product review
Separations in a monolith A column that is, in essence, one large particle has many advantages. Steve Miller
M
onolithic columns are relative youngsters in the realm of commercial separation columns, but their speed and high flow rates have caught the attention of scientists separating complex mixtures of materials. They are particularly attractive for the separation of large biomolecules, for which a single HPLC run can extend into hours. “There is a very high interest in the scientific community,” says Frantisek Svec of the University of California, Berkeley, and Lawrence Berkeley National Laboratory. “The number of papers on monolithic technology has grown, and now meetings always have a session on monoliths that is well attended.” Industrial users are also increasingly interested in monoliths, for both laboratory separations and purification of manufactured products. “The great advantage of monolithic columns is the low back pressure,” says Georges Guiochon of the University of Tennessee and the Oak Ridge National Laboratory. “It allows you to increase the flow rate and achieve a faster separation without losing resolution.” As manufacturers tackle more complex mixtures and as disciplines such as combinatorial chemistry generate large numbers of samples, the time required for a separation becomes more important. Representatives of monolithic column manufacturers generally indicate that their primary market currently resides in analysis, isolation, and processing of biomolecules and pharmaceuticals, although many are pursuing other applications as well. Monolithic columns cover the same range of column sizes as packed columns and are even extending to dimensions below the threshold of particle packing. Capillary monoliths with diameters as small as 100 µm are on the market for LC/MS; there are also monoliths for capillary electrokinetic chromatography applications. Small-scale monolithic columns eliminate the problems associated with frits, which are needed to contain conventional microparticulate packings, and the formation of channels, which are inevitable when a column diameter is only 100 times that of a particle or less. At the other end of the scale, BIA Separations, based in Slovenia, plans to introduce columns with capacities as large as 8 L within the next year. Table 1 lists selected commercial monolithic columns. This © 2004 AMERICAN CHEMICAL SOCIETY
list is not meant to be comprehensive; some vendors may offer products that are not listed here.
Monoliths vs packed columns The development of monolithic columns has followed a track opposite that of the more conventional HPLC column. In a packed HPLC column, separation occurs as molecules diffuse into and out of pores on the surface of the particles that make up the stationary phase. The driving force for the separation is diffusion, a relatively slow process. Reducing the particle size from the oncestandard 10-µm diam to, say, 5 or 3 µm, shrinks the size of the pores, which improves separation, but it comes at a cost. The back pressure on the system increases substantially as the particle size decreases. Although HPLC was originally known as high-pressure LC (and later renamed high-performance LC), pressure is not an advantage in typical chromatographic separations. Pumping systems, column materials, and even many analytes have a maximum operating pressure beyond which they fail. To avoid damage, the solvent flow and/or column length must be reduced when smaller particles are used—tactics that counter part of the gain. Monolithic columns, on the other hand, essentially create a single large particle that entirely fills the column. Within this monolith (literally, single stone), a series of connected pores M A R C H 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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product review
Table 1. Selected monolithic columns. Product line
CIM (Convective Interaction Media)
UNO
Swift Monolithic Columns
Monolithic Capillary Columns, Monolithic Nano Columns
Company
BIA Separations
Biorad Laboratories
ISCO, Inc.
LC Packings/Dionex
Contact information
Teslova 30 SI-1000 Ljubljana Slovenia 386-1-426-5649 www.biaseparations.com
Life Science Research Group 2000 Alfred Nobel Dr. Hercules, CA 94547 510-741-1000 www.bio-rad.com
4700 Superior St. P.O. Box 82531 Lincoln, NE 68504 402-465-3072 www.isco.com
Abberdaan 114 1046 AA Amsterdam The Netherlands 31 20 683 97 68 www.lcpackings.nl
Design
Disk, column
Column
Column, capillary column
Column
Chemistry
Polymethacrylate
Acrylamido-vinyl
Polymethacrylate
Polystyrene-divinylbenzene
Separation modes
Ion exchange, reversed phase, hydrophobic interaction, epoxy, immunoaffinity
Ion exchange
Reversed phase, ion exchange
Reversed phase
Target markets
Biomolecular separations on analytical to prep scale
Prep-scale biomolecules
Large-molecule separations on LC/MS analysis analytical to prep scale
(4.6 10 mm) to (15 68 mm)
(0.075 50 mm) to (35 250 mm) (100 µm 50 mm) to (200 µm 50 mm)
Disk: 16 3 mm; column: Dimensions (internal 8–800-mL volume (annular ring diameter column length or disk thickness) design)
creates a continuous skeleton, filled with interconnected pores that form flow channels of a consistent size. “All of the mobile phase flows through the pores of the stationary phase,” says Svec. “The driving force of mass transfer is convection, which is many times faster than diffusion.” Because of the high permeability of the monolith, these columns generate much less back pressure than packed columns. This means monoliths can handle much higher flow rates, which, in conjunction with the convective mass transfer, means much shorter separation times. Also, the geometry of the porous channels allows better contact between the analyte and the active sites of the stationary phase. Published comparisons routinely demonstrate 10-fold improvements in analysis time without a loss of resolution.
