" CHVROMATOGRAPHIC SCIENCE SERIES
`
`VOLUME 87
`
`' HPLC of Biological
`“ Macromolecules
`
`‘
`
`Second Edition, Revised and Expanded
`
`Fred E. Regnier.
`
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`edited by
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`MTX1 029
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`MTX1029
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`ISBN: 0-8247-0665-X
`
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`2
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`2
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`Organic Polymer Support Materials
`
`Frantisek Svec
`University of California at Berkeley
`Berkeley, California
`
`INTRODUCTION
`In the early days of chromatography, the pioneering work of Tsvett [1], Lederer
`[2], and Martin [3] focused on separations using beds of natural porous inorganic
`materials as the separation media. Polymeric supports based on modified natural
`polysaccharides were introduced in the mid-1950s and soon became standard for
`the separation of biomacromolecules in low-pressure size exclusion and ion-
`exchange chromatographic mode [4-6]. High-performance liquid chromatogra-
`phy (HPLC) emerged about three decades ago as a result of advances in the
`preparation of microparticulate silica [7,8]. This new separation technique was
`first used for the separation of small molecules. The remarkable speed of
`separation that could be achieved by using HPLC encouraged the development
`of a broad spectrum of separation media. These media were based on different
`materials such as natural and synthetic polymers, glass and inorganic oxides.
`Biological molecules were first separated using the HPLC technique in the mid-
`1970s when macroporous silica became available. Further rapid development of
`the HPLC of biopolymers was observed after the introduction of hydrophilic
`organic resins [9,10].
`
`17
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`3
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`

`

`18 (cid:9)
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`Svec
`
`The increasingly firm requirements of regulatory agencies to monitor and
`control biotechnology processes and the desire of the industry to reduce overall
`process time and cost are the most important driving forces in the development of
`techniques that allow rapid analysis and/or efficient isolation and separation of
`biomacromolecules. High resolving power makes HPLC the hottest candidate to
`become a completely routine tool for both analytical and process scale separa-
`tions in the very near future.
`It has been correctly pointed out that the most significant advances in
`chromatography have always followed the appearance of enhanced support
`matrices [11]. Therefore, much effort has been devoted to research and develop-
`ment on novel, highly efficient, and selective separation media. For example,
`bonded silicas started the era of reversed-phase HPLC, and this technique is now
`the most widely used chromatographic mode [8]. Development of rigid hydro-
`phobic porous polymers led to development of size exclusion chromatography
`(SEC) in nonaqueous media [12]. Later, with novel supports, aqueous high-
`performance SEC of water-soluble polymers and biopolymers also became
`possible. Polymeric stationary phases currently dominate some chromatographic
`modes such as ion-exchange, hydrophobic interaction, and affinity chromatogra-
`phy as a result of their stability over the entire pH range and their accessibility
`with very different surface chemistries. All of these chromatographic modes are
`of particular relevance to the subject of this book.
`Hundreds of original papers in scientific journals that are published every
`year, together with many excellent books and reviews [for example, 7,8,10,13-
`22], cover various aspects of the support technology for the separation of
`biomacromolecules. In addition, a plethora of commercial literature on HPLC
`separation media is available from the individual manufacturers and vendors.
`Therefore, this overview will focus on the organic supports that have been
`developed and marketed recently rather than the polymeric separation media in
`their entirety.
`
`TYPICAL SHAPES OF PARTICULATE SEPARATION MEDIA
`In the early days of chromatographic packings, the simplest way to obtain small
`particles was to crush bulk materials. For example, large pieces of porous silica or
`dried dextran gels were crushed in ball mills and then fractionated according to
`size. Although they are easy to prepare, irregular particles packed poorly in
`columns and created large voids, thus robbing the column of its efficiency. The
`many sharp edges of the particles also fractured easily, causing clogging of the
`systems. Therefore, spherical beads that by default do not suffer from these
`significant flaws were soon developed.
