biocompatible polymeric monoliths for protein and peptide separations

J. Sep. Sci. 2009, 32, 3369 –3378 Y. Li and M. L. Lee 3369

Yun LiMilton L. Lee

Department of Chemistry andBiochemistry, Brigham YoungUniversity, Provo, UT, USA

Review

Biocompatible polymeric monoliths for protein andpeptide separations

The concept of biocompatibility with reference to chromatographic stationaryphases for separation of biomolecules (including proteins and peptides) is intro-duced. Biocompatible is a characteristic that indicates resistance to nonspecificadsorption of biomolecules and preservation of their structures and biochemicalfunctions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide-and poly(meth)acrylate-based monoliths] used for protein and peptide separationsare reviewed in detail, with emphasis on size exclusion, ion exchange, and hydro-phobic interaction chromatographic modes. Biocompatible monoliths for enzymereactors are also included. The two main synthetic approaches to produce biocom-patible monoliths are summarized, i. e., surface modification of a monolith that isnot inherently biocompatible and direct copolymerization of hydrophilic mono-mers to form a biocompatible monolith directly. Integration of polyethylene glycolinto the poly(meth)acrylate monolith network is becoming popular for reduction ofnon-specific protein interactions.

Keywords: Monoliths / Biocompatibility / Non-specific adsorption / Proteins / Peptides / Poly(ethy-lene) glycol / HPLC / Capillary column /

Received: July 8, 2009; revised: August 25, 2009; accepted: August 25, 2009

DOI 10.1002/jssc.200900478

1 Introduction

The term “biocompatible” is typically used in referenceto medical devices. It indicates that the material of whichthe device is constructed does not produce a toxic, injuri-ous, or immunological response in living tissue [1, 2].Many materials have been identified that are compatiblewith tissue to varying degrees, e. g., titanium (implants),poly(2-hydroxyethyl methacrylate) (contact lenses),ceramics (dental prosthesis), polyurethanes (artificialheart), and pyrolytic carbon (heart valve implants) [3–7].

A good review concerning biocompatibility was recentlypublished [8].

During the last 20 years, polymer-based monolithicsupports have been introduced into separation science[9–12], as well as into the field of organic catalysis andbiocatalysis [13–17]. Major contributions have beenmade by Svec, Fr�chet, and co-workers [9–12], who ini-tially developed radical polymerization systems for thesepurposes [18]. Polymer monolithic media represent uni-formly structured matrices with large interpenetratingpores (usually in the low micrometer range), which pro-vide unique advantages such as fast kinetics, high reac-tivity, and high throughput. Other advantages of mono-liths are that they can be created in virtually any form bya simple molding process, and they demonstrate reason-able mechanical strength. All of these advantages qualifythe monolith format as excellent for biochemical andmedical applications if it possesses the added quality ofgood biocompatibility. For example, biocompatible,highly porous monolithic scaffolds for use in both cellcultivation and tissue engineering have been preparedfrom hydrophilic monomers by electron-beam-initiatedfree-radical polymerization and ring-opening metathesispolymerization [19]. A novel implantable, wireless,monolithic passive pressure sensor for ophthalmic appli-cation was microfabricated using parylene as a biocom-patible structural material [20]. A polyamide rate-modu-

Correspondence: Milton L. Lee, Department of Chemistry andBiochemistry, Brigham Young University, Provo, UT 84602, USAE-mail: [email protected]: +1-801-422-0157

Abbreviations: AAm, acrylamide; BuMA, butyl methacrylate;EDMA, ethylene dimethacrylate; FITC, fluorescein isothiocya-nate; GMA, glycidyl methacrylate; HEA, 2-hydroxyethyl acry-late; HEMA, 2-hydroxyethyl methacrylate; HIC, hydrophobic in-teraction chromatography; IEC, ion-exchange chromatography;LC, liquid chromatography; LIF, laser-induced fluorescence;MBAA, N,N9-methylenebisacrylamide; M-IPG, monolith with im-mobilized pH gradient; PEG, polyethylene glycol; PEGDA, poly-ethylene glycol diacrylate; PEGMA, poly(ethylene glycol) meth-acrylate; PEGMEA, polyethylene glycol methyl ether acrylate;polyAAm, polyacrylamide; RPC, reversed-phase chromatogra-phy; SCX, strong-cation exchange; SEC, size exclusion chroma-tography; VS, vinylsulfonic acid

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

3370 Y. Li and M. L. Lee J. Sep. Sci. 2009, 32, 3369 – 3378

lated monolithic drug delivery system with biocompati-ble and biodegradable properties was recently patented[21]. A monolithic pH-sensitive hydrogel valve for bio-chemical and medical applications was designed, con-structed and tested in a microfluidic device [22].

