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Review
Judit Horvath Vladislav Dolnk Molecular Dynamics Inc., Sunnyvale, CA, USA
Contents
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 . . 5.3.1.1 Poly(acrylamide) and its derivatives . 5.3.1.2 Epoxide-based coating . . . . . . . . . 5.3.1.3 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
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)
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.
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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].
CE and CEC
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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.
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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].
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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.
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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
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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 5.3.1.1 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
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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].
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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].
5.3.1.7 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.
5.3.1.5 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].
5.3.1.8 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.
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].
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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
7 References
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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
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