POLYVINYLALCOHOL COATED CAPILLARY ELECTROPHORESIS COLUMNS

Abstract: Coated capillary electrophoresis columns and methods for their use in electrophoretic separations are disclosed. The coated capillary columns include a length of tubing having an interior surface having an interconnected polymeric coating. The interconnected polymeric coating includes a hydrophobic hydrocarbon polymeric functionality covalently bound to the interior surface and polyvinylalcohol interconnected with the hydrophobic polymeric functionality. Exemplary columns are prepared by causing a Si-OH reactive compound, having a hydrophobic polymeric functionality, to react with Si-OH functionalities on the interior surface of capillary columns. Then causing vinyl acetate to polymerize in contact with the hydrophobic polymeric functionality and hydrolyzing the resulting polyvinylacetate forms a coating of interconnecting hydrophilic and hydrophobic polymers. Alternative exemplary coatings include a modified polyvinylalcohol such as a polyvinylether.

Description: POLYVINYLALCOHOL COATED CAPILLARY ELECTROPHORESIS COLUMNS BACKGROUND OF THE INVENTION l. Field of the Invention The present invention relates generally to capillary columns having interior surface coatings and electrophoretic separation methods for their use. More particularly, the present invention involves capillary columns having a neutral crosslinked hydrophilic coating on their interior wall surfaces. The neutral crosslinked hydrophilic coating reduces analyte interaction with the interior surface of the capillary column and simultaneously protects the interior surface of the capillary column during the electrophoretic separation of acidic and basic compounds. 2. Description of Relevant Art Electrophoretic separation techniques have been utilized for years to separate molecules according to differences in the effective charge of the molecules, and/or according to differences in the molecular size of the molecules. Up until recently electrophoretic separations were conducted in gel slabs or open gel beds which were typically fabricated of polyacrylamide gel material. More recently capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of molecules. High resolution separations of molecules having different effective charges have been achieved by applying electrophoretic principles to buffer filled or gel filled narrow capillary tubes. Typically, capillary columns used in capillary electrophoresis are fabricated of lengths of silica tubing having diameters on the order of 25 mm to 200 mm and lengths from about 10 to 200 cm. The buffer and gel separation mediums are pumped directly into the column interiors and electrophoretic techniques are used to separate numerous types of molecules including peptides, proteins, and oligonucleotides, nucleic acids and other charged molecular species. The field of electrophoretic separation technology is continually expanding with respect to the types and sizes of molecules which can be separated and detected using capillary electrophoresis procedures. The advantages associated with capillary electrophoresis are numerous. Quantitative information can be achieved with very small sample sizes, and the amount of gel or buffer consumed is minuscule. Furthermore, the time required for the separations is sharply reduced, and the technique lends itself to automation and electronic data storage and data manipulation. Significantly, capillary electrophoresis is associated with certain phenomenon which are not present in traditional slab gel electrophoresis. One of these is the now familiar electroosmotic flow phenomenon characterized by bulk flow of buffer solutions toward one of the electrodes. Electroosmotic flow is generated by the ionization of silanol functionalities on the surface of silica capillary tubing. The ionization results in a layer of protons in the electrophoretic buffer solution at the surface of the silica tubing. In the presence of an electric field the layer of protons resembles a positively charged column of fluid which migrates toward the cathode, causing a general bulk movement of the buffer medium. Advantageously, electroosmotic flow can be utilized in many applications to improve electrophoretic separations. For example, when the electrophoretic migration of the molecules being separated is in the opposite direction to that of electroosmotic flow, the net effect is an increase in column performance and improved separations. For many electrophoretic applications electroosmotic flow is undesirable and eliminating or substantially reducing the bulk flow is preferred. Generally, when electroosmotic flow is reduced to a minimum, electrophoretic sample components move only by electrophoretic migration, which improves analysis reproduci 3bility and mass recovery of sample components. One method to minimize or to control electroosmotic flow, is to utilize capillary columns fabricated of silica capillary tubing coated on the inside with a polymeric material. The polymeric coating eliminates or substantially reduces the degree of ionization of the surface silanol groups which causes at least a substantial reduction in the bulk flow within the electrophoresis column. In order to avoid unwanted hydrophobic-hydrophobic interactions between sample components and the coating, the polymeric coatings are traditionally hydrophilic. One problem associated with the nature of prior art hydrophilic polymeric coatings is their low physical integrity, their tendency to dislodge from the surface of the capillary, and the resulting short useful life. Another problem with prior art hydrophilic polymeric coatings relates to their photo stability. More particularly, these coatings tend to absorb uv radiation at wavelengths below 260 nm and are subject to photolysis which causes their eventual loss of functionality. This is particularly undesirable in analytical electrophoresis systems using uv detectors. Covalently binding hydrophilic polymers to the surface of the capillary tubing, helps reduce the coating's tendency to dislodge, however the physical integrity and photo stability of the coatings remains a problem. Additionally, even when covalently bound to the surface of silica capillaries through an Si-O-Si, prior art hydrophilic coatings promote aqueous interaction with Si-0- Si functionalities. This can result in the unwanted hydrolysis and the Si-O-Si bond and a resulting shorted use life time. As mentioned above, another problem associated with capillary electrophoresis techniques is the tendency for sample components to adhere to the wall of the capillary tubing, and in particular silica tubing. This is especially true in the case of small charged molecules which are easily attracted , to reactive silica functionalities. When small peptides and amines are present in electrophoretic separation mediums, they interact Yboth electrostatically and hydrophobically with the capillary wall. The result is a decrease in separation efficiency and undesirable band broadening which gives erroneous separation data. Like electroosmotic flow, providing electrophoresis capillaries which are capable of minimizing the degree of saitiple component and wall interaction have not been totally successful. Previous attempts include using a dynamic double layer coating. These bilayer coatings are not stable and require additives in the running buffers used during the electrophoretic process. Hydrophobic coatings are effective in protecting the Si-O-Si surfaces of silica columns, but cause unwanted hydrophobic-hydrophobic interactions between the capillary wall and analytes. As noted above, hydrophilic coatings are not effective in protecting the Si- O-Si surface. Accordingly, it is an objective of the present invention to provide novel coated capillary columns useful for electrophoresis separations and which minimize electroosmotic flow. It is additionally an objective of the present invention to provide capillary columns which reduce or eliminate interactions between sample components and the interior capillary wall. It is further an objective of the present invention to