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Electrophoresis 2001, 22, 644655

Judit Horvath Vladislav Dolnk Molecular Dynamics Inc., Sunnyvale, CA, USA

Polymer wall coatings for capillary electrophoresis

This review article describes the preparation of dynamic and static polymeric wall coatings for capillary electrophoresis. Properties of bare fused-silica surfaces and methods for the characterization of capillary coatings are summarized. The preparation and basic properties of neutral and charged wall coatings are considered. Finally, advantages and potential applications of various coatings are discussed.
Keywords: Capillary electrophoresis / Wall coating / Dynamic coating / Static coating / Electroosmotic flow / Adsorption / Review EL 4304

1 2 3 4 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 Introduction . . . . . . . . . . . . . . . . . Properties of the fused-silica surface Characterization of a capillary coating Dynamic wall coating . . . . . . . . . . . Polymeric surfactants . . . . . . . . . . Charged polymers . . . . . . . . . . . . . Neutral polymers . . . . . . . . . . . . . . Polysaccharides and their derivatives Poly(vinyl alcohol) . . . . . . . . . . . . . Poly(alkylene glycol) polymers . . . . Poly(ethylene oxide) . . . . . . . . . . . Poly(vinylpyrrolidone) . . . . . . . . . . Poly(dimethylacrylamide) homoand copolymers . . . . . . . . . . . . . . 4.3.7 Copolymers of acrylamide . . . . . . . 4.3.8 Biopolymer coating . . . . . . . . . . . . 5 Permanent wall coating . . . . . . . . . 5.1 Pretreatment of the capillary . . . . . . 5.2 Intermediate layer . . . . . . . . . . . . . 5.3 Polymer top layer . . . . . . . . . . . . . 5.3.1 Neutral, hydrophilic polymer layer . . Poly(acrylamide) and its derivatives . Epoxide-based coating . . . . . . . . . Diol-bonded capillaries . . . . . . . . . . ... ... .. ... ... ... ... ... ... ... ... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644 645 645 645 646 647 647 647 647 648 648 648 648 648 649 649 649 649 650 650 650 651 651 5.3.2 5.3.3 6 7

PEG coatings . . . . . . . . . . . . . . . . . PVA . . . . . . . . . . . . . . . . . . . . . . . Cellulose derivatives . . . . . . . . . . . . Dextran . . . . . . . . . . . . . . . . . . . . . PVP . . . . . . . . . . . . . . . . . . . . . . . Charged, hydrophilic polymer coatings Hydrophobic coatings . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1 Introduction
There are many reasons for chemical modification of the capillary wall in electrophoresis. Goals may include reduction or elimination of analyte-wall interactions, alteration of electroosmotic flow (EOF) to effect more rapid separation, improved reproducibility, or resolution of particularly difficult separation problems. The hydrophilicity of the coating is a very important factor. Strongly hydrophilic coatings are usually optimal for separation of biomolecules, though other applications may require hydrophobic properties. An ideal capillary wall coating is stable under conditions required for separation, preferably over a broad range of buffer pH. Performing coating reactions inside the capillary is an area of active research. This reflects the inherent inaccessibility and low surface area of the capillary lumen, and the limited availability of suitable analytical tools. Homogeneity and reproducible application of the coating are two areas of particular importance. Capillary wall coatings are described as dynamic or static, based on the attachment of the coating to the capillary wall surface. Dynamic coatings involve adsorptive secondary interactions, while static coatings are based on covalent bonding between the capillary wall and the coating material. Hybrid coat0173-0835/01/0404-644 $17.50+.50/0

Correspondence: Dr. Judit Horvath, Molecular Dynamics Inc., 928 East Arques Avenue, Sunnyvale, CA 94085, USA E-mail: judit.horvath@am.apbiotech.com Fax: +1-408-737-4887 Abbreviations: HEC, hydroxyethylcellulose; HPC, hydroxypropylcellulose; PEI, poly(ethyleneimine); PEO, poly(ethyleneoxide); poly(AEG), poly(acryloylethoxyethyl glucopyranose); PVA, poly(vinyl alcohol)

 WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001

Electrophoresis 2001, 22, 644655 ings are a combination of dynamic and static coatings, with one layer held by covalent forces while another layer is adsorbed to the adjoining surface.

