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 separat- ing complex mixtures of materials. They are particularly at- tractive 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 Berke- ley National Laboratory. “The number of papers on monolith- ic technology has grown, and now meetings always have a ses- sion 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 Ten- list is not meant to be comprehensive; some vendors may offer nessee and the Oak Ridge National Laboratory. “It allows you to products that are not listed here. increase the flow rate and achieve a faster separation without los- ing resolution.” As manufacturers tackle more complex mixtures Monoliths vs packed columns and as disciplines such as combinatorial chemistry generate large The development of monolithic columns has followed a track op- numbers of samples, the time required for a separation becomes posite that of the more conventional HPLC column. In a packed more important. Representatives of monolithic column manufac- HPLC column, separation occurs as molecules diffuse into and turers generally indicate that their primary market currently resides out of pores on the surface of the particles that make up the sta- in analysis, isolation, and processing of biomolecules and pharma- tionary phase. The driving force for the separation is diffusion, a ceuticals, although many are pursuing other applications as well. relatively slow process. Reducing the particle size from the once- Monolithic columns cover the same range of column sizes as standard 10-µm diam to, say, 5 or 3 µm, shrinks the size of the packed columns and are even extending to dimensions below pores, which improves separation, but it comes at a cost. The back the threshold of particle packing. Capillary monoliths with pressure on the system increases substantially as the particle size diameters as small as 100 µm are on the market for LC/MS; decreases. Although HPLC was originally known as high-pressure there are also monoliths for capillary electrokinetic chromatog- LC (and later renamed high-performance LC), pressure is not an raphy applications. Small-scale monolithic columns eliminate advantage in typical chromatographic separations. Pumping sys- the problems associated with frits, which are needed to contain tems, column materials, and even many analytes have a maximum conventional microparticulate packings, and the formation of operating pressure beyond which they fail. To avoid damage, the channels, which are inevitable when a column diameter is only solvent flow and/or column length must be reduced when small- 100 times that of a particle or less. At the other end of the er particles are used—tactics that counter part of the gain. scale, BIA Separations, based in Slovenia, plans to introduce Monolithic columns, on the other hand, essentially create a columns with capacities as large as 8 L within the next year. single large particle that entirely fills the column. Within this Table 1 lists selected commercial monolithic columns. This monolith (literally, single stone), a series of connected pores
Product line CIM (Convective Interaction UNO Swift Monolithic Columns Monolithic Capillary Columns, Media) Monolithic Nano Columns
Company BIA Separations Biorad Laboratories ISCO, Inc. LC Packings/Dionex
Contact information Teslova 30 Life Science Research Group 4700 Superior St. Abberdaan 114 SI-1000 Ljubljana 2000 Alfred Nobel Dr. P.O. Box 82531 1046 AA Amsterdam Slovenia Hercules, CA 94547 Lincoln, NE 68504 The Netherlands 386-1-426-5649 510-741-1000 402-465-3072 31 20 683 97 68 www.biaseparations.com www.bio-rad.com www.isco.com www.lcpackings.nl
Separation modes Ion exchange, reversed phase, Ion exchange Reversed phase, ion exchange Reversed phase hydrophobic interaction, epoxy, immunoaffinity
Target markets Biomolecular separations on Prep-scale biomolecules Large-molecule separations on LC/MS analysis analytical to prep scale analytical to prep scale
Dimensions (internal Disk: 16 3 mm; column: (4.6 10 mm) to (15 68 mm) (0.075 50 mm) to (35 250 mm) (100 µm 50 mm) to (200 µm diameter column 8–800-mL volume (annular ring 50 mm) length or disk thickness) design)
