Review
`
`Review
`
`Poly(Styrene-Divinylbenzene) Based Media for Liquid
`Chromatography
`
`By Christian W. Huck* and Günther K. Bonn
`
`The latest developments and in particular important synthetic aspects for the preparation of modern poly(styrene-divinylben-
`zene) (PS-DVB) based liquid chromatography (LC) supports are reviewed. In this context, the chemistry of particular porous
`and nonporous, functionalized, monolithic, coated silica and more specialized mixed organic PS-DVB media is covered. Spe-
`cial consideration is given to modern approaches such as micro-(l)-HPLC and coating techniques and their most important
`applications. Synthetic particularities relevant to the corresponding applications are outlined.
`
`face. Consequently, any functional group, which may be
`either monomeric or polymer-bound, is attached to the sur-
`face via covalent bonding. The other approach for changing
`the surface of a carrier is the simple deposition/adsorption
`of other elements (e.g., charged metals).
`
`2 Poly(Styrene-Divinylbenzene) Particles
`
`In the following chapters an overview of the use of unde-
`rivatized, functionalized, mixed organic PS-DVB media as
`well as PS-DVB based resins used for immobilized affinity
`chromatography (IMAC) is given.
`
`2.1 Properties of Poly(Styrene-Divinylbenzene) Particles
`
`Since 1967, when Horvµth synthesized pellicular column
`packings by coating solid glass beads with anion-exchange
`stationary phases [1], the use of nonporous organic poly-
`meric microparticulate packing materials for fast HPLC
`has gained increased interest. The main advantage of this
`packing is that stagnant mobile-phase mass transfer, which
`leads to band broadening and a consequent loss of efficien-
`cy and resolution, is eliminated [2]. These materials are
`pressure- and temperature-stable and efficient at much
`higher flow velocities than traditional column packings.
`Performance losses with these columns are nearly always
`attributable to the introduction of contaminants from sam-
`ples or from the mobile phase, not to the mobile phase it-
`self. Micropellicular polymeric sorbents made of spherical
`particles of a few lm in diameter were introduced for the
`rapid separation of proteins and peptides by reversed-phase
`[3, 4] and ion-exchange HPLC [5±8]. Maa and Horvµth [3]
`were the first to describe the analysis of proteins on a col-
`umn packed with nonporous PS-DVB within less than 20 s
`(see Fig. 1).
`
`1 Introduction
`
`Due to the steady progress in medicinal and natural
`sciences, analytical
`techniques
`for characterization and
`quantitation are required. Since solid-phase extraction
`(SPE) was introduced in the 1980s, different new techniques
`such as supercritical fluid extraction (SFC) and capillary
`electrochromatography (CEC) have been developed1). Be-
`sides these analytical tools, ªclassicalº separation techniques
`such as high-performance liquid chromatography (HPLC)
`have been improved and optimized. One main advantage
`was the reduction of the column inner diameter to only a
`few micrometers, which resulted in the so-called micro (l)
`or capillary HPLC technique. New inputs from synthetic,
`especially polymer chemistry and more efficient analytical
`tools for their characterization significantly enhanced the
`quality of LC supports in terms of stability, reproducibility,
`selectivity and efficiency.
`This review covers aspects of synthetic chemistry relevant
`to the characterization of new and highly efficient poly(sty-
`rene-divinylbenzene) (PS-DVB) based liquid chromatogra-
`phy (LC) supports. The main emphasis is on the develop-
`ment in the last 10 years. Principal synthetic concepts and
`key procedures for the preparation of particular porous
`and nonporous, functionalized, monolithic, coated silica and
`mixed organic PS-DVB media are summarized and cross-
`references for relevant applications in other separation tech-
`niques are given. For many applications underivatized
`PS-DVB supports are used. In principle, surface functional-
`ization of PS-DVB LC supports may be accomplished by
`two approaches. One entails the chemical transformation of
`functional groups of the carrier material located at the sur-
`
`± [
`
`*] Dr. C. W. Huck (christian.w.huck@uibk.ac.at), G. K. Bonn, Institute of
`Analytical Chemistry and Radiochemistry, Leopold-Franzens University
`Innsbruck, Innrain 52a, A-6020-Innsbruck, Austria.