Wall coatings for CE


2 Properties of the fused-silica surface

It is generally accepted that there are 45 silanol groups/ nm2 on the smooth, nonporous heat-stabilized amorphous silica surface. The exact number of silanol groups is determined by the silica treatment, and is smaller than a theoretically calculated value of 7.8 groups/nm2 [1]. In addition to isolated silanol groups, there are geminal and vicinal silanol groups present at the silica wall [2] that exhibit characteristic infrared (IR) spectra [3]. Silanol groups ionize in aqueous solutions, bringing a charge to the capillary surface and causing an EOF during capillary electrophoresis (CE). The acid-base ionization constant (pKa) of silanol groups ranges between 2 and 9, as determined by the curve of the dependence of EOF of an untreated capillary on pH [4]. This is not surprising if the silica surface is modeled as a polyanion. By analogy, the pKa of carboxylic groups varies widely in multiprotic carboxylic acids. Silica capillaries also exhibit a pH hysteresis effect because the electroosmotic mobility at the same pH differs, depending upon whether the pH value was obtained by acidification or alkalization [5]. At a pH above 12, EOF is reduced, probably because the formation of a soluble silicate polymer at the capillary surface increases the viscosity of the electric double layer [6].

Another method uses a CCD camera to follow the motion of a fluorescent neutral marker zone along the length of the capillary [12]. An alternative method of characterizing capillaries involves measuring their streaming potential, which is the potential generated by flow of a liquid along a charged surface [13]. Atomic force microscopy (AFM) can also be used to probe the coated inner surface. Barberi and co-workers [14] and Cifuentes and co-workers [15] used AFM to probe the inner surface of acrylamidecoated capillaries. They found a significant correlation between AFM and CE methods. Rough surfaces gave relatively poor CE performances compared to surfaces with homogeneous polyacrylamide coatings. In these studies, the thickness of the homogeneous coating was noted to be a few nanometers. Gas chromatography (GC) was used by Kohr and Engelhardt [7] to measure the dependence of analyte retention on temperature, to characterize the coating procedure for nonpolar coatings, and to differentiate between polar and nonpolar interactions. They found these values in good agreement with measurements of the EOF and its dependence on pH. Free-solution CE has been used in combination with mathematical modeling to estimate the surface density, the pK of surface chemical groups, and the coating thickness of neutral hydrophilic coatings [16].

4 Dynamic wall coating 3 Characterization of a capillary coating

Characterization of capillary coatings is extremely difficult because of the small size and relative inaccessibility of the surface involved. Characterization of the properties of coated capillaries is commonly performed to measure the EOF and investigate its dependence on the pH of the buffer [7]. Measurement of the residual EOF in the coated capillary can be obtained by photometric monitoring of the electrophoretic pattern of a neutral marker. This method, however, becomes time-intensive when the residual EOF is very low. Alternative methods have been developed to avoid long runtimes and related experimental difficulties. In one such method, the signal at the detection points is measured by electroosmosis after a neutral marker is injected, then rinsed by applying a low pressure [810]. The precision of this measurement can be increased by evaluating the relative position of three peaks of the neutral marker, during which two of the peaks are injected, pressure-mobilized, and separated, while the third is injected and pressure-mobilized past the detector [11]. The principles of this method are summarized in Fig. 1 [11]. Dynamic wall coating is an attractive coating method because it overcomes difficulties in conducting reproducible, homogeneous chemical derivatization reactions in the capillary lumen. Dynamic coating is typically prepared by rinsing the capillary with a solution containing a coating agent that is either a polymer or a small molecular-mass compound. Because the attachment of the coating to the wall is based on adsorption, a small amount of coating agent is usually added to the separation medium to keep the coating on the capillary wall surface. The lifetime of dynamic-coated capillaries can be extended by using an occasional, simple regenerating process. Many types of polymeric and small molecular mass buffer additives are used as dynamic coatings. This review examines only polymeric buffer additives. Dynamic coating prepared with small molecular mass additives are summarized in another review article (by Righetti) in this issue. Self-coating sieving matrices can also be used as an effective means of establishing the dynamic coating on the capillary wall. In this case, the capillary is filled with a polymer solution that partly adsorbs to the capillary surface, forming a dynamic coating and, simultaneously, a

CE and CEC


J. Horvath and V. Dolnk

Electrophoresis 2001, 22, 644655

Figure 1. Principle of highspeed determination of EOF. Reprinted from [11], with permission.

three-dimensional sieving matrix for separation of various biomolecules in CE. No separate coating process is necessary. In some cases, however, obtaining a reproducible surface coating requires that the capillary surface be reactivated after every run or after a brief period of consecutive runs. Numerous natural and synthetic, charged and neutral polymers have been tried in the pursuit of the best dynamic coating. Novel polymers have been synthesized and various coating methods have been developed for different types of analytes. Neutral polymers are used most often, as they eliminate electrostatic interactions between the analyte and the capillary wall. Charged surface coatings were developed for specific separation problems

such as fast separation, reversed EOF, and separation of complex analyte mixtures.

4.1 Polymeric surfactants

Adding a small amount of polymeric surfactant to the running buffer is a very simple, easy way to achieve dynamic coating on the capillary surface. Poly(n-undecyl-a-D-glucopyranoside) (PUG), a novel, nonionic micelle polymer, provides baseline separation of seven tricyclic antidepressants [17]. Polyoxyethylene ether (Brij-35), a well-known nonionic surfactant, is also a powerful tool for taxonomic investigations [18].