The definition of “biocompatible” when used todescribe a chromatographic property differs somewhatfrom its use to describe medical devices. To date, a spe-cific definition of a biocompatible stationary phase forchromatographic applications has not been proposed. Inthis review, we begin with a general description of bio-compatibility as it relates to a chromatographic station-ary phase. Then, the characteristics and molecular struc-tures of protein-resistant surfaces are explored. Finally,we review the polymeric monoliths which demonstratenegligible non-specific adsorption of proteins in chroma-tographic separations. Selected applications in liquidchromatography (LC) and microfluidics for protein andpeptide separations are described.

2 Biocompatibility in chromatography

2.1 Definitions

The word “biocompatibility” seems to have been men-tioned for the first time by Hegyeli and Homsy in theearly 1970s [23, 24]. Since then, biocompatibility hasbeen redefined several times for biomaterials, and its def-inition is very contextual [25, 26]. The most recent defini-tion was given by Williams in 2008: “Biocompatibilityrefers to the ability of a biomaterial to perform itsdesired function with respect to a medical therapy, with-out eliciting any undesirable local or systemic effects inthe recipient or beneficiary of that therapy, but generat-ing the most appropriate beneficial cellular or tissueresponse in that specific situation, and optimizing theclinically relevant performance of that therapy.” [8] Andmost importantly: “… the sole requirement for biocom-patibility in a medical device intended for sustainedlong-term contact with the tissues of the human body isthat the material shall do no harm to those tissues,achieved through chemical and biological inertness”.

The concept of biocompatibility can be transferred tochromatographic stationary phases such as monoliths.We define a biocompatible stationary phase material as amaterial that resists nonspecific adsorption of biomole-cules (including proteins and peptides) and does notinteract with them in a way that would alter or destroytheir structures or biochemical functions. For example,various chromatographic interactions or size exclusioneffects should allow high recoveries of proteins andenzymes and good retention of their enzymatic activitiesafter the chromatographic procedures [27–29]. The bio-compatibility of a stationary phase can be achievedthrough control of surface chemistry and protein adsorp-

tion. The closely related term “inertness” reflects theresistance of the stationary phase to protein adsorptionand denaturation [30]. An excellent review was publishedrecently by Nordborg and Hilder that summarizes recentadvances in polymer monoliths for ion-exchange chro-matography (IEC). This review emphasizes the impor-tance of reducing non-specific interactions between theanalyte and stationary phase due to relatively weak inter-actions involved in this chromatographic mode [31].

2.2 Protein adsorption

The 20 naturally occurring amino acids found in pro-teins/peptides vary dramatically in properties of theirside-chains (such as polarity and charge) [32]. Proteinmolecules can be characterized by their size (molecularweight) and shape, amino acid composition andsequence, isoelectric point, hydrophobicity, and biologi-cal affinity. Differences in these properties can be used asthe basis for separation methods applied in purificationstrategies [33]. However, proteins tend to adsorb nonspe-cifically to most solid surfaces due to various hydropho-bic domains, charged sites and hydrogen bond donor/acceptor groups. Protein adsorption occurs at differenttypes of interfaces, and the protein-surface interactionsare important in bioseparations and other biomedicalapplications.

The adsorption of a protein on a surface is a complexprocess. Both the kinetics of the adsorption and the struc-ture of the layer of adsorbed protein are important inthis process [34, 35]. The adsorption process generallyinvolves the following steps: transport towards the inter-face by diffusion or diffusion/convection, binding to thesurface, detachment from the interface, and transportaway from the interface [36–38]. When adsorbed at inter-faces, proteins may undergo conformational changes orstructural rearrangements. A significant conformationalchange would result in protein denaturation, i. e., thecomplete loss of activity.