provide capillaries having physically stable coatings. It is additionally an objective of the present invention to provide capillaries useful for the electrophoretic separation of a variety of charged molecules. It is another objective of the present invention to provide coated capillary columns having improved useful life when used in connection with uv detectors. It is further an objective of the present invention to provide capillary columns having coatings which are compatible with a wide variety of buffering compounds. SUMMARY OF THE INVENTION The present invention sati rsfies the above-identified objectives by providing capillary columns having interior surface coatings which contribute to the elimination or substantial reduction in the amount of interaction between sample constituents and the interior surface of the capillary column. The coated capillary columns of the present invention additionally contribute to minimizing the degree of electroosmotic flow during electrophoretic separations, thereby eliminating bulk flow and restricting sample movement to electrophoretic migration. Advantageously, use of the capillary columns of the present invention results in improved separations for a number of acidic and basic compounds including amines, amino acids, peptides, proteins, and nucleic acids. Moreover, the polymeric coatings described herein have considerable physical integrity and are highly stable, making the useful life of the columns substantially improved over prior art columns. As an added feature, preferred embodiments of the present invention utilize polymeric coatings which are stable to uv radiation at common uv detector wavelengths thus making them especially suitable for use in electrophoresis systems having uv detectors. The most preferred capillary columns have coatings which are stable in the presence of a wide variety of buffering compounds and do not form undesirable complexes. More particularly, the capillary columns of the present invention include a length of tubing having an interior surface. The interior surface has a crosslinked polymeric coating which includes a hydrophobic polymeric functionality covalently bound to the interior surface of the capillary tubing and a hydrophilic polymer copolymerized with the hydrophobic polymeric functionality. Preferably, the capillary tubing used to prepare the column is fabricated of silica and the hydrophobic polymeric functionality is covalently bound to the interior surface by Si-O-Si bonds which are formed by reacting the capillary surface Si-OH functionalities with a suitable reactant. In preferred embodiments the hydrophobic polymeric functionality is polybutadiene and the hydrophilic polymer is polyacrylamide. In most preferred embodiments, the hydrophilic polymer is hydrolyzed polyvinylacetate (polyvinylalcohol) . In alternative embodiments, the hydrolyzed polyvinyl acetate is further reacted with a hydroxy reactive compound to form modified polyvinylalcohol. The coated capillary columns of the present invention can be prepared by providing a length of silica tubing having an interior surface and Si-OH functionalities on the interior surface and causing a Si-OH reactive compound to react with Si-OH functionalities. Suitable Si-OH reactive compounds include silane and siloxane compounds having at least one neutral hydrophobic functionality and preferably the hydrophobic functionality is polymeric and capable of further reacting with polymerizable monomers. Then, causing a hydrophilic monomer to homopolymerize and react with the hydrophobic functionality, results in an interconnected polymeric coating of hydrophilic and hydrophobic polymers. In preferred processes the reactive compound is a polymeric polybutadienenyl triethoxy silane and the hydrophilic monomer is acrylamide. In most preferred processes the reactive compound is a polymeric polybutadienenyl triethoxy silane and the hydrophilic monomer is vinylacetate, and subsequent to polymerizing the vinylacetate, which coreacts with the polybutadiene functionalities of the silane, the resulting polyvinylacetate is hydrolyzed to polyvinylalcohol. Alternatively, to form a coating stable to buffers which may react with polyvinylalcohol a modified polyvinylalcohol can be formed by further reacting the polyvinylalcohol with a hydroxyl reactive compound to form a modified polyvinylalcohol. Most preferred modified polyvinylalcohols are those which are ether modified. Also disclosed herein are methods for methods for using the coated capillary columns of the present invention. More particularly, methods for using the herein disclosed capillary columns include methods for analyzing sample compositions for sample constituents by capillary electrophoresis. Such methods involve providing a capillary electrophoresis column having a length of tubing with an interior surface having an interconnected polymeric coating as described herein. The next steps involve immersing a first end of the tubing in an anodic reservoir, immersing said second end in a cathodic reservoir and introducing the sample composition into the length of capillary tubing at the first end or the second end. Then, applying an electric field capable of causing the sample constituents to migrate at different rates results in the sample constituent separation and subsequent detection and analysis. The interconnecting polymeric networks incorporated into the capillaries of the present invention have high physical integrity and extended useful life. In the most preferred embodiments the interconnecting polymeric network and particularly, the hydrophilic polymeric portion is photostable when used in connection with uv detectors. Moreover, these photostable coatings can be modified to incorporate chemical stable functionalities suitable for use in a wide variety of analyses and buffer systems. These and other advantages associated with the present invention will become apparent to those skilled in the art upon an understanding the invention as described in the detailed description of the invention taken in combination with the following drawings. BRIEF DESCRIPTION OF THE DRAWINGS Fig. l is a cross-sectional view of capillary electrophoresis column of the present invention illustrating a crosslinked hydrophobic polymer and hydrophilic polymer coating on the interior surface of a silica capillary column. Fig. 2 is a partial cross-sectional view of capillary electrophoresis column illustrating a preferred embodiment of the present invention. Fig. 3 is an electropherogram resulting from the electrophoretic separation of the Halll digestion of fC174 DNA. Fig. 4 is an electropherogram resulting from the electrophoretic separation of proteins in an aqueous solution in accordance with the present invention. Fig. 5 illustrates the reproducibility of the electropherograms obtained using an exemplary column of the present invention. Fig. 6 is an electropherogram obtain from the electrophoretic analysis of proteins in chicken egg white. Fig. 7 is a cross-sectional view of capillary electrophoresis column of the present invention illustrating a most preferred embodiment in which the hydrophilic polymer is hydrolyzed polyvinylacetate. Fig. 8 is an electropherogram resulting from the electrophoretic separation of lysozyme, cytochrome C, and ribonuclease A in an aqueous solution in accordance with the present invention. Fig. 9 is an electropherogram resulting from the 500th consecutive electrophoretic separation of the three proteins shown in the electropherogram of Fig. 6. Fig. 10 demonstrates the reproducibility of 500 consecutive injections of the sample used to obtain the electropherogram of Fig. 8 and Fig. 9. Fig. 11 is an electropherogram resulting from the electrophoretic separation of poly(A) oligonucleotides in a capillary of the present invention in which the hydrophilic polymer is polyvinylalcohol. Fig. 12 is an electropherogram resulting from the electrophoretic separation of 40 mer - 60 mer poly(A) in a column in which the hydrophilic polymer is hydrolyzed polyvinylacetate (polyvinylalcohol) . Fig. 13 is an electropherogram resulting from