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4.2 Charged polymers

The adsorption of charged polymers introduce positive or negative charges on the surface, thus changing the direction and/or the strength of the EOF, as well as the analyte-wall interaction in the separation capillary. This method is usually used to solve a specific separation problem, i.e., fast separation of positively charged analytes. Reversed EOF can be achieved by coating capillaries with strong cationic polymers such as polyamine (i.e., eCAPTM) [19, 20], poly(dimethyldiallylammonium chloride) (PDMAC) [2124], poly(ethyleneimine) (PEI) [25], Polybrene (PB) [26, 27], poly(arginine) (PA) [28] and a cationiccyclodextran [29]. Cationic capillary coatings effectively separate positively charged proteins, peptides, and drug samples. PEI is useful either as an immobilized or physically adsorbed layer. Adsorbed coated PEI capillaries are fast and easy to prepare, and have an excellent stability between pH 3 and 11 [25]. A comparison of Polybrene and polyarginine (PA) coated capillaries found that PA capillaries offer higher efficiencies, but inferior stability compared to Polybrene capillaries [28]. When compared to PDMAC-coated capillaries, however, Polybrene coatings are less reproducible and require more frequent application of coating material [24]. In another method, a polyelectrolyte multilayer coating was prepared by alternately rinsing the capillary with positively and negatively charged polymers. The resulting capillaries were stable under extreme pH and ionic strength for the separation of a series of basic proteins [30]. Highly stable, irreversibly adsorbed cationic capillaries can be prepared by coating the inner surface of the fused-silica capillary with 40140 nm polystyrene particles derivatized with aw diamines [31]. The original efficiency of these particle-coated capillaries was low, but improved dramatically after in-column derivatization of the amino functionality with 2,3-epoxy-1-propanol, which significantly increased the hydrophilicity of the coating. This method separated five basic and four acidic proteins in less than 7 min with efficiencies of up to 1 900 000 theoretical plates per meter [31]. A stable wall modification was accomplished by creating successive multiple ionicpolymer layers (SMIL) [3234]. An anionic polymer (i.e., dextran sulfate) was tightly fixed to the capillary wall by a cationic polymer (i.e., Polybrene) that attached to the uncoated fused silica capillary by noncovalent bonding. Reproducibility of this coating process was excellent, and the capillary demonstrated an acceptable lifetime. Capillaries modified with anionic dextran sulfate exhibited pH-independent EOF between pH 2 and 11 and supported efficient separation of acidic proteins and amino acids [34]. A comparison of two anionic coatings revealed

an important role for the hydrophilic nature of the coating on protein separations; better protein separation was obtained with the more hydrophilic dextran sulfate than the more hydrophobic poly(vinyl sulfonic acid) [23].

4.3 Neutral polymers

The goal of introducing neutral polymers to the capillary surface is usually to reduce or eliminate EOF as well as analyte-capillary surface interaction in the separation capillary. The hydrophilicity of the coating polymer is very important: increasing the hydrophilicity will reduce sample-wall interaction in biopolymer separations, however, it will also reduce the stability of the absorbed coating.

4.3.1 Polysaccharides and their derivatives

Several cellulose derivatives have been identified as coating-forming buffer additives that effectively mask surface silanols and suppress EOF. Cellulose acetate, cellulose triacetate and cross-linked hydroxypropylcellulose (HPC) coatings offer efficient separation of basic proteins [35]. Hydroxypropylmethylcellulose separates histone H1 subtypes at low pH [36], and hydroxyethylcellulose (HEC) can be used as a coating and sieving matrix for DNA restriction fragments [37]. Cellulose-based absorbed coatings, such as hydroxypropylmethylcellulose, can easily be washed off the capillary wall because of their hydrophilicity. These compounds must, therefore, be added to the buffer to replace polymer lost from the capillary surface [38, 39]. Epoxybutane-modified hydroxypropylcellulose (EB-HPC) has been used to form a hybrid coating for protein separations [40]. In this method, the capillary wall is chemically bonded with cationic surfactant moieties, then modified EB-HPC is adsorbed to the surface. Although the capillary surface is charged, the presence of the adsorbed polymer suppresses or, in most cases, eliminates the EOF. This adsorbed layer was stable for more than 8 h of operation without the addition of polymer to the separation buffer. Guaran, a natural polysaccharide, does not form a stable coating, but can be used as a buffer modifier to improve the separation of basic proteins and drugs by CE [41].

4.3.2 Poly(vinyl alcohol)

Poly(vinyl alcohol) (PVA) binds more strongly to silica surfaces than does HEC, so columns coated with this compound have improved stability. PVA-coated capillaries offer good separation of DNA restriction fragments with both HEC and acrylamide matrixes, though PVA cannot be used as a sieving matrix because of the formation of inhomogeneous networks after only a few separations [37].