The major types of interaction that are relevant in pro-tein adsorption are: (1) hydrophobic interaction betweenprotein and hydrophobic sites on the surface [39], (2) elec-trostatic interaction between charged protein moleculesand oppositely charged sites on the surface [40], (3)hydrogen bonding between protein and hydrogendonor/acceptor [41], and (4) affinity binding between pro-tein and specific functional groups on the surface [42].Hydrophobic interaction plays a major role in proteinadsorption, and increasing the surface hydrophilicity tosome extent is expected to lessen the adsorption. Mostpolymer surfaces have some hydrophobic characteristics.An adsorbed layer of protein typically contains moreions and water than protein, and the presence of a chaot-ropic agent not only destabilizes the structure of the pro-tein but also influences its adsorption properties [43]. For


J. Sep. Sci. 2009, 32, 3369 –3378 Liquid Chromatography 3371

example, detergents or chaotropic salts can weakenhydrophobic interactions [44]. Additionally, most surfa-ces acquire some charge when exposed to ionic solution.In such cases, electrostatic interaction becomes moreimportant, even dominating protein adsorption kinetics.Even if the overall charge of a protein is zero, i. e., at itsisoelectric point, electrostatic interaction between theprotein and a surface may still exist, because the chargedistribution on the protein surface is not uniform. Inmost cases, affinity binding contributes to the specificinteraction between a protein and immobilized ligand,whereas hydrophobic, electrostatic and hydrogen bond-ing interactions are nonspecific.

In the above-mentioned protein-surface interactions,the governing factors are determined both by the physi-cal and chemical states of the adsorbing material, pro-tein surface, and solution environment [45], includingthe hydrophobicities, electrostatic charges and poten-tials of the material and protein surface (which can becontrolled by varying the pH and ionic strength in thesystem), and the chemical composition of the material.Other factors that may also influence protein adsorptionby surfaces include intermolecular forces betweenadsorbed molecules, solvent-solvent interactions,strength of functional group bonds, and chemistry andmorphology of the solid surface. Additionally, theadsorption of proteins at the liquid-solid interface oftenexhibits features that are irreversible on experimentaltime scales. Results are negligible desorption, slow sur-face diffusion, or surface-induced conformational or ori-entational changes [46].

2.3 Non-specific adsorption of proteins

As the name suggests, specific adsorption indicatesadsorption of a target protein to an immobilized ligandor otherwise specially prepared surface due exclusivelyto biospecific interactions [47]. Non-specific adsorptionmay be regarded as “foreign”, leading to undesirableproperties or reactions [48, 49]. Consider a surface thatcontains specific functional groups but is inherently“sticky” and permits nonspecific adsorption. Such a sur-face may lead to excessive protein-surface interaction,resulting in the loss of activity. In addition, a substantialpercentage of protein molecules can adsorb on the sur-face with their active sites inaccessible to target mole-cules. Finally, a target molecule can also adsorb nonspe-cifically on the “sticky” surface, thus contributing to thebackground signal. Therefore, for technologies thatinvolve contact of surfaces with biomolecules, it is funda-mentally desirable to maintain inert surfaces and reducenon-specific adsorption of proteins. Examples include:(1) bioseparations using media that are resistant to bio-fouling [50], (2) sensitive solid-phase immunoassays thatretain selectivity even in the presence of high concentra-

tions of serum proteins [51], (3) quantitative optical bio-sensors for protein characterization [52], (4) long-termimplantable devices that are biocompatible [53], and (5)solid-phase supports for the growth of adherent cells [54].The mechanisms underlying nonspecific protein adsorp-tion are still not completely understood. Electrostaticinteraction and hydrophobic interaction are two mainmechanisms involved in non-specific protein adsorption[55]. Of course, a combination of these two effects mayoccur. Many studies and reviews have focused on proper-ties of surfaces that lead to hydrophobic adsorption ofproteins [56, 57].

2.4 Nondesirable adsorption in chromatography

Size-exclusion chromatography (SEC), IEC, reversed-phase chromatography (RPC) and hydrophobic interac-tion chromatography (HIC) are the most widelyemployed chromatographic techniques for separation ofproteins and peptides [58]. Ideal SEC occurs only whenthere is no interaction between the analytes and the col-umn matrix. Unfortunately, SEC columns may exhibitsome interaction with solutes, resulting in deviationfrom ideal size-exclusion behavior, i. e., non-ideal SEC [59,60]. Although these non-ideal, mixed-mode effects mayprovide increased selectivity, they must be suppressed ifpredicable solute elution behavior is required. Thus, elec-trostatic effects (or hydrophobic effects) between ana-lytes and the column matrix can be minimized by addingsalt to the mobile phase or changing its pH.