the electrophoretic separation of 40 mer - 60 mer poly(A) in a column containing ether modified polyvinylalcohol using the same electrophoretic conditions as Fig. 12. Fig. 14 is an electropherogram resulting from the electrophoretic separation of a ΦX174 HallI digestion in a capillary of the present invention having a hydrophilic polymer of polyvinylalcohol. Fig. 15 is an electropherogram resulting from the electrophoretic separation of the ΦX174 HallI digest of Fig. 14 using the same conditions as that used to obtain Fig. 13 except the hydrophilic p 1olymer is a ether modified polyvinylalcohol. Fig. 16 is an electropherogram resulting from the separation of histamine, lysozyme, cytochrome C, and myoglobulin using a borate buffer and a capillary of the present invention in which the hydrophilic polymer is an ether modified polyvinylalcohol. Fig. 17 is a cross-sectional view of capillary electrophoresis column of the present invention illustrating an embodiment in which the hydrophilic polymer is a modified polyvinylacetate (hydrolyzed polyvinylacetate) . DETAILED DESCRIPTION OF THE INVENTION The present invention provides coated capillary columns which are particularly useful in electrophoretic separation systems such as the P/ACE series Capillary Electrophoresis Systems manufactured and sold by Beckman Instruments, Inc. , Fullerton, California. More specifically, the coated capillary columns of the present invention are useful as separation mediums in applications in which a variety of molecular species are separated on the basis of their electrophoretic mobility. These molecular species include basic and acidic macromolecules such as proteins, charged polysaccharides and polynucleotides as well as smaller compounds such as basic drugs and nucleic acids. The coated capillary columns described herein contribute to eliminating or substantially minimizing electroosmotic flow during electrophoretic separations. As a feature of the present invention, the coating materials used for forming the coated columns are compounds joined by covalent bonds to each other and to the wall of the capillary column, thus forming an interconnecting polymeric network. This results in coatings having enhanced physical integrity which in turn provides columns with longer useful lives and improved reliability and precision in electrophoretic separation procedures. The enhanced physical integrity and the chemical properties of the coatings also contribute to a prolonged ability to prevent the interaction of different analytes with the wall of the capillary column. Furthermore, the uv stability of the coatings used in the columns and processes of the present invention contribute enhanced useable use life. Unlike many prior art capillaries which have coatings lacking stability in the presence of some electrophoresis buffers, certain embodiments of the present invention do not complex with or become chemically altered by the presence of electrophoresis buffers. The coated capillary columns of the present invention incorporate hydrophilic polymeric properties in the coating and thus substantially eliminate hydrophobic interactions between sample components and the capillary wall. Additionally, the capillary columns of the present invention incorporate hydrophobic functionalities covalently bonded to the surface of the capillary column. These hydrophobic properties reduce the interaction of the aqueous electrophoresis buffer environment with the interior wall of the capillary, thus minimizing hydrolysis of the covalently bound coating from the wall of the capillary tubing. More particularly, and referring to a representative partial longitudinal cross-sectional view of a column shown in Fig. 1, the coated capillary columns 10 of the present invention are fabricated of a length of capillary tubing 12 having an interior surface 14. The interior surface 14 has an interconnected polymeric coating which includes a hydrophobic polymeric functionality 16 covalently bound to the interior surface 14 of the capillary tubing 12 and a hydrophilic polymer 18 bonded to the hydrophobic polymeric functionality 16. Capillary tubing 12 suitable for use in the present invention can be fabricated from organic polymeric materials such as polyacrylates, polyvinyls, polymethacrylates or inorganic materials which include silica, quartz. Preferably, the selected organic or inorganic material has chemical moieties, or can be altered to have chemical moieties, which react under mild pH and temperature conditions with compounds having hydrophobic functionalities so that the hydrophobic polymeric functionality 16 is // covalently bonded to the surface of the capillary tubing. As illustrated in Fig. l, the hydrophobic polymeric functionality 16 additionally is capable of reacting with a hydrophilic compound 18 which is either a polymer or is polymerizable, thus providing a coating of a crosslinked network of hydrophobic polymer and hydrophilic polymer. The preferred material for fabricating capillary columns useful in the present invention is silica. Silica capillary columns have been used for decades in chromatography procedures and more recently have found utility in electrophoresis procedures. Accordingly, the fabrication and handling of these columns are credited to those skilled in the art and will not be discussed in greater detail herein. As discussed in more detail in the context of the present invention, advantageously, silica has Si-OH functionalities which readily react with compounds such as siloxanes and similarly reactive silanes. Since siloxanes and silanes having a variety of different chemical subs ituents are widely available commercially, their use in combination with silica columns is particularly advantageous in the practice of the present invention. In accordance with the present invention, preferred compounds having hydrophobic functionalities and capable of covalently bonding to the surface of the capillary tubing are silane or siloxane compounds with polymeric hydrocarbon moieties. Such hydrocarbon containing compounds are preferably additionally capable of reacting with selected hydrophilic monomers or hydrophilic polymers. This additional capability allows the formation of a bonded interconnecting network of hydrophobic polymers and hydrophilic polymers on the capillary interior surface. Accordingly, preferred hydrophobic polymeric hydrocarbon moieties are reactive hydrocarbons such as polybutadiene. Those skilled in the art will appreciate that these hydrocarbons have reactive vinyl moieties for reacting with vinyl hydrophilic monomers. Moreover, vinyl hydrophilic monomers can be polymerized in the presence of such vinyl containing hydrocarbons and react with the vinyl containing hydrocarbons to form a network of hydrophilic polymers covalently linked to hydrophobic polymeric functionalities which are in turn covalently bonded to the wall of the capillary tubing. Thus, in preferred embodiments of the present invention, compounds having hydrophilic functionalities are vinyl containing hydrophilic monomers and the compounds having hydrophobic functionalities are silanes or siloxanes modified to carry polybutadiene moieties which are capable of reacting with the vinyl hydrophilic monomers. Those skilled in the art will appreciate that once polymerized, the result is a homopolymerized and copolymerized network of hydrophobic polymeric functionalities and hydrophilic polymeric functionalities. The resulting coated electrophoresis capillary columns have substantially reduced amount of Si-OH functionalities on the interior surface of the capillary tubing. Thus, electroosmotic flow or bulk flow within the column while under the influence of an electric field is substantially eliminated. Moreover, the hydrophobic polymeric functionality bonded to the surface of the column reduces aqueous electrophoretic buffer interactions with the covalent bond formed to covalently link the coating. This reduced interaction eliminates hydrolysis of the covalent bond, which is typically an Si-O- Si bond, and contributes to an increased useful column life. Finally, the