the monolith to the capillary walls. In the past few years, a handful of compa nies have added monolithic columns to their product lines. For example, Dionex, Sunny vale, Calif., offers capillary columns (0.5mm diameter and smaller) based on styrene divinylbenzene monoliths and a line of larger diameter columns made of the same material under the trade name ProSwift. As Dionex Chief Science Officer and Vice President for R&D Christopher A. Pohl sees it, one of the key advantages of monoliths is their greater chromatographic efficiency (resolution) under mild experimental con ditions compared with packed columns. "Typically, environmental and biological
Packed columns have intrinsic characteristics that inevitably lead to limitations in chromatography.
Tanaka
be achieved with conventional equipment and one-third or one-fourth the pressure, Pohl stresses. That's the sort of finding Tanaka and coworkers just reported. By fine-tuning their synthesis method, the group prepared silica monoliths with per meabilities comparable with those of col umns packed with 5-um particles but with the separating power of columns packed with particles half that size (Anal. Chem. 2006,78,7632).
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