IEC is a very useful chromatographic mode for protein/peptide separations. Although IEC is based on electro-static interaction between analyte and matrix, thematrix may often exhibit significant hydrophobic char-acteristics, leading to mixed mode contributions to sol-ute separations [61]. Mixed-mode effects can enhancepeptide or protein separation because proteins are poly-ions with both hydrophobic and hydrophilic surfaces[62]. However, the removal of non-specific hydrophobicinteractions may be necessary in order to elute solutes inthe specifically desired separation mode. A low polarityorganic solvent such as acetonitrile can be added to themobile phase buffer to suppress hydrophobic interac-tions between the solute and the ion-exchange matrix[63]. The excellent resolving power of RPC has resulted init becoming the predominant LC technique for peptideand protein separations (particularly the former) inrecent years. Protein denaturation is a common problemin RPC due to the strong hydrophobic interactions. Simi-larly, HIC of proteins is based on the strength of hydro-phobic interactions between proteins and a non-polarstationary phase. However, compared to RPC, the mobilephase conditions utilized are milder, and the relativehydrophobicity and density of the bonded ligands arelower. Additionally, in HIC, the stationary phases and



operating conditions are designed to preserve proteinsin their native conformations. Thus, proteins in theirnative states are eluted in order of surface hydrophobic-ity [64].

It is important to minimize non-specific interactionsin chromatographic separations to (1) obtain predicablesolute elution behavior, (2) obtain good recovery of sam-ple mass and bioactivity, and (3) avoid slow adsorption-desorption kinetics that lead to important band broaden-ing. As is well known, denaturation of sample proteinsduring separation and irreversible attachment to thematrix results in poor recovery [65]. The preservation ofnative conformation (and bioactivity) is often a primaryconsideration in method development and may restrictthe separation conditions within certain limits.

3 Known materials that resist proteinadsorption

Soft gel media, typically crosslinked dextran, cellulose,polyacrylamide (polyAAm), agarose and polysaccharides,have demonstrated low non-specific interactions withpeptides and proteins, and have been widely applied ingel electrophoresis and gel permeation chromatography[66–69]. Composite materials, such as highly crosslinkedpolyAAm-polysaccharide, polyAAm-dextran, and dex-tran-agarose composites have been developed to providemore rigid backbones [70]. Other neutral hydrophilicpolymers were also investigated and found useful in pre-venting protein adsorption, such as polyvinyl alcohol[71], polyethylene oxide [72], polyvinylpyrrolidinone [73],and a copolymer of polyethylene glycol (PEG) and poly-propylene glycol [74]. All of these polymers are neutral(i. e., no ionic interactions possible) and hydrophilic, andtheir surfaces share the following structural features: (1)hydrophilic, (2) overall electrically neutral, and (3) hydro-gen bond acceptors, but (4) not hydrogen bond donors[30]. Other materials used in gel electrophoresis reportedby Zewert and Harrington are poly(hydroxyalkyl meth-acrylate), poly(hydroxyalkyl acrylate), polyethylene gly-col methacrylate (PEGMA) and polyethylene glycol acry-late. Successful electrophoresis of proteins demonstratedthe protein compatibility of such polymers [75]. How-ever, these inert polymers and gels are too soft to be usedfor LC. Biocompatible hydrogels can be prepared bycopolymerization of hydrophilic monomers, or cross-linking of hydrophilic polymers [76].

4 Biocompatible polymer monoliths forbioseparations

Usually, polymer monoliths are prepared by polymeriza-tion of monomer solutions, which are composed ofmonomer, crosslinker, porogen and initiator. They can

be initiated either by a redox system, e. g., tetramethyl-ethylenediamine and ammonium persulfate, or by a freeradical initiator, e. g., 2-dimethoxy-2-phenyl acetophe-none or azobisisobutyronitrile. An alternative way to ini-tiate the radical polymerization process is by high energyirradiation. Compared to silica monoliths, polymericmonoliths, in particular, offer the advantage that bychoosing the right functional monomer, monoliths for avariety of chromatographic purposes can be designed,such as for improved biocompatibility.