`In general, spherical particles are manufactured by suspension polymerisa-
`tion, which was invented in Germany in 1912 [23]. In aqueous suspension
`
`I
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`4
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`

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`Organic Polymer Support Materials (cid:9)
`
`19
`
`polymerization, a water-insoluble polymerization mixture containing a dissolved
`free radical initiator is stirred in a large volume of water containing a suspension
`stabilizers such as water-soluble polymers [cellulose derivatives, starch,
`poly(vinyl alcohol), poly(vinyl pyrrolidone)] or very fine inorganic powders to
`form small droplets of the dispersed organic phase in the continuous aqueous
`phase. These droplets adopt a strictly spherical shape to minimize their interfacial
`free energy, and their average size is roughly controlled by the rate of stirring and
`the amount and type of suspending stabilizer used. The free radical polymeriza-
`tion is initiated by increasing the temperature of the stirred mixture, and solid
`polymer beads are formed [24,25]. This technique is currently the method of
`choice for the preparation of separation media in a regular spherical shape for
`high-performance liquid chromatography.
`
`MORPHOLOGY
`
`Current separation media can be divided into two major categories: gels and
`macroporous materials. The former family is represented by polymers that
`typically do not exhibit any porosity unless swollen in water or another solvent.
`This solvation results in the separation of the polymer chains from one another to
`form "pores", which are solvent-filled voids. This type of porosity is temporary
`because of the reversible nature of solvation and exists only as long as the solvent
`remains within the polymer network. Lightly cross-linked synthetic polymer
`beads(cid:151)often termed "swellable" or "microporous" gels(cid:151)acquire porosity only
`upon swelling. Well-known examples of separation media without permanent
`porosity are natural polysaccharides such as dextran, guar, and agarose, and
`hydrogels such as acrylamide and hydroxyethyl methacrylate-based polymers.
`Once swollen, materials are usually soft and easily deform under pressure, which
`generally prohibits their use in tall packed beds and at the high flow velocities
`required for high-performance liquid chromatography columns.
`Unlike the preceding materials that require solvent swelling to become
`porous, macroporous polymers are characterized by a permanent porous structure
`formed during their preparation that persists even in the dry state. Their internal
`structure consists of numerous interconnected cavities (pores) of different sizes,
`and their structural rigidity is secured through extensive cross-linking. Macro-
`porous polymers emerged in the late 1950s as a result of the search for
`mechanically resistant ion-exchange resins with enhanced exchange kinetics
`[26]. In contrast to gels, columns packed with these resins generally exhibit
`good flow characteristics. Macroporous beads are widely used not only for the
`preparation of chromatographic media but also as catalysts, adsorbents, supports,
`carriers, and ion-exchange resins. All of these applications take advantage of their
`rigid porous structure that remains unaffected by changes in their environments.
`Although macroporous materials swell much less than microporous gels in any
`
`5
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`20 (cid:9)
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`Svec
`
`solvent regardless of its polarity, mass transport in and out of the pores is faster
`than in unswollen gels because they contain permanent pores available even in the
`dry state. Molecules can diffuse freely through these pores, which constitute a
`labyrinth of tortuous interconnected cavities of different sizes.
`Because none of these morphologies is perfect for the preparation of
`chromatographic separation media, numerous hybrid materials have also been
`developed in an attempt to preserve advantages of both gels and macroporous
`materials while trying to avoid their drawbacks. In addition, nonporous micro-
`pellicular beads are available for the rapid separation of biopolymers.
`
`"Rigid" Gels
`Gel-type separation media prepared from natural polysaccharides were introduced
`to chromatography in the mid-1950s [4]. The original supports were modified
`cellulose powder [4,27] and soft gels of cross-linked dextran [5] and agarose [6].
`Beads based on other polysaccharides such as chitosan, starch, and guaran were
`added to this group over time. Generally, these materials are not suitable for high-
`performance separation techniques because the back pressure in columns steeply
`increases at higher flow rates as a result of compressibility of the bed. However,
`they are readily available from renewable sources and provide both excellent
`hydrophilicity and chemical stability. Therefore, several attempts were made to
`improve the properties such as shape and mechanical strength ofpolysaccharide-
`based gel supports, thus making them useful even at higher flow rates. These
`efforts were most successful with cellulose- and agarose-based media.
`
`Cellulose Supports
`Cellulose is a polymer formed from /3-1,4-linked D-pyranose repeat units.