J. Horvath and V. Dolnk

Electrophoresis 2001, 22, 644655 viscosity. This medium gave approximately 600 bases read length in approximately 2 h of run time, and allowed 100 or more successive sequencing runs without reconditioning of the capillary surface. PDMA also efficiently reduces the EOF and DNA-wall interactions in uncoated capillaries. Stability of the coating improves dramatically when the separation pH is changed from 8.3 to 7.8 [51]. To improve thermal stability of the noncovalent coating, small amounts of high-molecular-mass PDMA must be added to the separation medium [38]. Although PDMA has excellent adsorptive properties, it is rather hydrophobic, which is undesirable for protein and DNA separations. To combine adsorbing properties with hydrophilicity, a copolymer of dimethyl acrylamide and allyl-glycidyl ether: poly(dimethylacrylamide-co-allyl glycidyl ether) (EPPDMA) was introduced recently [52]. The oxirane units of the hydrophilic allyl-glycidyl ether comonomer improve the overall hydrophilicity of the copolymer, while keeping the excellent adsorptive properties of the PDMA polymer. These new adsorbed coated capillaries offer excellent separations in DNA fragment analysis using various sieving matrixes [52] as well as in DNA sequencing, where read lengths over 600 bases were achieved routinely in 90 min of run time [53]. The EPPDMA capillaries showed excellent stability without the need to add polymer into the running buffer. No treatment of the capillaries was necessary for over 200 sequencing runs (300 h). After that, regeneration was achieved in less than 1 h by simply rinsing the column with an alkaline solution followed by the coating polymer solution.

4.3.3 Poly(alkylene glycol) polymers

Neutral, hydrophilic alkylene-glycol polymers efficiently coat the capillary walls and reduce EOF and wall adsorption. Various nucleotide mixtures were separated by using UCON (a poly alkylene glycol-based synthetic lubricant) coated capillaries [42] and PEG as buffer additives (13%) [43].

4.3.4 Poly(ethylene oxide)

Yeung and Fung introduced linear poly(ethylene oxide) (PEO) as a replaceable sieving matrix for DNA analysis. This compound has self-coating characteristics, so PEOcoated capillaries can be prepared using PEO solutions as sieving matrices [44] or coating agents [45]. Silanol groups must be fully protonated to achieve good coating characteristics, so capillaries must be washed before coating and between runs with hydrochloric acid followed by PEO solutions. The major disadvantage of the PEO coating/matrix system is that these capillaries need 1 h of reconditioning after every 30 runs. PEO is also an efficient dynamic coating and sieving matrix when used as a triblock copolymer with poly(propylene oxide) (PPO). The viscosity-adjustable property of the block copolymer is useful for automated separation of DNA samples with CE [46, 47]. Another copolymer of PEO, the grafted poly(Nisopropyl acrylamide)-g-poly-(ethylene oxide) has excellent self-coating properties and a slightly temperaturedependent viscosity-adjustable property, which is useful for the separation of relatively small sized DNA by CE [48].

4.3.7 Copolymers of acrylamide 4.3.5 Poly(vinylpyrrolidone)

Poly(vinylpyrrolidone) (PVP) is an effective sieving matrix in uncoated capillaries for genotyping and sequencing applications [49] using a simple column regeneration step between runs. Compared to PEO coating, PVP coating offers more efficient EOF reduction and significantly shorter regeneration times (3 min versus 2 h). PVPcoated capillary arrays can be used with a replaceable PEO sieving matrix in high throughput DNA sequencing [50]. Poly(acrylamide) is a very hydrophilic polymer and wellknown for its excellent permanent capillary wall coating and sieving properties. Because of its hydrophilicity, however, poly(acrylamide) does not bind well to the silica surface [38]. To include acrylamide polymer in adsorbed coatings for protein and DNA separations in CE, novel copolymers were synthesized using acrylamide and another monomer that has adsorptive potential [39]. The copolymers were designed to consist of highly hydrophilic monomers: an alkyl backbone bearing glucose and oxyrane units copolymerized with acrylamide. The resulting poly(acrylamide-co-allyl a-D-glucopyranoside) (poly(AGAA)) and poly(acrylamide-co-allyl a-D-glucopyranosideco-allylglycidyl ether) (epoxypoly(AG-AA)) are highly hydrophilic polymers that adsorb to the silica surface. The introduction of epoxy groups pendent from the polymer backbone dramatically improved the adsorption, and very stable columns and efficient protein separations were achieved [39].