Generally, there are two ways to obtain biocompatiblepolymer monoliths. One is through surface modificationto create a hydrophilic surface within the monolith thatresists the adsorption of proteins, including chemicalreactions of surface functionalities and grafting ofchains of functional polymers. Separation of the mono-lith synthesis and surface modification processes allowseach process to be optimized independently. Neutralhydrophilic polymers such as PEGMA monomer havebeen used to prepare protein-resistant surfaces on poly-mer monoliths via surface grafting [77]. Hydrophilic-grafted monoliths seem well-suited for use as inertenzyme-immobilization matrices for flow-through enzy-matic microreactors. The other more direct way to formbiocompatible polymer monoliths is by direct copoly-merization of hydrophilic monomers, leading to the for-mation of hydrophilic network structures. Most com-monly used monomers are hydrophilic (meth)acryl-amides and (meth)acrylates, as shown in Fig. 1. The chem-istry of a monolith is largely controlled by choice of themonomers used in its preparation. Polymerization ofvinyl monomers is most frequently initiated via radicalinitiators (peroxides and azo-compounds). Radicals aregenerated by heating, by the use of a redox initiator or aphotoinitiator.

4.1 Polyacrylamide-based monoliths

Acrylamide (AAm) is a moderately hydrophilic co-mono-mer which imparts some hydrophilicity in monolithicmaterials. It is typically polymerized with a crosslinkerto build some rigidity into the skeletal structure of thepolymer. The most common cross-linker is N,N9-methy-lenebisacrylamide (MBAA). Continuous rods made from acrosslinked polyAAm were initially the first monolithicmaterials for chromatography purposes [78, 79], andthey were based on solvent-swollen poly(acylic acid-co-MBAA). Despite the lack of a permanent pore structure,this material was used in the separation of proteins by anion-exchange mechanism. A more rigid poly(AAm-co-MBAA) monolith was prepared by copolymerization ofthe hydrophilic monomers in the presence of a poro-genic solvent for potential use in separation of biologicalpolymers, solid-phase extraction, or for immobilizationof proteins [80].



A novel poly(glycidyl methacrylate-co-ethylene glycoldimethacrylate) (GMA-co-EDMA) monolith with immobi-lized pH gradient (M-IPG) was reported for the analysis ofpeptides and proteins [81, 82]. An improved M-IPG, i. e.,poly(AAm-co-GMA-co-MBAA), was achieved by copolymer-izing more hydrophilic monomers (AAm and MBAA)with the functional monomer (GMA) [83, 84]. Comparedwith poly(methacrylate ester) based M-IPG, the hydrophi-licity of the new material was improved, and the adsorp-tion of proteins was avoided. A rigid, porous poly(AAm-co-butyl methacylate-co-MBAA) monolith containing upto 15% butyl methacrylate (BuMA) units was prepared bydirect polymerization and used as a separation mediumfor the rapid HIC of proteins [85]. Palm and Novotnydeveloped a porous polymer monolithic trypsin micro-reactor for fast peptide mass mapping, which offeredhigh flow permeability as well as biocompatibility [86].The monolith was prepared by polymerization of AAm,N-acryloxysuccinimide and MBAA in an electrolyte bufferwith PEG as molecular template. Incorporation of AAmand MBAA certainly helped to improve the biocompati-bility. The material did not irreversibly bind proteinsand, thus, did not foul easily, so that it could be reusedfor the analysis of human plasma without loss of effi-ciency. A continuous bed matrix from a monomer solu-tion (piperazine diacrylamide and methacrylamide) con-taining allyl glycidyl ether and 2-hydroxyethyl methacry-late (HEMA) was derivatized with C18 ligands for RPC ofproteins and peptides [87]. Furthermore, additionaldimethyldiallylammonium chloride could be added tothe monomers to create charged ligands for reversed-phase electrochromatography of proteins [88]. In conclu-sion, the highly crosslinked rigid polyAAm monolithscan serve as a platform for the preparation of specific sep-aration media for chromatographic modes such as HICand affinity chromatography of proteins, both of which

require biocompatible surfaces endowed with ligands orfunctionalities.

Additionally, due to their hydrophilicity in chromato-graphic behavior, polyAAm-based monoliths were suc-cessfully used in hydrophilic-phase electrochromatogra-phy of neutral (oligo)saccharides [89, 90]. However, themain use of the AAm networks has been in electrosepara-tion methods, mainly for SDS-PAGE in slightly cross-linked gels, and electrochromatography of small mole-cules in highly crosslinked gels [91–93]. The fundamen-tal polymerization procedure and its kinetics in AAmporous systems have been extensively discussed [94–96].