`Cellulose particles consist of chains in ordered, crystalline domains interspersed
`with amorphous regions. High crystallinity resulting from a regular linear
`structure and extensive hydrogen-bonding between the polymer chains make
`cellulose insoluble in standard solvents. In contrast, functionalized cellulose is
`more soluble because the newly introduced groups remove the chains from each
`other and break the hydrogen bonds. For example, cellulose ion exchangers swell
`in water and may even dissolve completely, depending on their degree of
`functionalization.
`Originally, cellulose was used in the shape of irregular fibrous particles
`(microcrystalline cellulose), which are not suitable for chromatographic applica-
`tions requiring high flow rates. This led to the development of porous cellulose
`beads. An extensive review of this topic was published by Stamberg [28]. In a
`typical procedure, a solution of cellulose is dispersed in an immisible liquid to
`small droplets, and these are solidified, regenerating the original cellulose.
`
`EL_ (cid:9)
`
`WONOMMMUM
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`6
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`

`Organic Polymer Support Materials (cid:9)
`
`21
`
`Even highly porous cellulose beads, which contain only 2 vol.% solid and
`as much as 98% of pores typically filled with water, are more rigid than their
`dextran- and agarose-based counterparts. Therefore, they do not easily deform
`under pressure and can be packed into high beds that exhibit essentially lower
`flow resistance compared with columns packed with hydrogels. The sizes of
`cellulose beads available on the market vary in a broad range from
`25 p.m up to a
`few millimeters. Obviously, even the smallest bead size is too large and
`disqualifies these cellulose beads from analytical HPLC separations; however,
`they are well suited for large preparative scale systems. Low-solid-content
`cellulose separation media with a widely opened porous structure allows rapid
`mass transfer and good separations, even in columns packed with rather large
`beads at very high flow velocities [29].
`Although. these supports can be used directly for size exclusion chromato-
`graphy (SEC) and desalination of protein solutions the major applications are
`expected in ion-exchange and affinity chromatography. To achieve this, the
`original matrix has been chemically modified [18,28-30]. Cellulose-based separa-
`tion media for SEC and ion-exchange chromatography are commercially avail-
`able under the trade names SephÆcel (Amersham Pharmacia Biotech, Uppsala,
`Sweden), Perloza (SCHZ, Lovosice, Czech Republic), Spherilose (ISCO,
`Lincoln, NE), and Macrobead Hydrogel (LigoChem, Fairfield, NJ).
`
`Agarose Supports
`Agarose is a linear alternating copolymer of galactopyranose and anhydrogalac-
`topyranose produced by seeweed. This polysaccharide dissolves in water only at a
`temperature higher than 50(cid:176)C; a swollen gel forms upon cooling to a lower
`temperature. Stirring ahot aqueous agarose solution in an apolar organic solvent,
`followed by cooling, leads to regular beads consisting of spontaneously aggre-
`gated polymer chains arranged in an open three-dimensional network structure
`[6]. The pore size depends on the concentration of agarose in the original solution
`and can be controlled during the preparation of the beads. As a rule, the higher
`the concentration, the smaller the pores. For example, an average pore size of
`30 nm is typical for a swollen 4% agarose gel prepared from a 4% agarose
`solution. This pore size allows size exclusion fractionation range of 30,000-
`6,000,000 as determined for dextran standards [16].
`Cross-linking of the polysaccharide chains using bifunctional compounds
`such as epichlorohydrin, divinylsulfone, and bis-epoxides during the solidifica-
`tion dramatically increases the mechanical stability of the beads and allows the
`manufacture of beads as small as 10 p.m. Although cross-linking does not
`significantly change the morphology of the beads, the number of hydroxyl
`groups is reduced to about 50% of the original value. Despite this, the number
`of hydroxyl groups remains large and the cross-linked agarose materials remain
`
`7
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`22 (cid:9)
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`Svec
`
`very hydrophilic. These beads are an intermediate for the production of other
`separation media.