hydrophilic polymeric portion of the coating shields the electrophoresis analytes from the hydrophobic portions of the coating, thereby eliminating unwanted hydrophobic-hydrophobic interactions between sample analyte components and the coating or capillary wall. Those skilled in the art will appreciate that a variety of compounds having hydrophobic hydrocarbon functionalities and a variety of hydrophilic monomeric compounds are suitable in the practice of the present invention. In addition to polybutadiene modified silanes or siloxanes (including polysilanes and polysiloxanes) , virtually any vinyl containing hydrocarbon modified silane or siloxane can be used. In particular, polybutadiene functionalities, or similar hydrocarbon backbone polymers, provided on reactive polysiloxanes or polysilanes having a wide range of /3 molecular weights are useful. These molecular weights range from 200 to over 2,000,000. The limiting factor in the molecular weight is the length of the polybutadiene chain which can cause steric hindrance and may interfere with bonding the silane or siloxane to the surface of the capillary column. Because these compounds have reactive vinyl functionalities, they are capable of copolymerizing with hydrophilic vinyl monomers including hydroxyethylmethacrylate, vinylpyrrolidone, acrylamides, vinylacetate and others to form a coating network which includes a hydrocarbon hydrophobic polymeric layer and a second network of hydrophilic polymeric layers. Similarly, esters capable of transesterification reactions on the surface of acrylate capillary tubing and having vinyl hydrocarbon functionalities can copolymerize with hydrophilic monomers to form similar networks. Referring again to Fig. l, in preferred embodiments of the present invention, capillary tubing 12 is fabricated of silica and, depending upon the particular analytical application, can vary in length and in diameter. Typically the column will be from between about 10 cm to 200 cm in length and from 25 - 200 mm in inner diameter. Advantageously, the surface of silica capillary tubing has SiOH moieties which are reactive with a number of organic and inorganic chemical functionalities and provide reactive sites for covalently attaching compounds having desirable chemical characteristics to the interior wall surface 14 of the capillary tubing 12. Further, and in accordance with preferred embodiments of the present invention, the hydrophobic chemical functionalities 16 are polybutadiene functionalities which are attached to the interior wall surface 14 via Si-O-Si bonds generated by the reaction of a siloxane with SiOH functionalities. In this embodiment, the hydrophilic polymer 18 is polyacrylamide, which, when formed by polymerizing acrylamide monomer in the presence of suitable initiators will also copolymerize with residual and free vinyl groups of the polybutadiene and contribute to the formation of the crosslinked polymeric coating. The partial longitudinal cross-sectional view of Fig. 2, illustrating one wall of a capillary column graphically shows a preferred coated column. More particularly, the coating of a preferred capillary column 30 of the present invention is a crosslinked polymeric network which is covalently bonded via a Si-O-Si 32 bond to the interior wall surface 38 of the capillary tubing 34. Polybutadiene forms a hydrophobic coating layer 36 adjacent the capillary interior wall surface 38 and polyacrylamide forms a hydrophilic outer layer 40 which is available for interacting with sample components and the aqueous buffer systems utilized in electrophoretic separations. Furthermore, the polyacrylamide acts as a bridge between lengths of polyacrylamide thereby forming a crosslinked network of polymers. Those skilled in the art will appreciate that the hydrophobic characteristics of the above-described coating minimizes the interaction of the aqueous buffer with the capillary wall surface. More particularly, hydrophobic-hydrophilic repulsions preclude interaction between aqueous media and the coating interface at the capillary wall surface, thereby eliminating hydrolysis of the SiOH bond. Thus, the polymeric coating network remains covalently bound to the capillary interior wall surface and contributes to a substantial increase in the useable life of the capillary column. Another feature of the preferred coated capillary column of the present invention is the hydrophilic characteristics of the coating. As shown in Fig. 1 and Fig. 2, the hydrophilic portion of the coating is available for interaction with the buffer system utilized during electrophoretic separations. Additionally, the hydrophilic polymeric portion acts as a buffer zone between the hydrophobic portion of the coating and the buffer media. This hydrophilic polymeric buffer prevents hydrophobic- hydrophobic interactions between the coating and electrophoretic sample components and thus minimizes sample absorption onto the surface of the capillary column. In accordance with the present invention a most preferred embodiment has all of the features of the above- described embodiments and additionally is capable of long term use in capillary systems utilizing uv detectors at wavelengths less than 260 nm. This most preferred embodiment takes advantage of the uv transparency of polyvinylalcohol, a hydrophilic polymer formed by hydrolyzing polyvinylacetate. More particularly, and referring to the partial longitudinal cross-sectional view coating of a most preferred capillary column 100 of the present invention is a crosslinked polymeric network which is covalently bonded via a Si-O-Si 110 bond to the interior wall surface 115 of the capillary tubing 120. Polybutadiene of Fig. 7, illustrating one wall of a capillary column, the forms a hydrophobic coating layer 130 adjacent the capillary interior wall surface 115 and polyvinylalcohol forms a hydrophilic outer layer 140 which is available or interacting with sample components and the aqueous buffer systems utilized in electrophoretic separations. Furthermore, the polyvinylalcohol acts as a bridge between lengths of polybutadiene thereby forming a crosslinked network of polymers. Those skilled in the art will appreciate that in addition to the advantages mentioned above, the capillary column of Fig. 7 has coating components which are stable to uv radiation at wavelengths less than 260 nm. This feature substantially increases the useful life of capillary columns of the present invention which are used in connection with uv detectors. Fig. 17 shows an alternative embodiment to those capillaries of the present invention in which the hydrophilic polymer is polyvinylalcohol. More particuarly, Fig. 17 illustrates a partial longitudinal cross-sectional view of capillary column 200 having a crosslinked polymeric network coating covalently bonded via a Si-O-Si bond 210 to the interior wall surface 215 of the capillary tubing 220. The coating includes a hydrophobic coating layer 230 adjacent the capillary interior wall surface 215 and a modified polyvinylalcohol forms a hydrophilic outer layer 240 which is available for interacting with sample components and the aqueous buffer In preferred embodiments, the modified polyvinyl alcohol is an ether (a J polyvinyl ether), preferably an alkyl ether (R in Fig. 17) prepared by reacting the polyvinylalcohol hydroxyl functionalities with an hydroxy reactive compound, such as an halide ether. Those skilled in the art will recognize that such modified polyvinylalcohols are particularly advantageous because the resulting coating is stable to electrophoresis buffer compounds which may complex or react with hydroxyl groups. For example borate buffers are commonly used in the electrophoretic separations for oligosaccharides and polysaccharides. Because borates complex with hydroxyl groups on the saccharides, they cause the otherwise neutral saccharides to become charged, thus providing them with a charge and electrophoretic mobility. Moreover, while polyvinylalcohol is attractive for its uv stability, when exposed to certain pH's they are characterized as leading to increased electroosmotic flows. Under