4.3.6 Poly(dimethylacrylamide) homo- and copolymers

Madabhushi [38] introduced a low-viscosity polymer solution, poly(dimethyl acrylamide) (PDMA) as a self-coating sieving matrix for DNA sequencing. Low-molecular-mass PDMA was introduced for this application to ensure low

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4.3.8 Biopolymer coating

An interesting idea to control protein adsorption was to develop a protein coating on the surface of the capillary [54]. The protein fibrinogen was tested and the adsorbed layers of fibrinogen were thermally treated to avoid desorption when exposed to an electric field.

layer [58] to form a resin in the deactivation reaction, which effectively covers any unreacted silanols. Most covalent binding procedures consist of several consecutive steps, making the capillary coating time-consuming, difficult to execute, and irreproducible. To simplify the process, Malik and co-workers [59] performed the entire coating process in one step. Capillaries were filled initially with a methylene chloride solution containing the coating polymer instead of the monomer (such as polyethers, HPC and PEI), the surface derivatization agent, and a free radical initiator. A brief heat treatment was then used to immobilize the polymer film on the capillary surface. This new method yielded efficient columns for protein separations, minimized lot variation and promoted reproducibility during consecutive runs. A variation of this method was published later, and involved separating the silica surface derivatization step from the bonding step, while still using a polymer solution in place of monomers [60]. This method, however, reduced the benefits of the original one-step method. The main disadvantage of using silanes to deactivate the silica surface is that the resulting siloxane bond offers unsatisfactory hydrolytic stability at alkaline pH. To improve the stability of the final coating, Cobb and co-workers [61] replaced the Si-O-Si bonds with Si-C bonds in a procedure using Grignard chemistry. This procedure has been modified later to allow large-scale production of capillaries to produce coated capillary arrays for the MegaBACETM DNA sequencer [62]. Nakatani and co-workers [63] modified the original Grignard chemistry method and confirmed the stability of the Si-C bond based coating. They found the Si-C coating was stable up to 30 days at pH 9.2 or at 30oC at pH 2.38.0, while the coating containing siloxane bonds was damaged at a pH > 4.6 [6466]. Figure 2 demonstrates the alkaline stability of various capillaries [63]. Another variation of the original Grignard surface modification used a more aggressive chlorination procedure (with SiCl4) to reduce the remaining EOF in the capillary [67]. Pesek et al. [68] introduced another method of forming a hydrolytically stable intermediate layer based on hydride-silica chemistry. The method uses catalytic hydrosilylation of terminal olefins on a SiH-containing substrate. Besides achieving superior hydrolytic stability olefin hydrosilylation provides the option of preparing g-methacryloxypropyl-modified surfaces suitable for copolymerization with acrylamide and its derivatives. When acryloyl amino ethoxyethanol was used, the coating had an excellent hydrolytic stability, and its lifetime at pH 8.5 was more than twice that of the conventional polyacrylamide [68

5 Permanent wall coating

A permanent wall coating is an attractive way to eliminate EOF and wall-analyte interaction in the separation capillary. Dynamic coated capillaries require occasional regeneration, and addition of the coating agent into the separation buffer might also be necessary. The use of uncoated capillaries with self-coating sieving matrixes is compelling, but the capillary performance deteriorates during repetitive runs and extensive rinsing is required between runs. Based on their separation performance, acrylamidecoated capillaries are still superior to any other type of coating and cannot be prepared by adsorptive methods. Consequently, there is a strong driving force to prepare hydrolytically stable, reproducible, covalently bonded capillaries for various separation purposes. Preparation of a permanent wall coating typically consists of three steps: capillary pretreatment, introduction of double bonds to the capillary wall, and binding of a polymer to this intermediate layer.

5.1 Pretreatment of the capillary

To achieve the best coating results, the capillary surface must be cleaned and activated by etching and/or leaching prior the coating process. The effect of each capillary preparation step (etching, leaching, dehydration, silylation, and coating) on the final performance of the column has been studied extensively [55]. Optimal coating reproducibility requires column etching with sodium hydroxide followed by leaching with hydrochloric acid. To improve the yield of the silylation reaction, all water has to be removed from the surface: 160oC overnight dehydration allows the best result [55].

5.2 Intermediate layer

Attaching a polymer coating to the capillary wall can be done easily using a reactive bifunctional silane such as gmethacryloxy propyl trimethoxy silane described by Hjertn [56, 57]. The surface silanols react with the silane group of the reagent, then the other functional group is used to attach and polymerize monomers to the capillary surface. A modified method uses a new polymer (polymethacryloxy propyl hydrosiloxane) as an intermediate