`List of abbreviations at the end of the paper.
`
`1)
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`Chem. Eng. Technol. 2005, 28, No. 12
`
`DOI: 10.1002/ceat.200500265
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` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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`Review
`
`The following approaches are used for the derivatization
`of PS:
`± copolymerization of monomers containing functional
`groups [18],
`± chemical modification of polymerized products [19],
`± chemical modification of polymerized products by graft
`polymerization [20].
`The basic assumption in all these approaches is that PS
`products consist of chemically pure PS, which is susceptible
`only to relatively harsh conditions such as those necessary
`for electrophilic substitutions leading to the required anchor-
`ing functions.
`
`Figure 1. Chromatograms of a protein mixture obtained on a 30 ” 4.6 mm
`i.d. column under two different conditions. Flow rate and temperature,
`(A) 3 mL/min, 25 C; (B) 7 mL/min, 80 C; linear gradient in 1 min from
`10 to 100 % acetonitrile in water containing 0.2 % TFA; samples, 1, 100 ng
`of ribonuclease A; 2, 50 ng of cytochrome c; 3, 10 ng of lysozyme; 4,
`100 ng of L-asparaginase; 5, 100 ng of b-lactoglobuline A; 6, 100 ng of
`ovalbumin [3].
`
`A major advantage of the pellicular configuration is that
`there are no significant intraparticular diffusion resistances
`[9], which is particularly useful for the rapid analysis of bio-
`polymers with high efficiency and resolution [10]. At the
`same time, the lack of internal pore structure offers certain
`other advantages such as good recovery of mass and biologi-
`cal activity in protein chromatography [11]. Theoretical and
`experimental studies have shown that the high speed and
`efficiency of chromatographic analysis with micropellicular
`sorbents can be further enhanced by operating the column
`at elevated temperature. Due to their solid, fluid-impervious
`core, pellicular sorbents are generally more stable at high
`temperatures than the porous silica-based stationary phases
`traditionally used in HPLC. With increasing temperature,
`both diffusivity and sorption kinetics of the eluted com-
`pounds are accelerated with a concomitant decrease in elu-
`ent viscosity. Hence, high flow velocities are possible and
`even columns packed with micropellicular sorbents can be
`operated below the upper pressure limit of the chromato-
`graph. These benefits can be especially significant in the rap-
`id chromatography of proteins, which have low diffusivities
`and sorption kinetics.
`Among the synthetic polymers, PS-DVB possesses partic-
`ularly good physical and chemical properties and has been
`used in both porous (350 m2/g, BET) [11±14] and nonpor-
`ous (4 m2/g, BET) [3, 12±17] forms (see Fig. 2), e.g., for
`HPLC of proteins, peptides, oligonucleotides, pharmaceuti-
`cals, and other biologically active materials. The nonporous
`polymers have played an important role in reversed-
`phase [3, 4] and ion-exchange [5±10] chromatography.
`
`Figure 2. Scanning electron micrographs of nonporous (a) and porous (b)
`PS-DVB particles. Nonporous PS-DVB, 2.5 lm, 4 m2/g; porous PS-DVB,
`5 lm, 350 m2/g [23].
`
`2.2 Synthesis of Poly(Styrene-Divinylbenzene) Particles
`
`Depending on the cross-linking degree and on the poly-
`merization process, two types of porous structures can be ob-
`tained, viz., PS-DVB copolymer matrices of the gel type and
`PS-DVB copolymer matrices of the macroporous type. Syn-
`thetic resins of the gel type usually contain 2±12 % divinyl-
`benzene as cross-linking reagent. In these resins, porosity is
`only present in the swollen state and micropores are formed
`(swelling porosity). If polymerization is carried out in the
`presence of at least 20 % or more of a nonpolymerizable
`compound, which dissolves the monomers but not the poly-
`mer, a product with a macroporous structure is obtained
`which is also existent in the dry state (permanent porosity).
`In such resins, DVB contents up to 65 % result in high cross-
`linking and higher mechanical stability.