`Agarose beads cross-linked using a two step procedure, which involves a
`reaction with butanediol diglycidyl ether during the preparation of beads followed
`by an additional modification with epichiorohydrin in an aqueous hydroxide
`solution, are marketed under the trade name Superose (Amersham Pharmacia
`Biotech). Rigidity, together with a small particle size, makes them suitable for
`SEC of proteins and nucleic acids at linear flow velocities of up to 185 cm/hr
`[22,31]. Highly cross-linked 6% agarose beads (Sepharose Fast Flow) are also
`used for the manufacture of high-capacity ion exchangers and activated matrices
`for the preparation of affinity chromatography media.
`Another family of new ion-exchange and affinity separation media
`designed for large scale purifications called Streamline (Amersham Pharmacia
`Biotech) is characterized by an average particle density of about 1.2 g/mL, which
`is significantly higher than that of plain beads. These beads consist of 6% cross-
`linked agarose with an embedded crystalline quartz core. In contrast to processes
`typical of packed columns, these beads are fluidized to an expanded bed in a
`column by an upward flow of the mobile phase. They tolerate easily the crude
`feedback that can contain cells, cell debris, and other solids which would be lethal
`for a packed column, because these contaminants pass freely through the
`expanded bed. After the beads are saturated with the target compounds such as
`proteins and peptides and the solid impurities are washed out, the flow of the
`liquid is reversed and the beads sediment and form a typical chromatographic bed
`from which the proteins are eluted. This procedure considerably simplifies protein
`recovery from crude streams because it requires only a single-pass operation
`without the need for prior clarification.
`Although advanced polysaccharide supports currently have high binding
`capacities and more favorable flow properties, their size and mechanical proper-
`ties still do not meet the requirements of rapid analytical HPLC separations.
`These remain the domain of rigid porous beads based on both inorganic and
`organic polymer materials.
`
`Macroporous Polymers
`In order to obtain a rigid material that exhibits macroporosity, the polymerization
`mixture for its preparation must contain not only the monovinyl monomer but
`also a substantial amount of a cross-linking divinyl monomer and a porogen. The
`porogen does not react during the polymerization process but remains within the
`newly formed beads, where it is surrounded by polymerized material in areas that
`will ultimately become the pores after the porogen is finally removed during
`work-up. In most cases, the porogen is a simple organic solvent with a sufficiently
`high boiling point. As a result of both cross-linking and solubility changes
`
`8
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`Organic Polymer Support Materials (cid:9)
`
`23
`
`associated with increased chain length, polymer molecules formed during the
`polymerization process precipitate from the surrounding medium of porogen and
`remaining monomers. This phase separation occurs at an early stage of the
`polymerization, leading to the formation of microscopic globular entities that
`continue growing but do not coalesce due to cross-linking. As the polymerization
`proceeds, these so-called microglobes come into contact with each other and
`associate to form clusters consisting of both interconnected globules and voids or
`pores (Fig. 1). Typically, the pore volume within a macroporous material
`correlates well with the volume of porogen used [26,32-34].
`Macroporous polymer beads are rigid and, therefore, well suited for HPLC.
`A well-defined pore size distribution is also important for the design of
`macroporous chromatographic separation media. Small pores and large surface
`areas are essential for the HPLC of small molecules, whereas the separation of
`large molecules such as proteins and nucleic acids requires significantly larger
`pores. As a rule, the larger the pores, the lower the overall surface area. In fact,
`development of some novel chromatographic techniques (perfusion chromato-
`graphy) and media (continuous monoliths) (see later) were based on the
`availability of porous materials with extremely large pores.
`Although the number of variables characteristic of a system for the
`preparation of macroporous materials is large, only a few are useful for the
`control of porous properties. These are the percentage of both the cross-linking
`monomer and the porbgen in the polymerization mixture, the composition of the
`porogen, and the reaction temperature. Changes in the first two variables also
`affect both the composition of the polymer and its mechanical properties. In
`contrast, temperature and type of porogen are useful for the preparation of porous
`polymers with a fixed chemical composition that differ only in their porous
`properties.
