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Cover Story

December 11, 2006
Volume 84, Number 50
p. 14-19

Monolithic Chromatography

Nontraditional column materials improve separations of biomixtures

Mitch Jacoby

They say you can't 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. Recently, however, some chemists have turned to an alternative type of column packing: one-piece 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 mixtures of biological molecules.

Merck KGaA
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.

"Packed-column chromatography is a mature technology," Frantisek Svec says, "but it's not flawless." Svec, who is a research 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 separation. 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 chromatography 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 packing materials. When a sample solution is injected into the column, the effects of 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 transferred 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 permeability. Low permeability means that fluids 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 always an option.

That's where monolithic packing materials come in. Using chemical methods to polymerize liquid precursors into a continuous porous mass of coalesced particles, scientists can control two sets of parameters simultaneously. 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.

Courtesy of Barry Karger
Sensitive Kind At Northeastern University, Quanzhou Luo (holding chromatography column), Shiaw-Lin (Billy) Wu, and Karger push chromatography's limits of detection.

To date, several monoliths based on organic and inorganic polymers have been prepared and shown to be efficient in separating proteins, peptides, oligonucleotides, and other analytes. In a number of cases, researchers have shown that the new materials outperform traditional columns in terms of chromatographic resolution, separation speed, and other factors. For example, experiments have shown that under similar operating conditions, test mixtures of proteins, pharmaceutical agents, and other compounds can be separated by monoliths in one-half to one-third the time required for particle-packed columns.

The history of monoliths in chromatography can be traced back five decades to Richard L. M. Synge, who was honored with the 1952 Nobel Prize in Chemistry for his contributions to separations science. As Svec notes, Synge's proposal in the 1950s for the novel structure was ahead of its time. That's because the materials available in his day for preparing what later came to be known as monoliths were unable to stand up to the pressures needed to drive the flow of the mobile phase. As a result, more than 30 years passed before real progress was made in developing the new types of materials for chromatography.

Details of the technique's history and development are discussed in a recent review paper coauthored by Svec and Christian G. Huber, a professor of analytical chemistry at Saarland University, in Germany (Anal. Chem. 2006, 78, 2100).

Image Title Courtesy of Frantisek Svec
Svec

By the late 1980s, researchers looking for alternatives to traditional packing materials had access to a variety of polymers with wide-ranging properties. One scientist working in that area was Boris G. Belenkii, a professor at the Russian Academy of Sciences, in St. Petersburg, and a colleague of Svec's. At that time, Svec was at the Institute of Macromolecular Chemistry, in Prague.

Belenkii's interest in novel media for separations stemmed from his work in new methods development. The Russian scientist was searching for ways to separate analytes by using ultrashort columns and found that inorganic packing materials were unsatisfactory for the job. In contrast, he found that a membrane made of cross-linked polymers based on monovinyl and divinyl methacrylate gave promising results.

Merck KGaA (top)
Courtesy of Frantisek Svec (bottom)
Take Your Pick Chromatography monoliths are one-piece porous solids made of fused micrometer-sized globules of silica (top) or an organic polymer (bottom) that can be synthesized directly inside a chromatography tube (visible in lower left corner).

That success spurred further work with one-piece separation media. Within a few years, Svec and other researchers had shown that effective monolithic columns (with conventional dimensions) could be prepared from styrene, divinylbenzene, and other monomers. The investigations extended the list of organic building blocks from which monoliths could be synthesized and led to a convenient preparation method in which monomers were polymerized directly in chromatography-compatible tubes. That procedure bypassed the awkward task of inserting the monolith in the tube after it was formed by polymerization.

While science was advancing on the organic monolith front, developments were also under way in the inorganic arena. At Kyoto Institute of Technology, in Japan, biomolecular engineering professor Nobuo Tanaka and coworkers Hiroyoshi Minakuchi and Naohiro Soga demonstrated in the mid-1990s that polymeric silica monoliths could be used effectively in chromatographic separations. Tanaka points out that the monoliths were prepared via a novel sol-gel (colloidal suspension) synthesis developed by Kyoto's Kazuki Nakanishi.

A key feature of that method is its propensity for making products riddled with pores in two size ranges: tiny ones that provide high surface area and access to the silica surfaces on which separations occur and large ones that facilitate easy flow of material through the monolith.