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`PS-DVB particles are normally produced by suspension
`polymerization in a wide size distribution. For getting parti-
`cles with a narrow size distribution a size classification pro-
`cess has to follow. This can be achieved by either sedimenta-
`tion or centrifugation and normally a yield of about 3 % is
`reached. However, by using the method of activated swelling
`during synthesis it is possible to produce monosized particles
`[21]. In this process, monodisperse 1 lm seed particles of
`pure polystyrene are produced by emulsion polymerization.
`During the activation step the seed particles are swollen with
`a highly water-insoluble compound of lower molecular mass
`(e.g., 1-chlorododecane) which is added as a very fine emul-
`sion in order to ease mass transport into the seed particles.
`The activated seed is grown in the third step with a mono-
`mer or a mixture of monomers such as styrene and di-
`vinylbenzene. Because of the differences in their solubility
`(1-chlorododecane is much less soluble in water than styrene
`or divinylbenzene), the only possibility of diffusion is that of
`the monomer into the activated particles, resulting in a con-
`siderable increase in diameter of the seed (3±20 lm) [22].
`In the last step, the grown seed is polymerized by raising
`the temperature. If the growing step is carried out with a
`mixture of monomers and an inert diluent, e.g., toluene or
`heptane, permanent porosity will be introduced into the par-
`ticles. Through variation of the inert diluent (e.g., toluene,
`alcohol, heptane) as well as the ratio between inert diluent
`and monomer and of the temperature during polymerization
`the pore size and the specific surface area of PS-DVB parti-
`cles can be adjusted. The chemical structure of a cross-linked
`PS-DVB copolymer is illustrated in Fig. 3 [23].
`
`2.3 Functionalized Poly(Styrene-Divinylbenzene) Particles
`
`For certain applications it is sufficient to adsorb (physi-
`sorb) the desired molecule onto the surface of PS-DVB [21].
`Nevertheless, for most HPLC applications, a covalently
`bound, yet well-defined attachment of any functionality to
`the support is highly desirable. In general, derivatizations of
`PS-DVB are restricted to the chemistry of aromatic systems.
`Besides the well-known procedures for the derivatization of
`
`Review
`
`PS-DVB resins such as chloromethylation [24], amination,
`nitration [25], sulfation, Friedel-Crafts alkylation and acyla-
`tion [26±28] or copolymerization of chloromethylstyrene
`and some simple derivatives [29], few reports exist on the in-
`troduction of more complex ligands or on more straightfor-
`ward synthetic routes.
`A representative example is the preparation of PS-DVB
`based chiral stationary phases from chloromethylstyrene
`and divinylbenzene by a stagged-templated suspension poly-
`merization step. Subsequent amination of the chloromethyl
`moieties and acylation of the benzylic amines leads to the
`desired CSPs. Liang et al. [30] described the synthesis of ep-
`oxide-functionalized PS-DVB beads via suspension polymer-
`ization starting from monosized PS-DVB. Access to further
`functionalization was achieved by swelling techniques and
`subsequent cross-linking with epoxypropyl vinylphenyl ether
`and divinylbenzene. In a similar approach, vinylphenol-co-
`divinylbenzene based monosized beads for use in size-exclu-
`sion chromatography and RP chromatography have been re-
`ported [31]. Itsuno et al. reported on a straightforward and
`highly attractive halomethylation avoiding the highly toxic
`chloromethyl methyl ether. Instead, trioxane and tin tetra-
`chloride were used as a source for the chloromethyl group
`[32]. In analogy, bromomethylated species are accessible by
`using tin tetrabromide instead of tin tetrachloride.
`Seubert and Klingenberg [33] introduced the preparation
`of sulfoacylated resins. Starting with a Friedel-Crafts acyla-
`tion employing x-halogen acyl chlorides, the corresponding
`x-halogene ketones were formed under AlCl3 catalysis.