certain conditions, the high electroosmotic flow is desirable. Those skilled in the art will recognize that because hydroxyl functionalities are capable of reacting with a wide variety of reagents, modified polyvinylalcohols can have a variety of different structures. More particularly, because hydroxyl functionalities can react with halides, amines, esters, acids, acid halides, and, as known in the art, many other types of reagents, the polyvinylalcohol portion of the coatings describes herein can be tailored to provide any of a number of different physical and chemical properties. In accordance with the present invention, processes for preparing coated capillary columns include the general steps of providing a length of silica tubing having an interior surface with Si-OH functionalities on the interior surface and then causing a siloxane compound to react with the Si-OH functionalities. The siloxane compound is further characterized as having a neutral polymerizable functionality which provides reactive sites for crosslinking or copolymerization with monomers which are reactive with the polymerizable functionality. When hydrophilic monomer polymerizable functionality the result is a crosslinked coating of hydrophilic and hydrophobic polymers. Prior to causing the siloxane to react with the SiOH functionalities, it is preferable to first prepare the interior surface of the capillary by successively washing the surface with an inorganic acid, an inorganic base and finally with a volatile lower organic alcohol. Causing the silane to react with the SiOH functionalities typically involves siiηply contacting the interior surface of the capillary with a suitable silane compound, such as polybutadieneyl triethoxy silane. The use of a inert gas under pressure, such as helium at about 10 psi, enhances the effectiveness and reduces the length of time required to contact the silane with the interior wall of the capillary column. As known in the art, the silane functionality readily reacts with SiOH moieties to form Si-O-Si bonds. Once the silane compound is secured to the interior wall of the capillary tubing, a hydrophilic monomeric compound is brought into contact with the covalently bound silane compound. As already mentioned, the hydrophilic monomeric compound is characterized by its hydrophilicity and its ability to react with a hydrophobic functionality on the covalently bound silane compound. In preferred embodiments the hydrophilic monomer is acrylamide which readily homopolymerizes and copolymerizes with the vinyl functionalities of polybutadiene. Those skilled in the art are credited with applying standard polymerization chemistry techniques known in the art for polymerizing acrylamide under conditions in which the acrylamide reacts with the vinyl functionalities available on the polybutadiene. In one embodiment of the present invention the polyacrylamide formed during the polymerization step is further crosslinked in order to enhance the physical integrity of the coating material. Standard crosslinking methodologies can be utilized including the use of divinyl monomers such as N,N' -methylene-bis-acrylamide during the polymerization step. An alternative crosslinking method involves utilizing formaldehyde in pos -polymerization crosslinking step. The two aldehyde protons of formaldehyde are available for reacting with the acrylamide protons and when the reacted acrylamide protons are located on different polyacrylamide strands, they become crosslinked. Those skilled in the art are credited with applying techniques for crosslinking acrylamide. Additionally, examples of conditions for crosslinking are provided below in the form of working examples. Following the homopolymerization of the hydrophilic monomer and the coreaction of the hydrophilic monomer with the hydrophobic polymeric functionality, excess polyacrylamide and acrylamide is removed from the interior of the capillary column using a water wash under pressure. The coated column is then suitable for use in a variety of electrophoretic separations. In most preferred embodiments of the present invention in which the hydrophilic polymer functionality is polyvinylalcohol, processes for preparing coated capillary columns involve the same general steps described above for attaching a silane or siloxane to the surface of a capillary column wall. The step of providing hydrophilic monomer for copolymerization with the polymerizable functionality involves providing vinyl acetate which homopolymerizes to form polyvinylacetate and copolymerizes with the hydrophobic functionality. Subsequently the polyvinylacetate is hydrolyzed in a basic system to form polyvinyl alcohol. Those skilled in the art will recognize that while a preponderance of the acetate functionalities of polyvinylacetate are hydrolyzed, a small portion will not hydrolyze. Thus, the resulting hydrophilic polymer is substantially polyvinylalcohol which includes a small number of acetate functionalities. In order to prepare capillary columns of the present invention in which modified polyvinylalcohol is the hydrophilic polymer of choice, a polyvinylalcohol containing capillary is prepared in the general manner just described. Subsequently reacting the polyvinylalcohol with a suitable hydroxyl reactive reagent results in the formation of a modified polyvinylalcohol. Preferably the reagent is an ether halide which reacts with the hydroxyl to form an ether modified polyvinylalcohol (polyvinylether) . /f The coated capillary columns of the present invention are easily formed and adapted for use in any capillary electrophoresis system. Moreover, their use requires no special handling procedures and typical electrophoretic techniques apply to their use. Accordingly, once positioned in standard capillary electrophoresis equipment, the columns of the present invention can be used in processes for analyzing sample compositions for sample constituents by capillary electrophoresis. In accordance with the present invention, these processes typically include the steps of immersing one end of the coated capillary column in an anodic reservoir and immersing a second end of the coated column in a cathodic reservoir. Then, introducing a sample composition into the interior of the coated column at one end and applying an electric field across the reservoirs will cause the sample constituents to differentially migrate within the capillary column. When a suitable detector, for example a uv-visible detector or fluorescence detector, is appropriately positioned at least one end of the coated column, the separated sample constituents are detected and an electropherogram is generated. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. EXAMPLE 1 The following describes exemplary first steps for the preparation of a coated capillary column of the present invention for use in electrophoresis procedures. These first steps involve preparing the interior surface of a capillary column and covalently bonding modified hydrophobic polybutadiene functionalities to the prepared interior surface. A fused silica capillary column having a length of 65 cm and a diameter of 50 mm (available from Polymicron of Phoenix, AZ) was prepared for the initial reaction by rinsing the interior surface walls with a solution of IN HCl A? for 15 minutes. The rinsing action was achieved by pumping the HCl solution through the capillary tubing. Next HCl solution was removed from the capillary tubing and the interior surface was rinsed in the same manner with IN NaOH for 15 minutes. Finally, after removing the NaOH solution, the capillary interior was rinsed with methanol for 15 minutes. After removing residual methanol solution from the capillary tubing, the prepared tubing was rinsed with a 50% toluene solution of a triethoxysilyl modified polybutadiene (50% polybutadienenyl triethoxy silane in toluene purchased from HulsAmerica, Inc. Piscataway, New Jersey. The polybutadienenyl triethoxy silane is a polymeric compound having multiple reactive triethoxy silane functionalities bonded to polymeric polybutadiene. As known in the art, the polybutadiene