PolyAAm-based supermacroporous monolithic cryogelbeds can be prepared by radical cryogenic copolymeriza-tion under freezing-temperature conditions [97].Recently, semi-interpenetrating polymer networks usingbiocompatible polyurethane and AAm were synthesizedfrom different isocyanate-terminated polyurethane pre-polymers derived from polytetramethylene ether glycol.Compared to a pure polyAAm network, the mechanicaland thermal properties were enhanced due to highercrosslinking density imparted by the hard segments [98].However, the resulting structure was not suitable forchromatographic applications.

4.2 Poly(meth)acrylate-based monoliths

Polymethacrylate-based materials comprise the largestand most examined class of monolithic sorbents. Themain advantage of current macroporous (meth)acrylate-based monoliths is their suitability for separation oflarge molecules, such as proteins and polynucleotides, aswell as large particles such as viruses [99]. Excellentreviews were recently published that summarize thepreparation and the application of polymethacrylate-based monolithic columns for separation, purification


Figure 1. Monomers and crosslinkers usedin preparing biocompatible monoliths.


and analysis of low and high molecular mass compoundsin micro- and large-scale HPLC formats [100, 101].

PolyHEMA is a known biocompatible material used inthe production of contact lenses. 2-Hydroxyethyl acrylate(HEA) is also a biocompatible monomer, which can beintroduced into the monolith backbone in order toincrease its biocompatibility. For example, increasing theHEA monomer content in the comonomer mixture (up to18 vol%) in the preparation of the hydrophilic poly(HEA-co-diethylene glycol dimethacrylate) monolith by radia-tion-induced polymerization [102, 103], resulted in mono-liths with increased hydrophilic character. However, fur-ther increase of HEA monomer content transformed themonolith into a soft gel. The resulting monoliths weretested as chromatographic columns, and were found suit-able for separation of nucleic acids and amino acids. Animportant advantage of separation using this copolymermonolith is that it is environmentally friendly, sincewater instead of organic solvent can be used.

As mentioned above, weakly hydrophobic monolithicmicrocolumns are suitable for separation of proteins inthe HIC mode. Neither poly(BuMA-co-glycerol dimeth-acrylate) nor poly(BuMA-co-butanediol dimethacrylate)was useful for HIC of proteins due to the significanthydrophobicities of the monoliths. To achieve thedesired hydrophilicity of the stationary phase, HEMAwas added to the polymerization mixture with the inten-tion of decreasing retention in dilute aqueous buffers toa level useful for HIC [104]. The resulting poly(BuMA-co-HEMA-co-butanediol dimethacrylate) monolith was usedfor the separation of a protein mixture consisting of myo-globin, ribonuclease A and lysozyme under linear gra-dient conditions and at a flow rate of 2.5 lL/min. Thefunctionalized monolith by direct copolymerization ofEDMA, HEMA, and 2-vinyl-4,4-dimethylazlactone wasused to prepare microreactors for immobilization oftrypsin and assay of enzymatic activity [105 –107]. As aresult of their high digestion rate, these reactors couldbe easily coupled in-line with liquid chromatography orelectrochromatography to enable the separation ofdigested proteins. A highly porous (up to 80% pore vol-ume) open-cellular monolithic polymer was preparedfrom HEMA and MBAA (or EDMA) using a high internalphase emulsion. This property may be particularly valua-ble in chromatographic applications involving biomole-cules such as proteins [108].

PEG is a neutral, non-toxic polymer that has an affinityfor water, which helps to create a microenvironmentconducive for protein stabilization and improved biomo-lecular interactions [109]. Oligoethylene glycol is themost common functional group used to resist non-spe-cific adsorption of proteins and other biological materi-als. This protein-resistant characteristic is probablycaused by a steric stabilization effect or low van derWaals interaction with proteins [110, 111].

Poly(GMA-co-EDMA) is one of the most prevalentcopolymer systems, since the epoxy groups can be easilymodified with functionalities for specific interactions,such as hydrophobic, ion exchange, affinity and reversedphase separations. Additionally, the remaining epoxygroups can be hydrolyzed into diol functionalities toincrease the hydrophilicity of the monolith. However,even if the epoxy groups are blocked, there is not suffi-cient hydrophilicity to minimize non-specific proteinadsorption in an aqueous mobile phase without anorganic additive. Recently, Krenkova et al. grafted a poly-(GMA-co-EDMA) monolith with PEGMA in order to elimi-nate non-specific adsorption of proteins and peptidesbefore reacting with azlactone functionalities forenzyme immobilization [112]. Figure 2 shows the dra-matic difference in adsorption of fluorescently labeledBSA before and after PEGMA grafting.