`Treatment with dimethyl sulfide (Me2S) resulted in the corre-
`sponding sulfinium salt. Addition of aqueous sodium hydro-
`2-re-
`gen sulfite finally yields the sulfoacylated species in a SN
`action (see Fig. 4). The resulting materials are claimed to
`possess properties superior to standard sulfonated PS-DVB
`resins in ion chromatography. Li and Fritz [34] recently de-
`scribed the preparation of a novel polymeric PS-DVB based
`resin by treating chloromethylated PS-DVB with diethylene
`triamine. Interestingly, the charge of the resin may be varied
`from +1 to +3 by pH. Some of the first reports on the prepa-
`ration of functionalized supports using functional monomers
`were by Buchmeiser et al. [35]. They described the prepara-
`
`Figure 3. Chemical structure of PS-DVB resins.
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`Figure 4. Sulfoacylation of PS-DVB resin.
`
`tion of a carboxylic acid derivatized PS-DVB copolymer.
`Thus, precipitation polymerization [35] of maleic anhydride
`with DVB in MEK-heptane (60 : 40) yields porous monosized
`particles in the range of 0.5±1.8 lm.
`Similarly, precipitation copolymerization of chloromethyl-
`styrene and DVB in acetonitrile-toluene was found useful
`for the preparation of monodisperse chloromethyl-function-
`alized polymer beads of 4±6 lm. Despite this attractive
`approach, a restricted access to the functional groups (car-
`boxylic acid and chloromethyl groups, respectively) must be
`assumed, as a major part of the functional monomer is lo-
`cated in the interior of the beads due to the continuous in-
`corporation of the functional monomer. A general method
`for the controlled functionalization of PS-DVB particles has
`been reported by Lochmann and FrØchet [36]. Metallation
`with a superbase (2-ethyl hexyl lithium-potassium 1,1-di-
`methylporpoxide) results in the formation of the corre-
`sponding polypotassium salt. Subsequent reaction with elec-
`trophiles yields the desired derivatives (see Fig. 5).
`
`Figure 5. Metallation of PS and reaction with electrophiles.
`
`It is worth noting that metallation occurs both at the aro-
`matic ring system and the benzylic position. The general
`advantage of this approach lies in the comparably high
`amounts of functional groups that may be attached onto the
`surface. Nevertheless, since alkyl lithium compounds and po-
`tassium tert-butylate represent rather hazardous compounds,
`this approach has not attracted much interest so far. Pore
`size specific functionalizations have been described for the
`separation of proteins and small hydrocarbons [37, 38]. For
`that purpose, large polymer pores were provided with phe-
`nyl groups in the presence of hydrophilic groups. In contrast,
`
`small pores were provided with a much higher phenyl con-
`tent [39, 40]. Consequently, relevant applications have been
`reported in the direct analysis of drugs from blood samples
`[41]. Jagodzinski et al. [42] described the functionalization of
`the surface with cyano and diol groups, which resulted in
`enhanced selectivity and capacity of the base resin for the
`separation of aniline, pyridine and phenol derivatives by
`using nonpolar solvents.
`Casillas et al. [43] described the use of Amberlite XAD-2,
`XAD-4, XAD-2-CH2-CH2-Br and XAD-4-CH2-CH2-Br res-
`ins, traditionally used in solid-phase extraction (SPE), for the
`analysis of amino acids and dipeptides resulting from fermen-
`tation products. Fritz and coworkers [44] demonstrated that
`-C(CH3)3, -CH2OH, -COCH3, -COCH2CH2COOH, -CH2CN
`functional groups introduced into PS-DVB resins have an ap-
`preciable effect on the retention times and k values obtained
`in reversed-phase liquid chromatography for the separation
`of alkyl benzenes and phenols.
`
`2.3.1 Poly(Styrene-Divinylbenzene) Based Ion Exchangers for
`HPLC
`
`Copolymers of styrene and divinylbenzene have many ad-
`vantages as matrices for ion-exchange resins. The copoly-
`mers have excellent physical strength and are not easily
`subject to degradation by oxidation, hydrolysis, or elevated
`temperatures. The aromatic ring can be reacted with re-
`agents producing ion exchangers as a result of the incorpora-
`tion of ionogenic groups. Electrophilic aromatic substitution,
`e.g., sulfonation, is the most common derivatization reaction
`for PS-DVB. Polymeric ion exchangers are usually finely
`sized spherical beads of PS-DVB with a mean diameter of
`8±12 lm. As already described above, depending on the
`cross-linking degree and on the polymerization process, co-
`polymer matrices of the gel type and PS-DVB copolymer
`matrices of the macroporous type are used.