functionality has pendant vinyl groups which are reactive under vinyl polymerization conditions. The polybutadienenyl triethoxy silane solution had a viscosity of 25 - 50 centistokes which is an indication of its molecular weight. The rinsing step was carried out for 30 minutes after which the two open ends of the capillary tubing were capped with silicon plugs and the capped tubing was allowed to stand for 24 hours. EXAMPLE 2 The following example illustrates the final steps in preparing a coated capillary column of the present invention. Generally, preparing this coated capillary column involves utilizing the polybutadiene bonded interior wall column prepared as described in Example 1 and synthesizing linear polyacrylamide in the presence of the bonded polybutadiene. The result is a layer of hydrophilic linear polyacrylamide surrounding the bonded hydrophobic polybutadiene coating. The polybutadiene bonded capillary column described in Example 1 was prepared. Then following the 24 hour aging period, the polybutadiene bonded capillary tubing was rinsed with toluene for 30 minutes. After 30 minutes, the residual toluene was removed and the capillary tubing was rinsed with methanol for 15 minutes. Next, a 5% aqueous solution of acrylamide, available from ICN Biomedical, Inc., Irvine, CA. , was prepared. The solution was degassed for 30 minutes under 100 mbar vacuum to reduce the solution oxygen content. Simultaneous with degassing the 5 wt% acrylamide solution, a separate 100 Ml volume of water was deoxygenated by bubbling helium through the water to reduce the oxygen content below 10% of the original oxygen content. Three minutes prior to removing the 5 wt% aqueous acrylamide solution from vacuum, the deoxygenated water was utilized to prepare a solution of 10 wt% N,N,N' ,N' -tetramethylenediamine (TEMED) , purchased from ICN Biochemicals, Irvine, CA, and a solution of 10 wt% ammonium persulfate (APS), purchased from ICN Biochemicals. Then, immediately, 10 mL of the 10 wt% TEMED solution and 10 mL of the 10 wt% APS solution were added to 2 Ml of the degassed acrylamide solution to form a polymerization mixture. This polymerization mixture was pushed into the polybutadiene bonded capillary tubing using 10 psi of helium pressure. Following a 3 minute reaction period, the 10 psi helium pressure was removed and two minutes later, the two ends of the capillary tubing were capped and the tubing was allowed to stand for 24 hours. Next the end caps were removed and residual polyacrylamide which was not reacted with the polybutadiene and residual acrylamide were pushed from the capillary column interior using water at 100 psi. The result was a thin layer of linear polyacrylamide coreacted with residual vinyl groups on the bonded polybutadiene. The coated capillary column prepared according to the foregoing procedure was ready for use in capillary electrophoresis separations. EXAMPLE 3 The following example is illustrative of a procedure for preparing an alternative coated capillary column of the present invention. The coated column described below differs from the coated column described in Example 2 in that the hydrophilic portion of the coating is a crosslinked polyacrylamide. The crosslinked polyacrylamide provides the coating with increased physical integrity and a resulting longer and more useful life. To prepare this alternative coating, the polybutadiene bonded capillary column described in Example 1 was prepared. Then following the 24 hour aging period, the polybutadiene bonded capillary tubing was rinsed with toluene for 30 minutes. After 30 minutes, the residual toluene was removed and the capillary tubing was rinsed with methanol for 15 minutes. Then, a 5 wt% aqueous acrylamide solution was prepared and degassed as described in Example 2. Sufficient N,N' -methylene-bis-acrylamide was added to the degassed 5 wt% acrylamide solution to make the solution l wt% N,N'- methylene-bis-acrylamide. Then a 10 wt% aqueous TEMED and 10 wt% aqueous APS solution were prepared as described in Example 2. A polymerization solution was prepared by adding 10 mL of the TEMED solution and lOmL the APS solution to 2 mL of the acrylamide and N,N' -methylene-bis acrylamide solution. This polymerization solution was brought into contact with a capillary column, prepared as described in Example 1, by pushing the solution under 10 psi of helium for 2 minutes followed by reducing the helium pressure to 0 psi. Four minutes was allowed to elapse and the polymerization solution was pushed a second time under the same conditions and for the same length of time. This procedure was repeated four additional times and the capillary was rinsed with double distilled and ionized water following the cycle. The resulting capillary column incorporated a coating which included a layer of polybutadiene covalently bonded to the interior surface of the capillary through a Si-O- Si bond and a network of crosslinked polyacrylamide which additionally reacted with residual vinyl groups of the polybutadiene. The resulting coating was securely attached to the interior wall of the capillary tubing and was additionally provided with enhanced physical integrity through the crosslinked network. EXAMPLE 4 The following example is illustrative of a procedure for preparing a second alternative coated capillary column of the present invention. The coated column described below differs from the coated column described in Example 2 in that the hydrophilic portion of the coating is a crosslinked polyacrylamide. The coated column differs from the column described in Example 3 in that the crosslinked polyacrylamide was formed utilizing different crosslinking reagents and procedures. The crosslinked polyacrylamide provides the coating with increased physical integrity and a resulting longer and more useful life. To prepare this alternative coating, the polybutadiene bonded capillary column described in Example 1 was prepared. Then following the 24 hour aging period, the polybutadiene bonded capillary tubing was rinsed with toluene for 30 minutes. After 30 minutes, the residual toluene was removed and the capillary tubing was rinsed with methanol for 15 minutes. Then, a 5 wt% aqueous acrylamide solution was prepared and degassed as described in Example 2. The solutions of TEMED and APS and the polymerization solution were prepared as described in Example 2. Following the preparation of these solutions the polymerization solution was forced into a capillary column, prepared as described in Example 1, under the same conditions as described in Example 2. After the acrylamide polymerized to form a linear polymer and the residual polyacrylamide which did not react with polybutadiene and excess acrylamide were removed from the column, the coated column was filled with a 37% formaldehyde solution having 2% IN NaOH. The formaldehyde solution was allowed to stand in the column for 24 hours after which the capillary column was washed with double distilled water. The resulting coated column was suitable for electrophoretic separations of a number of different types of compounds. EXAMPLE 5 The following illustrates exemplary applications for the coated capillary columns of the present invention. In particular, as described below, the coated columns of the present invention are useful in the separation of small charged particles such as nucleic acids. A 30/37 cm (37 cm overall and 30 cm to uv window) coated capillary prepared as described in Example 2 above was positioned in a P/ACE capillary electrophoresis instrument (manufactured by Beckman Instruments, Inc. , Fullerton, CA) . The column was filled with a buffer which included 3% acrylamide in 50 mM tris-HCl and 2mM EDTA. A sample of Halll digested fC174 DNA (200 mg/mL DNA) solution, containing an internal standard of orange G, was injected into one end of the column using a 10 sec 0.5 PSI pressure injection and 200 V/cm or 7.4 kV was applied across the electrophoresis reservoirs at a temperature of 20° C. The separated basic DNA fragments were detected with a uv detector at 254 nm. Fig. 3 shows the electropherogram which was obtained