creates a continuous skeleton, filled with interconnected pores a porogen, which controls the size of the pores, is washed out that form flow channels of a consistent size. “All of the mobile with solvent. The chemistry of the column is controlled by the phase flows through the pores of the stationary phase,” says Svec. choice of monomer, so columns can be produced to target spe- “The driving force of mass transfer is convection, which is many cific applications, such as protein or nucleic acid separations. In times faster than diffusion.” Because of the high permeability of addition to standard analytical and prep-scale columns, several the monolith, these columns generate much less back pressure manufacturers use this technology to produce capillary HPLC than packed columns. This means monoliths can handle much monolithic columns. Because even very small particles can cre- higher flow rates, which, in conjunction with the convective mass ate non-functional channels due to the limited number of par- transfer, means much shorter separation times. Also, the geome- ticles across a column diameter, monolithic columns have a try of the porous channels allows better contact between the large advantage over narrow-bore packed columns. Monolithic analyte and the active sites of the stationary phase. Published columns with diameters as low as 100 µm are available com- comparisons routinely demonstrate 10-fold improvements in mercially, and diameters as low as 20 µm have been reported analysis time without a loss of resolution. in the literature. Although most monolithic columns currently available are Column materials and configurations based on organic polymers such as polymethacrylate, polyacryl- Although research on polymer gel monoliths as stationary phas- amide, and polystyrene–divinylbenzene, silica supports offer es began in the 1970s, monolithic columns first reached the some advantages. Unlike polymer monoliths, the skeleton of market in the 1990s in the form of short disks rather than the silica monolith contains both an array of large pores and columns. These disks, which are still produced, take advantage nanometer-scale pores that provide a large surface area, which of the monolith’s ability to separate in a short distance. Typical- can be bonded to form reversed-phase columns. According to ly, the monolith is prepared in a mold and cut into disks several Svec, the silica base is particularly suited to the separation of millimeters thick. A disk is mounted in a cartridge with fittings small molecules, such as peptides and drug candidates, while for mobile-phase flow. One advantage of the short flow path the polymer monoliths are generally preferable for larger mole- and low pressure is that a number of cartridges can be mounted cules such as proteins, nucleic acids, and synthetic polymers. in series in the HPLC system, allowing separation based on a se- This distinction is emphasized, as well, by the types of sample quence of column functionalities, such as reversed phase, ion ex- applications provided by column manufacturers. change, and bioaffinity. “An additional benefit of the monolith Silica monoliths initially had problems with voids that formed is that it is adaptable to the user’s needs,” says Darryl Glover after manufacture, as the monolith pulled away from the column of BIA Separations. “The material can be shaped to specific wall. The voids produced a “wall effect”, a loss of separation applications, such as syringes or well plates.” caused by solvent flowing through “channels” within the voids. Unlike disks, monolithic polymer columns are polymerized A procedure developed by Merck encases the dried silica mono- in situ in a plastic or stainless steel tube. After polymerization, lith in PEEK shrink-wrap, which prevents void formation. Cur-
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Table 1. Selected monolithic columns (continued).
Product line Chromolith Seprasorb
Company Merck KGaA Sepragen Corp.
Contact information Frankfurter Str. 250 14500 Doolittle Dr.
D-64293 Darmstadt San Leandro, CA 94577 Germany 510-667-1004 49-6151- 72-0 www.sepragen.com www.chromolith.com
Design Column, capillary column Disk
Chemistry Silica Modified cellulose
Separation modes Reversed phase, normal Ion exchange
phase
Target markets Analytical separations Separation of biomole-
of small and medium- cules from complex sized molecules; drug matrixes molecules
Dimensions (internal (4.6 25 mm) to 25 20 mm
diameter column (4.6 mm 100 mm) length or disk thickness)
rently, Merck is the only manufacturer marketing a monolith-
ic 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. Accord- ing to industry sources, several leading column manufacturers who are not yet in the monolith market have expressed an in- terest 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 ap- plications and into a broader range of industries will hinge on cost. “These columns will take the market, if the price is com- petitive,” says Guiochon. “For general use, though, the ad- vantages 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 elec- trochromatography, a relatively new technique which features both electrophoresis and chromatography, people have switched almost exclusively to monolithic technology.” Small-scale tech- niques are growing in importance because of the tiny amounts of material involved in biological separations, combinatorial chem- istry, 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.
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