`
`PS-DVB Based Cation-Exchange Stationary Phases
`At pH < 11.0, most carbohydrates are uncharged, thus ex-
`hibiting retention on the polymer resin via hydrophobic in-
`teractions with the support. AT pH > 11.0, carbohydrates
`are not retained because of their negative charge which re-
`pulses them from the negatively charged cation-exchange
`± modify the reten-
`resin. Therefore, ionic groups such as SO3
`tion properties of the hydrophobic resins. Furthermore,
`loading of sulfonated resins with various cations causes sub-
`stantial changes in the retention of neutral carbohydrates.
`Low cross-linked resins with a high swelling porosity are
`used to optimize the separation of oligosaccharides, higher
`cross-linked resins are mainly applied to high resolution sep-
`arations of mono- and oligosaccharides.
`While diluted mineral acids such as sulfuric acid are used
`for cation-exchange resins in the H+ form, deionized water is
`the mobile phase of choice for metal-loaded cation-ex-
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`change resins. Organic modifiers such as acetonitrile may be
`added to the eluent up to a concentration of 30 % (v/v) with-
`out causing damage to the PS-DVB matrix.
`
`PS-DVB Based Anion-Exchange Stationary Phases
`High-performance anion-exchange chromatography (HPAEC)
`using polymer-based stationary phases and high pH in combi-
`nation with pulsed amperometric detection (PAD) is a power-
`ful tool for carbohydrate separations. Elution at high pH al-
`lows the separation of carbohydrates as their oxyanions. PAD
`is characterized by high sensitivity and relative specifity for
`compounds with hydroxyl groups.
`Dionex introduced a range of polymeric nonporous Mi-
`croBead pellicular resins [45±47]. Fig. 6 shows the configura-
`tion of a pellicular anion-exchange resin with the superficial
`anion-exchange layer. These beads exhibit rapid mass trans-
`port, fast diffusion, high pH stability and excellent mechani-
`cal stability (> 4000 psi). Different resins of sulfonated
`PS-DVB agglomerated with 350 nm MicroBead quaternary
`amine functionalized latex (Carbo Pac PA1) or sulfonated
`ethyl vinyl benzene/divinylbenzene particles agglomerated
`with 350 nm MicroBead quaternary amine functionalized
`latex (Carbo Pac PA-100).
`An anion-exchange stationary phase, based on nonporous
`highly cross-linked (65 % divinylbenzene) PS-DVB matrix,
`derivatized by direct nitration of the spherical particles, fol-
`lowed by reduction with tin metal and quaternization with
`methyl iodide, was described by Corradini [26]. The nitrated
`PS-DVB particles (PS-DVB-NO2) are reduced with tin/hydro-
`chloric acid in DMF. Subsequently, quaternization is carried
`out with pentamethylpiperidine and methyl iodide to obtain
`+I±). Jochum
`quaternized PS-DVB particles (PS-DVB-N(Me3)3
`et al. [48] reported on the use of this phase for the separation
`of mono-, di-, and oligosaccharides. The separation of oligo-
`saccharides is strongly affected by the accessibility of oxyan-
`ions to functional groups attached to the stationary phase, and
`
`Review
`
`the differences in the retention times can be related to the dif-
`ferent acidity of the substituted hydroxyl groups or the differ-
`ent configuration of the glucosidic bond, which causes a differ-
`ent orientation when adsorbed by the stationary phase.
`Generally, oligosaccharides show higher retention times
`compared to di- and monosaccharides, which can be ex-
`plained on the basis of an increasing number of negative
`charges on the saccharides owing to the increasing number
`of hydroxyl groups. To demonstrate the suitability of the
`quaternized PS-DVB anion-exchange sorbent, the separa-
`tion of oligosaccharides from starch (Dextrin 10) up to more
`than 20 glucose residues is depicted in Fig. 7.