J. Horvath and V. Dolnk

Electrophoresis 2001, 22, 644655 roles in controlling the properties of the final coated capillary. Polymerization is usually done in situ in the capillary and, since the polymerization cannot be driven to a 100% yield, residual unreacted monomer can be left entangled in the polymer layer. Another potential problem with free radical polymerization performed within the capillary tube is that polymerization also proceeds in the solution and not only on the surface. This process removes monomer from the surface coating, causing variability in the coating thickness. It also increases the viscosity of the solution, impeding removal of the polymerization solution from the capillary. The use of surface confined living radical polymerization was proposed to bind both linear and crosslinked polymer films to the silica surface in a controlled manner [76]. In this method, polymerization is intrinsically confined to the surface, enabling control of film thickness and eliminating the danger of clogging the capillaries. Righetti and co-workers [81] found that the column performance improves dramatically after the addition of 3% short chain polyacrylamide to the running buffer. They concluded that there may have been uncoated patches on the surface of the capillaries, creating naked sites that could be dynamically coated by small molecular weight polymer added to the run buffer. In this way, covalent coating can be complemented by dynamic coating of the uncoated sites.

Figure 2. Time course of electroosmotic mobility (cm2/ Vs) as a function of number of days of exposure to pH 8.0. (*) Uncoated capillary; (&) coated capillary through siloxane linkages; (~) coated capillary through Si-C linkages. Reprinted from [63], with permission. 70]. The excellent hydrolytic stability of the poly(acryloylethoxyethyl glucopyranose) (poly)-coated capillaries is illustrated in Fig. 3 [70]. Other methods for improving the stability of the olefinic sublayer used long-chain alkoxy-silane with terminal olefinic group [71] or active silane transfer agents like acetylacetone vinyldimethylsilylenolate [72]. Another method uses a sol-gel process to prepare a stable, homogeneous sublayer with high density of polymerizable olefinic groups. Formation of this sublayer was independent of the initial surface properties of the fused-silica capillary, and the process was robust and easy to use. The resulting layer is very stable: at pH 10, the EOF remains constant on a low level over a long period of time [73]. A similar sol-gel process was used to prepare a hydrolytically stable amino-silica glass coating [74] and UCON-coated columns [75].

5.3.1 Neutral, hydrophilic polymer layer Poly(acrylamide) and its derivatives
Poly(acrylamide) has been used in many wall coatings [57, 61]. An extensive study evaluated the effect of polymerization conditions on properties of the final acrylamide coated capillary [77]. Leftover, unreacted acrylamide monomer in the wall coating can substantially increase the background absorbance (even at 254 nm) and leave potentially harmful reacting species in the background electrolyte. Consequently, a chemical scavenging method using cysteine was proposed to remove such material [78]. Poly(acrylamide) is a superb capillary coating, but is rather unstable at alkaline pH. Tremendous effort has, therefore, been dedicated to studying and improving its hydrolytic stability. One way of improving this stability is to cross-link the linear polymer chains. When a cross-linked polyacrylamide (acrylamide + bisacrylamide) coating was used instead of linear chains, the lifetime of the coating improved significantly, reaching 90 days in the range of pH 210 [79]. Efficient protein separations and hundreds of consecutive runs were achieved by using cross-linked sub- and toplayers [80]. First poly(vinylmethyl siloxanediol) was attached and cross-linked to the surface of a fused-silica column, then linear polyacrylamide was grafted

5.3 Polymer top layer

The attachment of the polymer layer to the wall can be achieved without the use of the intermediate layer by using heat or other treatments. This is true with PVA or polysaccharides, for example. In general, however, for static coatings, the attachment of the top polymer layer is done using a free radical polymerization reaction with the established reactive olefinic sublayer on the silica surface. Polymerization conditions including the concentrations of the monomer, initiator, and catalyst as well as their relative ratios, temperature, and time, play crucial

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Figure 3. Reproducibility of separation of (1) trypsin inhibitor, (2) b-lactoglobulin A, (3) blactoglobulin B, (4) L-asparaginase and (5) a-lactalbumin in a poly(AEG)-coated capillary at pH 8.5: 1st, 150th and 300th runs. Reprinted from [70], with permission. to the top of this polymeric sublayer. Finally, the linear poly(acrylamide) (LPA) toplayer was cross-linked with formaldehyde. Another means of improving stability is to use a monomer with increased hydrolytic stability in place of acrylamide. Poly(N-acryloyl aminoethoxyethanol) (poly AAEE) [81] combined very high hydrophilicity with extraordinary stability to alkaline hydrolysis [8284]. Unfortunately, the monomer can autopolymerize [85]. Another polymer, poly(Nacryloylaminopropanol) (polyAAP), was introduced [85, 86] and used successfully as a column coating and sieving matrix for electrophoresis of DNA fragments [8789]. A polymer wall coating made of poly(AEG) showed superior efficiency and migration time reproducibility for basic protein separations. The lifetime of this material was over 300 runs at pH 9.0 [70]. Poly(acryloyldiethanolamine) wall coating was prepared for capillaries in DNA sequencing and showed superb stability and a lifetime of over 500 runs [90]. diamine compounds as a cross-linker [93, 94]. This modified coating was stable between pH 2 and pH 12, but showed residual EOF. High-efficiency protein separations were achieved using Pluronic polymers (polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer) for the chemical modification of the capillary, which blocked protein adsorption to the surface [95]. Successful application of PEO-based surface modification has been developed for the separation of the 12 most common ribonucleotides, but requires that the silica surface first be deactivated with hexamethyldisilazane, then coated with PEO [96]. Diol-bonded capillaries