`
`Macroporous Separation Media
`Specifics of the macroporous polymer stationary phases for HPLC have been
`thoroughly discussed in several review articles [34-39]. Macroporous styrene-
`divinylbenzene resins were first used for SEC in the mid-1960s [12]. Because
`they can be used in organic solvents, they significantly facilitated the determina-
`tion of molecular weight distributions of organic polymers. In addition, their
`hydrophobicity makes them suitable for separations in the reversed-phase mode
`[10,34,40,41]
`Lee [42] correlated porous properties with the chromatographic behavior of
`macroporous poly(styrene-divinylbenzene) beads PRP- 1 and PRP-3 (Hamilton
`Co., Reno, NV) with specific surface areas of 100 and 400 m 2 /g, respectively,
`and observed less than a 5% increase in retention for PRP-3. Obviously, high
`surface area alone appears not to be the most important feature affecting the
`separation. In order to achieve both high selectivity and capacity, the polymer
`
`9
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`24 (cid:9)
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`Svec
`
`FIGURE 1 Scanning electron micrograph of inner part of a macroporous polymer.
`
`support must have a sufficiently large number of pores with a size capable of
`accommodating the proteins. Polymer Laboratories (Amherst, MA) developed a
`series of micrometer-sized rnacroporous poly(styrene-divinylbenzene) beads
`PLRP-S with pore diameters of 100, 300, 1000, and 4000A. Beads with 100
`pores are not suitable for the separation of large molecules, but the others exclude
`only proteins with molecular weight larger than 3 x 10 5 , 106, and 107 , respec-
`
`10
`
`

`

`Organic Polymer Support Materials
`
`25
`
`OH
`
`r,,(cid:151)OH
`
`3 (cid:9)
`2 (cid:9)
`1 (cid:9)
`FIGURE 2 Examples of beads with typical hydrophilic functionalities.
`
`4
`
`(OH
`OH
`
`tively. Those pores are adequate for the separation of very large biomolecules.
`The retention of proteins in PLRP-S columns was reported to be similar to that of
`columns packed with C 18 silica [36].
`The quest for more hydrophilic macroporous beads suitable for aqueous
`mobile phases resulted in several highly cross-linked resins prepared by a
`copolymerisation of 2-hydroxyethyl methacrylate 1, glycidyl methacrylate 2,
`vinyl alcohol 3 (actually, hydrolyzed vinyl acetate), and N-tris(hydroxymethyl)
`methylacrylamide 4 monomers shown in Fig. 2. Obviously, the epoxide rings of
`poly(glycidyl methacrylate) have to be hydrolyzed first to obtain the hydrophilic
`diol flinctionalities (Scheme I ).These materials were commercialized under trade
`names such as Biosphere (Labio, Prague, Czech Republic), Fractogel (EM
`Separation Technology, Gibbstown, NJ), OHpak (Showa Denko K.K., Tokyo,
`Japan), PL-aquagel-OH (Polymer Laboratories, Amherst, MA), Suprema (Poly-
`mer Standard Service, Mainz, Germany), Trisacryl (Biosepra, Marlborough,
`MA), and TSK gel PW (Tosoh Biosep, Montgomeryville, PA). A broad range
`of particle sizes is available extending from 5 urn for analytical columns to 30-
`60 tm (230-400 mesh) for the preparative scale separations. These beads often
`have well-controlled porous properties and therefore also analyte size selectivities
`defined as SEC exclusion limits. As a result of advanced polymerization
`processes, modem SEC media yield columns with high efficiency. For example,
`Tosoh Biosep guarantees more than 45,000 theoretical plates/rn for columns
`packed with 6-urn polymer beads such as TSKgel G3000PWXL. Many of these
`beads also serve as intermediates for the preparation of functionalized separation
`media.
`
`2(cid:176)
`
`(cid:149)-e
`
`0 (cid:9)
`
`HO OH
`
`Scheme 1
`
`11
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`(cid:9)
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`

`26 (cid:9)
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`Svec
`
`Separation Media with Surface-Modified Pores
`There are two major reasons for modification of pore surfaces in some polymeric
`resins: (1) hydrophilization of hydrophobic surfaces and (2) an increase in
`loading capacity and kinetics of ion-exchange resins. The former is typical of
`styrene-divinylbenzene copolymers that are too hydrophobic to be used directly
`for the separation of biopolymers in some modes. In this case, hydrophilic
`polymers such as dextran, poly(oxyethylene), poly(ethylene imine), and poly-
`(vinyl alcohol) are adsorbed and cross-linked on [35,43] or covalently linked to
`the pore surface to form a thin "biopolymer friendly" barrier on the hydrophobic
`surface. Regnier [44] was one of the first to develop a covalently attached
`hydrophilic coating that substantially decreased the nonspecific irreversible
`adsorption of proteins.