`
`Figure 7. Separation of oligosaccharides from Dextrin 10. Stationary phase,
`+ anion exchanger, column II (5 lm, 250 ” 4 mm i.d.); gradi-
`PS-DVB-N(CH3)3
`ent elution, from 96 mM sodium hydroxide in 60 min to 96 mM sodium hy-
`droxide with 400 mM sodium acetate; flow rate 1.0 mL/min; detection, PAD;
`temperature, 30 C; sample volume, 5 lL; peak identification: numbers indi-
`cate degree of polymerization (d.p.).
`
`Figure 6. Schematic illustration of a pellicular anion-exchange resin bead.
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`Coupling of Cation-Exchange PS-DVB Columns with Differ-
`ent Counterions
`The quality of carbohydrate separation on cation-exchange
`resins is affected, among other things, by the cross-linking de-
`gree and the type of counterion. Furthermore, it has been
`shown that a cation-exchange resin in the protonated form
`with a cross-linking degree of 8 % is particularly suited for the
`separation of alcohols, ketones, aldehydes, acidic sugars, and
`cabrohydrates [49]. While the same resin loaded with Ca2+ or
`Pb2+ has been succesfully applied to the separation of mono-
`and oligosaccharides, Ag+ is the preferential counterion for
`the analysis of oligosaccharides. Depending on the counter-
`ion, cation-exchange resins are more suited for the separation
`of oligosaccharides but less for monosaccharides and sugar
`degradation products. Ca2+ loaded exchangers are more
`appropriate for the analysis of monosaccharides and various
`sugar degradation products such as aldehydes and ketones.
`However, by means of coupling various differently loaded
`cation-exchange columns it becomes possible to analyze
`both oligo- and monosaccharides and their degradation
`products in one single run [50]. In this coupling of different
`cation-exchange resins, the length of the columns can be
`adjusted to achieve an acceptable elution order. Fig. 8
`
`shows the separation of various saccharides and degradation
`products by means of a Ca2+ loaded sulfonated PS-DVB
`(7.5 % cross-linking, 8 lm, 300 ” 7.8 mm i.d., Spherogel Car-
`bohydrate N, Beckman) coupled to an Ag+ loaded resin
`(4 % cross-linking, 25 lm, Aminex HPX 42A, Bio-Rad,
`Richmond, CA, USA) at a column temperature of 95 C
`with deionized water as mobile phase. The flow rate was
`1.2 mL/min and detection of the analytes was accomplished
`by means of a refractive index detector.
`
`2.3.2 Poly(Styrene-Divinylbenzene) Based Ion Exchangers for
`Low Pressure LC
`
`Separation on Cation Exchangers
`Over 1,000 publications on the separation of rare-earth and
`transuranium elements document the great applicability of
`ion exchangers in this field. Rare-earth elements were sepa-
`rated on a Dowex 50 column (97 cm, 0.26 cm2) with a rela-
`tively fine particle diameter (270±350 mesh) and a mobile
`phase consisting of citric acid. Furthermore, the separation
`of transplutonium elements, alkali metals, Ca-Ti, Cd-Co-Ni,
`Cd-Ag, Fe-Co-Ni, Fe(II)-Fe(III), Au(III)-In-Cd, Ba from
`other ions, Ca and Sr from K and Rb, Mg from Al, and trace
`amounts of Ga from other metals can be separated [51].
`
`Separation on Anion Exchangers
`For this purpose mainly anion exchangers with strong base
`functional groups (e.g., quaternary ammonium ions) and a
`styrene-divinylbenzene resin matrix (Dowex 1 and 2, Dow
`Chemicals Co.; Amberlite, IRA 400, 401, 410, 411, Rohm
`and Haas Co.; AG 1, Bio-Rad, Labs.) are used. A typical ex-
`ample is the formation of uranium chloride complexes for
`the separation from many other ions.