A simplified diol-bonding reaction was described recently [97] in which the capillary was treated with aqueous 3-glycidoxy propyl trimethoxysilane (GPTMS) at room temperature in a one-step procedure. The reaction involved a simple dynamic coating procedure at room temperature instead of the more typical static coating methods that require vacuum pumps and ovens. In the coated capillaries, there was still residual EOF which was low between pH 3 and pH 5 and slightly increased at pH > 6. Proteins and nucleotides were separated with high efficiency using this material, and columns demonstrated good stability at pH < 8. Epoxide-based coating

Epoxide monomers and polymers are attractive for use as capillary coatings because of their hydrophilicity and alkaline resistance. With this type of coating, however, a significant residual EOF is left in the capillary. Crosslinked epoxy polymer covalently attached through glycerylpropylsilane using multistep procedures resulted in columns with reduced protein adsorption and a strong enough residual EOF to carry both positive and negative protein analytes past the detector [91, 92]. This coating is stable between pH 4 and pH 10. Properties of the epoxide-coated capillary could be improved further by using PEG coatings

Hydrophilic PEG coatings are also useful for reducing nonspecific protein adsorption and EOF, and increasing wetting of the capillary surface. Fuzzy and interlocked


J. Horvath and V. Dolnk

Electrophoresis 2001, 22, 644655 pH 12 with SDS-containing buffer did not harm the capillaries [105]. A disadvantage of this method, however, was that the quality of the coating was strongly influenced by the actual silica surface, compromising reproducibility.

poly-ether-coated capillaries were introduced with various levels of EOF [98]. The fuzzy coating, which had an additional cross-linked polysiloxane sublayer, exhibited lower EOF than the interlocked coating which lacked this sublayer. The same group developed ionic poly-ether coatings which demonstrated reversible anodal/cathodal EOF [99]. When terminally activated PEG derivatives were grafted onto activated silica surfaces, it was found that the polymer conformation influences the grafting density [100, 101] and exhibits greatly reduced EOF between pH 2 and 11 [101]. Surprisingly, when PEG-coated capillaries were used with a methanolic electrolyte, the EOF was reversed (directed to the anode). As expected, cathodic EOF can be observed in aqueous electrolytes [100]. Dextran
Dextran is a very hydrophilic, neutral polymer, and a candidate for hydrophilic coatings for biopolymer separations. A covalently attached dextran coating subsequently cross-linked with PEG chains exhibited strong hydrophilicity, reduced EOF, and good chemical stability [105, 106]. It also gave high-resolution separations for acidic and basic proteins and mono- oligosaccharides. PVA
The strongly hydrophilic and neutral polymer, PVA, can be used either as a dynamic coating (buffer additive), or as a water-insoluble covalent coating on fused-silica surfaces. Permanent PVA coating can be readily achieved by thermal immobilization [100, 102]. The resulting coating was stable for a series of separations over a wide range of pH and also suppressed EOF even at higher pH values [100, 102]. PVP
In addition to its use as a dynamic coating, the very hydrophilic PVP can be covalently attached to the derivatized capillary surface [107, 108]. PVP coating can also be successfully used for acidic and basic protein separations.

5.3.2 Charged, hydrophilic polymer coatings

Introduction of positive surface charges can significantly reduce interaction with basic proteins, but irreversible adsorption of acidic proteins may occur. Binding weak cationic polymers to the surface creates a pH-dependent positive charge on the surface. Such polymers include copolymers of vinylpyrrolidone and vinylimidazole [107] or a homopolymer of poly(2-aminoethyl methacrylate hydrochloride) (PALM) [109]). The pH dependence of EOF in fused-silica capillaries can complicate CE method development. Strong cationic polymers such as a reactive polyamide resin [110], poly(vinylamine) [111], and PEI coating can be used to develop permanent positive coatings for separation of basic analytes. In a multilayer coating, the PEI layer was further coated with a neutral, top polyether layer [104]. This minimized electrostatic interactions between the permanent positively charged PEI polymeric layer and the negatively charged biomolecules, but kept the anodal flow constant, regardless of the electrolyte pH. The positively charged monomer can be combined with a neutral monomer such as acrylamide to modify the rate of the reversed EOF in the column. Trimethylammonium styrene chloride bonded to the vinyl-derivatized silica surface gave capillaries with reversed EOF up to pH 6.0 [112]. By adding acrylamide to the polymerization mixture, the resulting capillaries exhibited zero EOF above pH 7.0 and reversed EOF at lower buffer pH. Capillaries coated with the strongly anionic polymer, sodium-2-acrylamido-2-methylpropanesulfonate (NaAMPS), exhibited pH independent EOF [113]. Similar to the cati- Cellulose derivatives