`In typical macroporous supports, only the functionalities that are exposed at
`the "rigid" pore surface (Fig. 3a) can interact with analytes. This not only limits
`the number of functional groups but also restrains their motion and prevents them
`from adopting the optimal spatial configuration required for the most efficient
`interaction. Attachment of a linear polymer containing segments of opposite
`polarity (hydrophilic and hydrophobic) affords a "fimbrinated" stationary phase
`[45] with loops and trains similar to those shown in Fig. 3b. This arrangement
`increases the mobility of functional groups and, indeed, improves the chromato-
`graphic properties. Another process that provides the surface with loose chains
`is the preparation of functional polymers in situ within the pores by graft
`polymerization. The resulting "brushlike" structure is schematically depicted in
`Fig. 3c. Willer [46] used free radicals generated from hydroxyl groups of porous
`Fractogel TSK (EM Separations Technology) beads using cerium (IV) ions and
`grafted functional monomers shown in Fig. 4 such as dimethylaminoethyl
`acrylamide 5, diethylaminoethyl acrylamide 6, tnmethylaminoethylacrylamide
`7, 2-acrylamido-2-methyl-l-propanesulfonic acid 8, and acrylic acid 9. This
`technique allows the control of the density of functionalities by simple dilution of
`
`- x
`
`x
` )-x
`
`111_No
`
`VX_r0_ e-r
`
`5 (cid:9)
`FIGURE 3 Examples of architectures of surface-modified pores.
`
`: (cid:9)
`
`:
`
`x (cid:9)
`
`x
`
`
`
`4
`
`12
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`

`Organic Polymer Support Materials
`
`27
`
`0 N 0 N ONH 0 N 0"NH 2 O"NH
`1 (cid:9)
`I (cid:9)
`I (cid:9)
`10
`
`NHO
`
`(N (cid:9)
`
`0 OH
`
`FIGURE 4 Examples of common acrylamide-based monomers.
`
`(10 in Fig. 4)
`the functional monomers with an inert monomer such as acrylamide
`and obtaining structures similar to that shown in Fig. 3d. Grafted ion exchangers
`for protein separations featuring both higher dynamic binding capacity and faster
`kinetics compared with "classical" materials are available under the trade name
`EMD Tentacle Ion Exchangers (EM Separations Technology).
`
`Composite Packings
`The major advantage of soft gel separation media is the accessibility and mobility
`of the functionalized chains constituting the matrix. On the other hand, rigid
`macroporous materials are characterized by much higher mechanical strength. A
`combination of benefits of both categories of materials led to the development of
`hybrid (composite) packings consisting of a rigid macroporous polystyrene-silica
`composite scaffold that contains pores filled with polymerized acrylamide
`monomers like 7 and 8 shown in Fig. 4 slightly cross-linked with methylenebi-
`sacrylamide (11 in Fig. 4) [47]. Both strong acid (S-HyperD) and strong base
`(Q-HyperD) media are commercially available from Biosepra. These separation
`media exhibit very high dynamic loading capacities of about 150mg BSA/mL
`and faster mass transfer kinetics compared with typical macroporous beads [47].
`Although the mechanism of mass transfer within the gel phase in the pores is not
`yet completely explained, some preliminary results indicate that the mass
`transport is not controlled by diffusion [48].
`
`"Gigaporous" Materials
`The transport of molecules within the pores of a standard macroporous material is
`controlled by diffusion. Small entities such as gases, small organic molecules, and
`ions move relatively quickly, whereas the transfer of large molecules such as
`proteins, nucleic acids, and synthetic polymers is considerably slower because
`their diffusion coefficients are several orders of magnitude smaller than those oT
`low-molecular-weight compounds. This effect is detrimental to processes in
`which the speed of the mass transfer limits the overall rate, as is the case in
`chromatography.