`
`2.4 Mixed Organic Poly(Styrene-Divinylbenzene) Particles
`
`Wagner and Schulz [52] described the synthesis of unfunc-
`tionalized polystyrene and polymethacrylate resins cross-
`linked with divinylbenzene for the characterization of ad-
`sorption properties following the isotherms of Redlich-Pe-
`terson by the separation of phenols. For the separation of
`C60 and C70 Zha et al. [53] prepared a 1-methylnaphthalene
`modified PS-DVB resin. As a result of the derivatization,
`the interaction force between fullerene and resin was so
`strong that the strong solvent o-xylene could be selected as
`mobile phase. The solubility of C60 and C70 in the mobile
`phase strongly increased and the column loading capacity
`improved considerably, which enabled baseline separation
`of C60 and C70.
`
`2.5 Immobilized Affinity Chromatography
`
`Figure 8. Separation of mono- and oligosaccharides using coupled columns.
`Columns, (A) = Ca2+ loaded cation exchanger, Spherogel, Carbohydrate N,
`Beckman, (B) = Ag+ loaded cation exchanger, HPX 42A, Bio-Rad.
`
`Suzuki et al. [54] described the use of a PS-DVB based res-
`in containing iminodiacetic acid homologues (IDA resins)
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`with spacer arms of different lengths. To elucidate the selec-
`tivity profile of the resin, the chromatographic retention
`behavior of rare-earth elements (REEs) was evaluated.
`Thereby, chelating resins having longer spacer arms between
`the ligand group and the polymer matrix showed a better cor-
`relation with the corresponding monomeric ligands in solu-
`tion. This result can be attributed to an increase in the steric
`flexibility of the chelating group. The ligand density on the
`polymer surface affected the structure of the metal-IDA
`complex.
`
`3 Monolithic Poly(Styrene-Divinylbenzene)
`Columns
`
`In the following chapters the main properties of mono-
`lithic PS-DVB media, their synthesis, application and func-
`tionalization are summarized.
`
`3.1 Monolithic Poly(Styrene-Divinylbenzene) Capillary
`Columns
`
`3.1.1 Properties of Monolithic Poly(Styrene-Divinylbenzene)
`
`Although stationary phases based on microparticles have
`been successfully utilized as separation media for HPLC
`for more than three decades [55, 56], the relatively large void
`volume between the packed particles represents a significant
`factor limiting the separation efficiency of conventional
`granular packing materials especially for the coupling to
`methods enabling structural analysis such as mass spectrom-
`etry (MS). A possibility to enhance mass transfer is the
`use of monolithic separation media [57, 58]. The chromato-
`graphic bed consists of a single piece of a rigid polymer
`which has no interstitial volume but only internal porosity
`consisting of micro-, meso-, and macropores (see Fig. 9)
`
`Figure 9. Scanning electron micrographs of a 200 lm i.d. monolithic poly(sty-
`rene-divinylbenzene)-capillary column.
`
`Review
`
`[59±64]. Because of the absence of intraparticular volume,
`all of the mobile phase is forced to flow through the pores
`of the separation medium [65]. According to theory, mass
`transport is enhanced by such convection [66±68] and has a
`positive effect on chromatographic efficiency.
`A general problem with all kinds of porous packing mate-
`rials having diffusive pores is the slow mass transfer of so-
`lutes into and out of the stagnant mobile phase present in
`the micro- and mesopores of the stationary phase, resulting
`in considerable band broadening particularly with high mo-
`lecular analytes [69, 70]. This drawback can be addressed by
`the elimination of diffusive pores, which restricts the mass
`transfer to a thin, retentive layer at the outer surface of
`the stationary phase, resulting in so-called micropellicular
`phases [71]. A monolithic column configuration lacking dif-
`fusive micro- and mesopores may be adequately described
`as a micropellicular monolith [72] and has been shown to en-
`able the separation of analytes over a very broad size range
`with efficiences significantly better compared to those of
`columns packed with micropellicular granular stationary
`phases. Miniaturized chromatographic separation systems
`applying capillary columns of 10±500 lm inner diameter are
`frequently the method of choice for the separation and char-
`acterization of peptides, proteins and nucleid acid mixtures,
`because very often the amount of available sample material
`is limited.