Surface-modified macroporous poly(BuMA-co-EDMA)monoliths that resist adsorption of proteins can be pre-pared using either single- or two-step photografting of aseries of hydrophilic monomers [77, 113], such as AAm,HEMA, vinyl pyrrolidinone, and PEGMA. Photograftedlayers of PEGMA reduced protein adsorption to less than2% relative to unmodified surfaces. The sequential two-step photografting process consisted of (1) formation ofcovalently bound surface photoinitiator sites followedby (2) surface-localized graft polymer. Monomer concen-tration and irradiation time during photografting werefound to be the most important parameters for optimiza-tion of the two-step process. Surface modification using atwo-step sequential photografting technique leads to asubstantial increase in surface hydrophilicity and a largereduction in protein adsorption. This photografting tech-


Figure 2. Assay of the sorption of fluorescently labeled BSAon poly(GMA-co-EDMA) monolith with hydrolyzed epoxyfunctionalities and after its grafting with PEGMA using amobile phase containing different percentages of acetonitrile.Reprinted with permission from Ref. [112].


nique should allow monoliths to be used for a variety ofbioanalytical applications that require minimal nonspe-cific adsorption of biomolecules. Hydrophilic photograft-ing should also be useful for creating nonfouling surfa-ces in applications such as mixers, valves, and filters.

Oligo(ethylene glycol) dimethacrylates with differentlengths of PEG bridging moieties between methacrylateunits have been used to copolymerize with BuMA for RPCof proteins. However, the hydrophilic crosslinkers do notsignificantly change the retention behavior of the col-umn since the hydrophobicity of the stationary phaseoriginates mainly from the butyl groups in the monomerand the hydrocarbon polymer backbone [114]. However,three PEG molecules (PEG-methacrylate, -diacrylate and-dimethacrylate) could be incorporated into galactose-based polyacrylate hydrogels to reduce non-specific pro-tein binding [115].

Recently, PEG-acrylates and PEG-diacrylates contain-ing three or more moieties in the PEG chains were usedto synthesize monoliths with improved biocompatibil-ity. The monomers polyethylene glycol methyl etheracrylate (PEGMEA) and polyethylene glycol diacrylate(PEGDA) were copolymerized for a porous monolith thatresists adsorption of both acidic and basic proteins whenusing an aqueous buffer without any organic solventadditives [116]. Compared to EDMA-based monoliths,clean fluorescent images obtained after flushing PEGDA-based monoliths loaded with fluorescein isothiocyanate(FITC)-labeled BSA were demonstrated to show negligiblenonspecific adsorption of proteins (see Fig. 3). Based onthese results, SEC monoliths with various molecularweight separation ranges for peptides or proteins wereprepared [116, 117]. These monoliths are expected to beuseful for biological sample preparation and purifica-tion, such as removal of nonprotein contaminants (DNA,viruses), protein aggregate removal, the study of biologi-cal interactions, and protein folding.

A strong cation-exchange (SCX) column that possessesnegligible non-specific interactions (i. e., hydrophobicinteractions) was prepared from copolymerization ofvinylsulfonic acid (VS) and the biocompatible cross-linker, PEGDA [118]. Excellent peak shapes for peptidesand proteins were obtained without addition of acetoni-trile in the eluent (see Fig. 4). The reduced hydrophobic-ity of the stationary phase originates mainly from thelow hydrocarbon content of the functional monomer(VS) and the PEG units in the biocompatible crosslinker.The hydrophobicities of the monoliths were comparedunder RPC conditions, yielding retention factors ofalmost zero for a poly(PEGDA) monolith and 4.4 for apoly(EDMA) monolith. EDMA is the most widely usedcrosslinker used for synthesizing polymethacrylate-based monoliths. When EDMA was replaced by PEGDA,the new poly (GMA-co-PEGDA) monolith provided betterresolution between conalbumin and ovalbumin, largely

due to the reduced tailing of the conalbumin peak [119].High recovery of proteins could be obtained on themonoliths incorporating PEGDA as crosslinker. Addition-ally, two novel polymeric monoliths for anion exchangecapillary LC of proteins were prepared in a single step bya simple photoinitiated copolymerization of two amine-containing monomers with the biocompatible cross-linker, PEGDA, in the presence of selected porogens.