after less than 20 minutes of migration. Each of the DNA fragments was separated from the other DNA fragments by a baseline separation with the order of migration being the following: 1st peak: internal standard Orange G; 2nd peak: 72 base pair DNA fragment; 3rd peak: 188 base pair; 4th peak: 194 base pair; 5th peak: 234 base pair; 6th peak: 271 base pair; 7th peak: 281 base pair; 8th peak: 310 base pair; 9th peak: 603 base pair; 10th peak: 872 base pair; 11th peak: 1078 base pair; 12th peak: 1353 base pairs. EXAMPLE 6 The following illustrates an exemplary application for the coated capillary columns of the present invention. In particular, as described below and shown in Fig. 4, the coated columns of the present invention are useful in the separation of proteins. An aqueous solution containing 1 mg/mL each of an internal standard (histamine) , lysozyme, cytochrome C and ribonuclease A was prepared and a 20/27 cm coated capillary column prepared according to the procedures described in Example 4 above was placed in a P/ACE capillary electrophoresis instrument (manufactured by Beckman Instruments, Inc., Fullerton, CA) . The column was filled with a 20 mM MES (buffer purchased from Sigma Chemical, St. Louis, MO) and 20 mM citrate buffer adjusted to a pH of 6.0. A sample of the aqueous protein solution was injected into one end of the column using a 2 sec 0.5 psi pressure injection and 500 V/cm was applied across the electrophoresis reservoirs at a temperature of 25° C. The separated proteins were detected with a uv detector at 214 nm. Fig. 4 shows the electropherogram which was obtained after less than 12 minutes of migration. Each of the proteins was separated from the other proteins by a baseline separation with the migration order left to right on the electropherogram being the internal standard (histidine) , lysozyme, cytochrome C and ribonuclease A. EXAMPLE 7 In order to demonstrate the multi-use and long term use stability and reproducibility of assays using the coated column of the present invention, the protein assay performed in Example 6 was repeated 210 times. For each of the repeated assays the electrophoretic migration time for each protein was detected. The results of this reproducibility test are illustrated in Fig. 5. The reproducibility of the internal standard (histamine) migration time is shown at line 1 (migration time 1.99 minutes) . The reproducibility of the lysozyme migration time is shown at line 2 (migration time 4.6 minutes). The reproducibility of cytochrome C migration time is shown at line 3 (migration time of 5.77 minutes) . Finally, the reproducibility of the ribonuclease migration time is shown at line 4 (migration time of 9.73 minutes) . The relative standard deviations for the repeated histamine assay, lysozyme assay, cytochrome C assay, and ribonuclease assay are 0.74%, 1.08%, 1.12%, 1.63%, respectively. These data clearly demonstrate the exceptional physical stability and long term use characteristics of an exemplary column of the present invention. A EXAMPLE 8 The following example illustrates a further application of an exemplary column of the present invention. More particularly, a column prepared according to the procedure described in Example 4, was used to separate the proteins in chicken egg white. The assay conditions and sample concentration were the same as described in Example 5. The results of the assay are shown in Fig. 6. The peak at migration time 5.46 was identified as lysozyme, the peak at 5.89 as conalbumin, and the peak at 7.18 ovalbumin. EXAMPLE 9 The following example illustrates the preparation of a most preferred coated capillary column of the present invention. The initial steps for the preparation of the coated capillary column involve preparing the polybutadiene bonded interior wall column as described in Example 1 except that the polybutadienenyl triethoxy silane rinse was for a period of 2 hours at 40 psi and the plugged capillary was allowed to stand at room temperature for 72 hours. After 72 hours the unreactive silane was pushed out by toluene at 40 psi and then the capillary was rinsed for 10 minutes with toluene and finally rinsed for 10 minutes with methanol. The remaining steps included first preparing a vinyl acetate and benzoyl peroxide solution by transferring 100 ml of a 5% benzoyl peroxide in ethyl acetate solution to a 40% vinyl acetate in ethyl acetate solution. The benzoyl peroxide and vinyl acetate solution was pushed into the polybutadiene bonded capillary column and heated to 80° C for 24 hours. After the 24 hour reaction period, unbonded polyvinylacetate was pushed out of the column using methanol at 60 psi. Next a solution of 10% sodiummethoxide in methanol was allowed to react with the polyvinylacetate in the capillary column for a period of 15 minutes. This causes the polyvinylacetate to hydrolyze to polyvinylalcohol. The result is a layer of hydrophilic linear polyvinylalcohol surrounding the bonded hydrophobic polybutadiene coating. The capillary is now read for use in capillary electrophoresis applications. EXAMPLE 10 The following example illustrates a further application of an exemplary preferred capillary column of the present invention. More particularly, a capillary column prepared according to the procedure described in Example 9 was used to separate 3 proteins -lysozyme, cytochrome C, and ribonuclease A. The assay conditions were as follows: A 20/27 cm (27 cm overall and 20 cm to uv window) capillary column having the coating prepared as described in Example 8 above was positioned in a P/ACE capillary electrophoresis instrument (manufactured by Beckman Instruments, Inc., Fullerton, CA) . The column was filled with a buffer which included 20mM citrate/MES adjusted to a pH of 6.0. An aqueous sample containing lysozyme, cytochrome C, and ribonuclease A was injected into one end of the column using a 1 second pressure injection and then 500 V/cm was applied across the electrophoresis reservoirs at a temperature of 20° C. The separated proteins were detected with a uv detector at 214 nm. Fig. 8 shows the electropherogram which was obtained after less than 15 minutes total migration time. Each of the proteins was separated from the other by a baseline separation. The order of separation is histidine (internal standard) , lysozyme, cytochrome C, and ribonuclease A. EXAMPLE 11 In order to demonstrate the multi-use and long term use stability and reproducibility of assays using a coated capillary column of the present invention, the protein assay performed in Example 10 was repeated 500 times. Fig. 9 is the electropherogram which was obtained for the 500th injection. For each of the 500 repeated assays the electrophoretic migration time for each protein was documented. Fig. 10 is a plot of migration time vs. injection number for each of the 500 injections. When the first 20 injections and the last 20 injections are analyzed statistically, the migration time for lysozyme has an average of 6.05 minutes, a standard deviation of 0.06 minutes, and a % relative standard deviation of 0.93. For the same injections cytochrome C has an average migration time of 7.64 minutes, a standard deviation of 0.08, and a % relative standard deviation of 1.01. The same analysis for ribonuclease A gives an average migration time of 12.96 minutes, a standard deviation of 0.21 minutes, and a % relative standard deviation of 1.6. Clearly, the performance of the coated capillary of the present invention is exceptional even after 500 injections using the same assay conditions. EXAMPLE 12 The following illustrates the electrophoretic analysis of nucleic acids using a coated capillary column of the present invention prepared as described in Example 9 above. A 20/27 cm (27 cm overall and 20 cm to uv window) capillary column having a coating prepared as described in Example 8 above was positioned in a P/ACE capillary electrophoresis instrument (manufactured by Beckman Instruments, Inc. , Fullerton, CA) . The column was filled with a buffer which included 100 mM TRIS-Borate, 45% v/v DMSO and 5% v/v ethylene glycol. A solution of poly(A) nucleic acids including 