Column materials and configurations Although research on polymer gel monoliths as stationary phases began in the 1970s, monolithic columns first reached the market in the 1990s in the form of short disks rather than columns. These disks, which are still produced, take advantage of the monolith’s ability to separate in a short distance. Typically, the monolith is prepared in a mold and cut into disks several millimeters thick. A disk is mounted in a cartridge with fittings for mobile-phase flow. One advantage of the short flow path and low pressure is that a number of cartridges can be mounted in series in the HPLC system, allowing separation based on a sequence of column functionalities, such as reversed phase, ion exchange, and bioaffinity. “An additional benefit of the monolith is that it is adaptable to the user’s needs,” says Darryl Glover of BIA Separations. “The material can be shaped to specific applications, such as syringes or well plates.” Unlike disks, monolithic polymer columns are polymerized in situ in a plastic or stainless steel tube. After polymerization, 100 A
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a porogen, which controls the size of the pores, is washed out with solvent. The chemistry of the column is controlled by the choice of monomer, so columns can be produced to target specific applications, such as protein or nucleic acid separations. In addition to standard analytical and prep-scale columns, several manufacturers use this technology to produce capillary HPLC monolithic columns. Because even very small particles can create non-functional channels due to the limited number of particles across a column diameter, monolithic columns have a large advantage over narrow-bore packed columns. Monolithic columns with diameters as low as 100 µm are available commercially, and diameters as low as 20 µm have been reported in the literature. Although most monolithic columns currently available are based on organic polymers such as polymethacrylate, polyacrylamide, and polystyrene–divinylbenzene, silica supports offer some advantages. Unlike polymer monoliths, the skeleton of the silica monolith contains both an array of large pores and nanometer-scale pores that provide a large surface area, which can be bonded to form reversed-phase columns. According to Svec, the silica base is particularly suited to the separation of small molecules, such as peptides and drug candidates, while the polymer monoliths are generally preferable for larger molecules such as proteins, nucleic acids, and synthetic polymers. This distinction is emphasized, as well, by the types of sample applications provided by column manufacturers. Silica monoliths initially had problems with voids that formed after manufacture, as the monolith pulled away from the column wall. The voids produced a “wall effect”, a loss of separation caused by solvent flowing through “channels” within the voids. A procedure developed by Merck encases the dried silica monolith in PEEK shrink-wrap, which prevents void formation. Cur-
product review
Table 1. Selected monolithic columns (continued). Product line
Chromolith
Seprasorb
Company
Merck KGaA
Sepragen Corp.
Contact information
Frankfurter Str. 250 D-64293 Darmstadt Germany 49-6151- 72-0 www.chromolith.com
14500 Doolittle Dr. San Leandro, CA 94577 510-667-1004 www.sepragen.com
Design
Column, capillary column Disk
Chemistry
Silica
Separation modes
Reversed phase, normal Ion exchange phase
Target markets
Analytical separations of small and mediumsized molecules; drug molecules
(4.6 25 mm) to Dimensions (internal (4.6 mm 100 mm) diameter column length or disk thickness)
Modified cellulose
Separation of biomolecules from complex matrixes 25 20 mm
rently, Merck is the only manufacturer marketing a monolithic silica column. But other column manufacturers report that they continue to research silica monoliths because they provide a different geometry and range of applications than polymer columns of the same dimensions.
The future looks smaller Although most monolithic columns are currently marketed for biomolecular separations, the advantages of low pressure and fast separation are assets to any lab using HPLC. According to industry sources, several leading column manufacturers who are not yet in the monolith market have expressed an interest in pursuing this technology, both to expand into new markets and provide an alternative to existing technologies. As with many new products, acceptance beyond specialty applications and into a broader range of industries will hinge on cost. “These columns will take the market, if the price is competitive,” says Guiochon. “For general use, though, the advantages will not pay if they are too expensive.” The most promising new market for monolithic columns may take advantage of their ability to perform in capillary columns. “The future of these materials is in the small scale, in capillaries and possibly microfluidic chips,” says Svec. “For capillary electrochromatography, a relatively new technique which features both electrophoresis and chromatography, people have switched almost exclusively to monolithic technology.” Small-scale techniques are growing in importance because of the tiny amounts of material involved in biological separations, combinatorial chemistry, and drug discovery. Monolithic columns should grow in importance as they shrink in size. Steve Miller is a freelance writer based in State College, Pa. M A R C H 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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