`
`13
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`(cid:9)
`

`

`28 (cid:9)
`
`Svec
`
`Consider what happens when a liquid is forced through a tube packed with
`standard macroporous particles. The liquid flows readily through the interstitial
`( voids between the particles where resistance to its flow is smallest. In contrast, the
`) liquid present in the pores does not move and remains stagnant. If a small amount
`of a substance or a mixture of compounds is injected into the stream of flowing
`liquid (the mobile phase), these compounds will also be carried through the voids.
`However, because of the concentration gradient between the solution in the voids
`and the stagnant liquid within the pores, diffusion enables transport of these
`compounds into the pores until their concentration is equal in both stream and
`pores. Once the concentration "pulse" has passed by the bead, the amount of
`compound in the main stream decreases steeply and the concentration gradient is
`reversed. The compound then diffuses back from the pores into the surrounding
`liquid and eventually only the original stagnant phase remains within the pores.
`Because the diffusion rate for small molecules is quite high and the
`equilibrium concentration within the pores is reached quickly, their concentration
`in the pores is almost identical to that in the pulse. The situation is quite different
`in the case of macromolecules. The slow diffusional mass transfer of macro-
`molecules in macroporous media result in severe peak broadening. The efficiency
`of the whole separation system deteriorates rapidly as the flow rate increases. As a
`result, longer columns or slower flow rates must be used to achieve the desired
`separation [8].
`A considerable improvement in catalytic activity was observed when
`heterogeneous catalysts were used with large pores allowing convection of
`reactants through the particles [49]. In contrast to diffusion, in which the
`concentration gradient is the driving force, convection driven by flow dramatically
`accelerates the mass transfer. However, most pores found in typical macroporous
`polymers are much too small (<100 nm) to allow convection. According to the
`Hagen-Poiseuille equation, the pressure needed to force a liquid through a straight
`tube increases exponentially as the tube cross-section decreases. In the case of
`macroporous beads, the pressure needed to achieve convection through typical
`pores with a diameter of 100 run or less would be too high to be realistic with
`today’s equipment. Therefore, Regnier [50] used polymer beads with pores as
`large as 600 to 800 nm (Poros, Applied Biosystems, Foster City, CA) and
`achieved some flow through these pores. He also coined the term perfusion
`chromatography for this technique [11,51]. The extent of convection depends on
`the overall pressure drop along the column, which in turn is a function of flow
`rate. The higher the flow rate, the more mobile phase flows through the pores.
`This manifests itself in very good separations of biomacromolecules even at flow
`rates that were deemed inconceivable in the past without considerable lost of
`column efficiency [52,53]. Perfusion chromatography is easily scalable and the
`high flow rates translate into high-throughput processes. The perfusion approach
`has also been extended to diagnostics and catalysis using immobilized enzymes.
`
`14
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`

`

`Organic Polymer Support Materials
`
`29
`
`Nonporous Beads
`The use of nonporous microparticles which are functionalized only at their
`external surface (micropellicular beads), is the ultimate solution to the problem of
`diffusion within thecores. Although silica-based micropellicular materials for the
`separation of biopolyhiers were introduced in the mid-1960s by Horvªth et al.
`[54], the real advantages for rapid separation of large molecules were demon-
`strated only after very small beads became available almost two decades later
`[55]. Both theoretical calculations and experimental data document that rapid
`mass transfer between the mobile and stationary phases and the absence of
`intraparticular diffusion allow the separations of biomacromolecules to be
`finished within a few seconds [55]. In addition, a higher working temperature
`that’ may easily exceed 100(cid:176)C further accelerates mass transfer and increases
`column efficiency [56,57].
`Hamilton Co. developed nonporous 5-[Lm poly(styrene-divinylbenzene)
`beads PRP-oc in the late 1980s. However, their chromatographic properties
`were inferior to those found for porous supports [42]. This is most likely due
`to their surface chemistry and relatively large size that translates into very small
`surface area. Typical macroporous polymers exhibit surface areas in the range
`from tens to hundreds of square meters per gram. To achieve such surface areas
`with nonporous beads, extrem