A poly(HEMA-co-PEGDA) monolith was incorporated inan electric field gradient focusing microfluidic device toreduce the bandwidths of focused proteins in the chan-nel. The focused protein bands were symmetrical due tonegligible interaction between the monolith and pro-teins [120]. A poly(methacrylic acid-EDMA) monolith wasshown to be biocompatible when used for in-tube solid-phase microextraction (SPME) of several antibiotics andangiotensin II receptor antagonists [121, 122]. The SPMEcolumns could be reused without loss of extraction effi-ciency since they did not foul easily.

Recently, Courtois et al. proposed the utilization of PEGas a constituent of the porogen solution to obtain a more


Figure 3. LIF images of monoliths before, during and afterloading of FITC-BSA. An LIF image was first recorded beforeloading of FITC-BSA, for which a dark background wasobtained for all monoliths. Each monolithic column wasloaded with 0.01 mg/mL FITC-BSA and the fluorescenceimage was taken. Then each was flushed with 100 mM (pH7.0) phosphate buffer containing 0.5 M NaCl for 5 min at alinear velocity of ~4 mm/s before taking the LIF image again.(A) PEGMEA/EDMA monolith; (B) EDMA monolith; (C)PEGDA monolith; (D) PEGMEA/PEGDA monolith. Reprintedfrom J. Chromatogr. A 2005, 1079, 382–391, Gu, B.,Armenta, J. M., Lee, M. L., Preparation and evaluation ofpoly(polyethylene glycol methyl ether acrylate-co-polyethy-lene glycol diacrylate) monolith for protein analysis, with per-mission from Elsevier.


biocompatible monolithic support [123]. The idea was toproduce a monolithic support with large protein-com-patible pores characterized by a protein-friendly surface,which would not cause unfolding or denaturation of theseparated proteins. The authors used 1.5–20 kDa PEGsdissolved in 2-methoxyethanol and a series of co-poro-gens as a porogen system.

5 Conclusions

The concept of “biocompatibility” was applied success-fully to chromatographic stationary phases. A biocom-patible stationary phase material should be able to resistnonspecific adsorption of biomolecules and preserve thebioactivity of the target biomolecules. The characteristicsof biocompatible materials that resist protein adsorptionwere also summarized. Generally, there are two methodsto obtain a biocompatible stationary phase: surface mod-ification of a suitable polymer and direct copolymeriza-tion of hydrophilic monomers. The two main classes of

biocompatible monoliths were reviewed, including poly-acrylamide-based and poly(meth)acrylate-based mono-liths. Integration of PEG into the polyacrylate or poly-methacrylate monolith network proved beneficial in thereduction of non-specific protein interaction.

Two of the most significant advantages of monolithsare easy fabrication and abundance of chemistry. How-ever, the surface modification approach, which is themost complex, is the most widely used. Recent introduc-tion of surface-reactive monomers or biocompatiblemonomers could simplify this process. It is anticipatedthat in addition to developing more convenient andeffective surface modification techniques for monolithfabrication from conventional commodity polymers, theuse of functional monomers with inherent propertiessuitable for specific chromatographic applications,including improved biocompatibility, will receive moreattention in the future.

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Figure 4. (A) SCX chromatography of synthetic peptidesusing a poly(VS) monolith. Conditions: 16 cm675 lm i.d.poly(VS) monolithic column; buffer A was 5 mM phosphate(pH 2.7) and buffer B was buffer A plus 0.5 M NaCl, both buf-fers containing 0% acetonitrile; 2 min isocratic elution of 1%B, followed by a linear AB gradient (2.5% B/min) to 100% B;24 lL/min pump master flow rate; 8 min gradient delay time;102 nL/min column flow rate. (B) SCX chromatography ofproteins. Conditions: 16 cm675 lm i.d. poly(VS) monolithiccolumn; buffer A was 5 mM phosphate (pH 6.2) and buffer Bwas buffer A plus 1.0 M NaCl; 24 lL/min pump master flowrate; column flow rate was 104 nL/min; gradient delay timewas 8 min; linear gradient from 1 to 50% B in 20 min, rampedto 100% B in 2 min, and held for 20 min; analytes, (1) 1.14mg/mL cytochrome c, (2) 1.60 mg/mL a-chymotrypsinogenA, (3) 1.10 mg/mL ribonuclease A, and (4) 1.50 mg/mL oflysozyme. Reprinted with permission from Ref. [118].


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biocompatible polymeric monoliths for protein and peptide separations

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