`The concept of monolithic stationary phases is especially
`suitable for the fabrication of capillary columns because the
`chemical immobilization of the monolith at the wall of fused
`silica capillaries has a positive effect on column stability and
`eliminates the rather difficult preparation of a frit to retain
`the stationary-phase particles in the capillary tube. In the
`case of nucleic acid analysis, femtomol to attomol amounts
`are separable and detectable in such miniaturized systems.
`Moreover, the low flow rates ranging from a few nanoliters
`to microliters per minute characteristic of capillary HPLC
`are well suited for directly interfacing the separation process
`with electrospray ionization mass spectrometry (ESI-MS).
`Such hyphenation adds another dimension to the analytical
`process by providing molecular mass and structural data
`about the separated analytes [73].
`Monolithic separation media also offer distinctive advan-
`tages for preparative scale separations [74]. Highly perme-
`able monoliths enable high percolation flow rates at low
`column backpressure without loss in column efficiency, re-
`sulting in fast loading and elution times [75]. Moreover, very
`stable and uniform chromatographic beds can be manufac-
`tured reproducibly even in large-diameter preparative col-
`umns [76, 77], which is very difficult to achieve with conven-
`tional, granular packing materials. Finally, the technology is
`readily scalable from laboratory devices to multigram load-
`ing levels required for manufacturing [78]. The mentioned
`advantages render monolithic technology a real complement
`to conventional separation columns packed with granular
`stationary-phase materials.
`
`Chem. Eng. Technol. 2005, 28, No. 12
`
`http://www.cet-journal.de
`
` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`1463
`
`7
`
`

`

`efficiency was observed using 0.1 % TFA as mobile phase
`additive. Generally, using the monolithic phase injected
`amounts of proteins are 10 to 40-fold lower than in the case
`of the particular PS-DVB column and, due to the low flow
`rate of 3 lL/min, no splitting for the hyphenation with MS is
`necessary. For the wheat germ lectin, the change from non-
`porous PS-DVB-C18 particles (2.1 lm, 50 ” 4.6 mm i.d.) to
`the monolithic phase allowed the partial resolution of two
`peaks, which can be deduced from Fig. 11 (peak 1a and 1b).
`Injection of the lentil lectin resulted in three peaks (peak 2a,
`2b, 2c). Peaks 3a, 3b and 3c belong to concanavalin A.
`
`Review
`
`3.1.2 Synthesis of Monolithic Poly(Styrene-Divinylbenzene)
`
`For the synthesis of monolithic PS-DVB phases the fol-
`lowing procedure is carried out: 1 m of a fused silica capil-
`lary (Polymicro Technologies, Phoenix, AZ, USA; 200 lm
`i.d., 350 lm o.d.) is silanized with (trimethoxysilyl)propyl
`methacrylate. Finally, a 20 cm piece of this capillary is filled
`with 100 lL styrene, 100 lL divinylbenzene, 280 lL 1-de-
`canole, 20 lL dioxane and 4 mg azo-bis-isobutyronitrile
`(AIBN). Polymerization is carried out at 70 C within 24 h.
`After that, the capillary is flushed with acetonitrile for one
`hour and cut into pieces of 7 cm. The proportion of mono-
`mer/porogen is kept constant at 2 : 3 (v/v). 1-Decanol is used
`as a macroporogen. Tetrahydrofurane, nitromethane and
`dioxane are used as microporogens. To optimize permeabil-
`ity, the influence of dioxane in the porogen mixture was
`investigated. Therefore, the amount of dioxane was varied
`between 2±12 % (v/v). An increase in the amount of dioxane
`caused a decrease in flow rate, which means that the pore
`size is strongly influenced by the concentration of the micro-
`porogen. Optimum permeability was achieved at an amount
`of 4 % dioxane. Finally, optimization of the nitromethane
`content (see Fig. 10) in the same manner allows the syn-
`thesis of monolithic PS-DVB phases with low permeability
`which means that low flow-rates already cause a high back-
`pressure.
`
`Figure 11. LC-UV of a lectin mixture using monolithic PS-DVB. Column,
`PS-DVB monolith (7 cm ” 200 lm i.d.); mobi