To immobilize the cellulose coating on the wall (HEC and HPC), thermal treatment at 140oC can achieve a chemical reaction between the hydroxyalkyl-substituted cellulose and the fused-silica capillary inner wall [103]. The resulting immobilized cellulose-coated columns are stable between pH 3 and 10, allowing them to be used for isoelectric focusing, unlike adsorbed cellulose-coated columns which are unstable under isoelectric focusing conditions. Cellulose acetate, cellulose triacetate and cross-linked HPC are polymers with good film-forming properties. Using this characteristic, the polymers first were dynamically coated onto the silica surface and then physically adhered (skin coating) by helium purging [35]. Unfortunately, the stability of this skin coating is very limited, so it can only be used at pH < 7.5. Use of large-molecularmass HPC in a multilayered coating process, produced capillaries with negligible EOF. These were further modified with additional functionalization using a PEI layer to which a top polyether layer was attached [104]. Chemical bonding of the silica surface using the reaction between methacryl groups, activated silica surface, and derivatized allyl methyl cellulose (and also allyl-dextran) reportedly gave stable capillaries. Even short washes at

Electrophoresis 2001, 22, 644655 onic coating, capillaries with a particular EOF rate could be synthesized by using acrylamide as co-monomer and changing the molar ratio of NaAMPS to neutral acrylamide in the copolymerization mixture [113].

Wall coatings for CE


5.3.3 Hydrophobic coatings

One of the goals of capillary coating is usually to make the hydrophobic silica surface more hydrophilic. However, more hydrophobic coating may be desired for certain applications. Also, to increase hydrophilicity, a hydrophobic sublayer can be used to introduce the hydrophilic top layer. A strongly hydrophobic C-18 coating was prepared and used to adsorb the surfactant Brij-35 as well as amphiphobic copolymers and hydrophilic homopolymers with hydrocarbon backbones, including methylcellulose (MC), PVA, and PVP [114]. Similarly, a highly crosslinked layer was prepared from the copolymer of HPC and 2-hydroxyethyl methacrylate by static coating onto a wall that was previously treated to increase hydrophobicity using 7-oct-1-enyl trimethoxy silane. This formed a stable self-assembled alkyl-silane monolayer [115]. In another case, an inert, hydrophilic capillary coating, suitable for highly efficient protein separations, was prepared by adsorbing a nonionic surfactant hydrophilic polymer, or a polymer surfactant onto a poly(alkyl siloxane) bonded coating layer, utilizing hydrophobic interactions [116]. Linear hydrocarbons and ethylbenzene modified silica surfaces greatly improved the electrophoretic separations of organic amines [117]. Calixarene-coated capillaries exhibited special selectivity on phenolic compounds [118]. A tube-in-the-tube design used the fused-silica capillary as a mold in the in situ formation of, and as a sheath for, a highly cross-linked poly(styrene-divinylbenzene) inner tubing [119]. The thin-walled (35 mm), fluid-impervious, strongly hydrophobic polymer inner tubing is extremely stable against highly alkaline solutions. To confer hydrophilicity on the inner surface of the polymer tube, a poly(oxyethylene) oligomer was grafted to its inner wall and subsequently cross-linked. The hydrophilic, grafted coating was stable to hydrolytic attack by 1 M NaOH and was successfully used to separate basic proteins between pH 3 and 6.

procedure that is simple, reproducible and that yields chemically stable coatings with minimized interaction between the surface and the analyte. With the introduction of high-throughput capillary array electrophoresis separtions, the importance of a reproducible coating process increased dramatically. Covalently bonded capillaries still exhibit longer lifetimes and require less maintenance than dynamic coated ones, but the reliable preparation of these coatings can be challenging. Dynamic coated capillaries are attractive because of their ease of preparation, but more work is needed to reduce required capillary maintenance such as regeneration, washing between runs, and other recursive steps. Another potentially favorable application for the dynamic coating method is the preparation of microchips and microarrays, where filling and emptying microchannels with low viscosity polymer solutions can be done easily. Increased research efforts should further expand the repertoire of the available dynamic coating methods. This work was supported by NIH grant 2R44 HG0156302.
Received November 20, 2000

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6 Conclusions
Chemical modification (covalent or adsorptive) can dramatically change the properties of capillary surfaces and allow silica capillaries to solve various separation problems in CE. By choosing appropriate chemical modification of the inner surface of the capillary, the EOF and the hydrophobicity of the capillary column can be manipulated. Enormous effort has been made to find a coating


J. Horvath and V. Dolnk

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