The reaction is based on hydrolysis and polycondensation of tetramethoxysilane in the presence of polyethylene glycol, urea, and other reagents. As the reaction proceeds, spontaneous phase separation leads to a structure with silica-rich regions and aqueous regions, Tanaka explains. Heating the product yields an alkaline solution, which etches tiny holes into the silica walls. As a result, when the aqueous phase is removed, the final product is a silica skeleton marked with micrometer-wide winding channels and walls that are pocked with 10- to 20-nm-sized pores.

According to Tanaka, one of the main concerns in making monoliths is ensuring that the monolithic material remains in intimate contact with the walls of the surrounding tube. If gaps form between the monolith and the tube's walls, the pressures encountered, for example, in typical high-performance liquid chromatography (HPLC) experiments (hundreds of bar), will cause the analyte solution to leak past the monolith.

Image Title Courtesy of Nobuo Tanaka
Tanaka

One strategy for ensuring the requisite contact is to bond the materials covalently. That approach works well when silica monoliths are formed from precursors directly inside quartz capillary tubes, Tanaka points out. The reason for the success, he explains, is that SiO2 groups are ubiquitous 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 attaching the monolith to the capillary walls.

In the past few years, a handful of companies have added monolithic columns to their product lines. For example, Dionex, Sunnyvale, Calif., offers capillary columns (0.5-mm 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 conditions compared with packed columns.

"Typically, environmental and biological samples are extremely complex," Pohl says. To aid in separating mixtures with thousands of components, 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 packing materials, however, ever greater operating pressures are needed to overcome the resulting low column permeability. But the extreme pressures (up to 1,000 bar) require specialized and expensive equipment.

With monoliths, such separation can 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 permeabilities comparable with those of columns packed with 5-µm particles but with the separating power of columns packed with particles half that size (Anal. Chem. 2006, 78, 7632).

Karin Cabrera adds that the higher separation efficiency goes hand-in-hand with higher permeability and higher flow rates, which leads to shorter run times on monolith columns compared with traditional ones. Cabrera is a senior scientist at Merck KGaA in Darmstadt, Germany, which offers a line of silica-based monoliths under the trade name Chromolith. The products are available in a number of column sizes and configurations, such as pretreated with the industry standard C18 surface layer, which renders the material hydrophobic, as required in many applications.

Initially, the high flow rate was the selling point for the monolith-packed columns, Cabrera says. But then, on the basis of customer feedback, Merck learned that the columns were more stable and longer lived than particle-packed columns. Cabrera attributes the enhanced robustness to the monoliths' rigid one-piece structure.

As laboratory outfitters broaden their lines of off-the-shelf monolith items, some analytical chemists continue to push the technology toward new limits and in new directions. For example, the research group of Barry L. Karger, director of Northeastern University's Barnett Institute, demonstrated that low-attomole (10-18 mole) and even zeptomole (10-21 mole) detection sensitivities of peptides can be achieved by following monolithic capillary-column separation with detection via electrospray ionization mass spectrometry. Such high sensitivity is particularly useful for analyses with limited sample quantities such as biopsy tissues.

Courtesy of David Hage
Gearing Up University of Nebraska chemists Hage (from left), John Schiel, and Sony Soman discuss chromatography experiments.

Another type of separation material is porous-layer open tubular packings, which are monoliths with a large open central channel. Karger and coworkers have prepared such materials inside columns with 10-µm diameters and investigated their performance. In one test run, the group demonstrated that 3,046 unique peptides could be separated from a 50-ng protein digestion sample and detected at the sub-attomole level. The results were just accepted for publication in Analytical Chemistry.

An emerging area in separations science known as affinity monolith chromatography relies on immobilizing selective affinity ligands in monolithic supports and using those agents to retain analytes via specific, reversible biological interactions. Such interactions include binding between an enzyme and a substrate or an antibody and an antigen. As David S. Hage, a chemistry professor at the University of Nebraska, Lincoln, explains, the ligands can be attached to monoliths through covalent immobilization, formation of coordination complexes, or other ways.

Hage's group uses the technique to study processes that cannot be probed via conventional chromatographic methods. For example, by carrying out separations in just seconds (or faster), the team can probe equilibrium phenomena such as binding interactions between drugs and serum proteins. Hage recently reviewed the technology and its use in bioaffinity and immunoaffinity chromatography, chiral separations, and other applications (J. Sep. Sci. 2006, 29, 1686).

The field of chromatography is more than 100 years old, "yet monoliths are still teenagers," Svec says. As new kids on the block, they haven't been around long enough to make the kind of impact on the chromatography community that some practitioners anticipate. "I'm convinced their popularity will grow," Pohl remarks. Svec agrees. It's only a question of time, he says, until monolithic columns successfully compete with well-established separation technologies.

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