40 mer through - 60 mer and a 20 mer and 40 mer poly(T) nucleic acids was injected into one end of the column using a 5 second lOKv electrokinetic injection. Then 200 V/cm was applied across the electrophoresis reservoirs at a temperature of 30° C. The separated lengths of DNA were detected with a uv detector at 254 nm. Fig. 11 shows the electropherogram which was obtained after less than 26 minutes of migration. The first two peaks are the poly(T) 20 mer and poly T 40 mer, respectively. The remaining peaks are poly(A) 20 mer through 40 mer. Clearly, each of the DNA fragments was separated from the other DNA fragments by a baseline separation. EXAMPLE 13 The following describes an exemplary method for preparing a an alternative embodiment capillary of the present invention. The alternative embodiment capillary incorporates a modified polyvinylalcohol (modified hydrolyzed polyvinylacetate) coating which is especially suitable for electrophoretic separations which utilize hydroxyl complexing buffers such as borates in combination with analytes having hydroxyl moieties (e.g. oligosaccharides, oligonucleotides, etc.). The initial steps for the preparation of the coated capillary column involve preparing the polybutadiene bonded interior wall column as described in Example 1 except that the wash following removal of excess unbonded triethoxysilyl modified polybutadiene silane included a 10 minute wash with methanol followed by a 10 minute wash with ethyl acetate. The remaining steps included rinsing the capillary with a solution of 1% w/v benzoyl peroxide and 40% v/v vinylacetate in ethyl acetate for 10 minutes. After sealing the capillary end, the vinyl acetate was allowed to polymerize in an oven at 85 °C for 24 hours. Then, after the capillary cooled to room temperature, the capillary end was trimmed and unbonded polyvinylacetate was pushed out of the column using ethylacetate at 60 psi. In order to remove traces of unreacted vinyl acetate, the capillary was washed lo minutes with ethylacetate and then washed 10 minutes with methanol. Next a solution of 5% sodiummethoxide in methanol was allowed to react with the polyvinylacetate in the capillary column for a period of 30 minutes. After rinsing the column with methanol for 30 minutes the capillary was allowed to air dry. The result is a layer of hydrophilic linear polyvinylalcohol surrounding the bonded hydrophobic polybutadiene coating. To prepare the modified polyvinylalcohol coating the just prepared capillary was rinsed for 10 minutes with a solution containing 40% (v/v) acetonitrile, 40 % (v/v) 2- methoxy ethoxy-methyl chloride (purchased from Aldrich Chemical, cat. no. 19,354-2) and 20% (v/v) diisopropylamine. The capillary was placed in an oven at 85 °C for one hour. After washing the interior of the cooled capillary for thirty minutes with methanol and allowing the capillary to air dry, it is ready for use in electrophoretic separations. EXAMPLE 14 The following demonstrates the difference in the electroosmotic flow properties characteristic of a capillary of the present invention in which the hydrophilic polymer is polyvinylalcohol coating and one in which the polyvinylalcohol is modified as described in EXAMPLE 13. A coated capillary having a polyvinylalcohol hydrophilic polymer (hydrolyzed polyvinylacetate) was prepared as described in EXAMPLE 9. A second coated capillary column having a modified polyvinylalcohol polymer was prepared as described in EXAMPLE 13. The capillaries had an overall length of 27 cm (20 cm to the detector window) and a 100 μm i.d.. Each capillary was installed on a P/ACE electrophoresis instrument with a running buffer of 100 mM Tris-borate at a pH of 8.3. An aqueous sample of benzyl alcohol was allowed to electrophorese in each capillary at 20 °C and 13.5 KV. The migration time of the benzyl alcohol is a measure of the electroosmotic flow characteristic of the capillary. Table I tabulates the migration time and the calculated electroosmotic flow (EOF) associated with each capillary. Significantly, there is a substantial decrease in electroosmotic flow associated with the modified polyvinylalcohol coating compared with the polyvinylalcohol containing coating. This is attributed to the deactivation of the polyvinylalcohol hydroxyl functionalities. TABLE I COLUMN MIGRATION TIME EOF Polyvinylalcohol 3.69 Min. 6.32 E-5 Ether Modified Polyvinylalcohol 22.63 Min 1.03 E-5 7/ EXAMPLE 15 The following demonstrates the differences in electrophoretic separation characteristics of a polyvinylalcohol containing capillary column as prepared in EXAMPLE 9 and the column prepared in EXAMPLE 13 in the separation of charged moieties. Poly(A) oligonucleotides having lengths ranging from a 40 mer to a 60 mer were synthesized on an automated DNA synthesis instrument. The two columns utilized in the experiments of EXAMPLE 14 were installed in P/ACE instruments having a running buffer of 7M Urea in 89 mM Tris-borate. The poly(A) oligonucleotides were electrophoresed in each column at 30 °C and 8.1 KV. Fig. 12 is the electropherogram obtained using the column prepared as described in EXAMPLE 9 (hydrophilic polymer is polyvinylalcohol or hydrolyzed polyvinylacetate) . Fig. 13 is the electropherogram obtained using the column prepared as described in EXAMPLE 13. Since the polyvinylalcohol containing coating has a greater EOF flow (which moves in the direction opposite to the electrophoretic migration) , the poly(A) separation time is substantially longer than the full separation obtained with the modified polyvinylalcohol containing column. EXAMPLE 16 The following demonstrates the separation of a φXl74 Halll digest sample utilizing a column in which the hydrophilic polymer is hydrolyzed polyvinylacetate (polyvinylalcohol) and a column having a coating containing ether modified polyvinylalcohol. The two columns utilized in EXAMPLES 14 and 15 were fitted separately on P/ACE instruments containing running buffers of 50mM Tris-borate at pH 8.3. Identical samples of a ΦX174 Halll digest were pressure injected into each capillary and the digests were allowed to electrophoretically migrate at 20 °C and 11.1 KV. Fig. 14 is an electropherogram of the migration in the hydrolyzed polyvinylacetate containing column (polyvinylalcohol) . Fig. 15 is an electropherogram of the migration in the ether modified polyvinylalcohol (a polyvinylether) containing column. These two electropherograms dramatically demonstrate the effect of the difference in electroosmotic flow associated with the two columns. The modified polyvinylalcohol containing column is able to separate the digest components in a short time while the polyvinylalcohol containing column does not provide the same efficient separation. EXAMPLE 17 The following experiment describes the characteristics of the separation of a sample of histamine, lysozyme, cytochrome C, and myoglobulin The column prepared in EXAMPLE 13 was fitted on a P/ACE instrument with a running buffer of 100 mM Tris-borate at pH 8.3. A sample containing the aforementioned four proteins was pressure injected and the proteins were electrophoresed at 30 °C and 13.5 KV. Fig. 16 is the electropherogram which resulted from the electrophoretic migration. Because the separation of these four proteins is improved in the presence of electroosmotic flow, the migration time is extended and in fact myogobulin does not elute under these conditions. Thus, the use of polyvinylalcohol containing as opposed to modified polyvinylalcohol is a preferred choice of column for these separations. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims: What is claimed is: 1. A capillary column for electrophoretic separations of sample components, said capillary column comprising: a length of tubing having an interior surface, said interior surface having an interconnected polymeric coating, said interconnected polymeric coating comprising a hydrophobic polymeric functionality covalently bound to said interior surface and polyvinylalcohol copolymerized with said hydrophobic polymeric functionality. 2. The capillary column of claim l wherein said tubing is fabricated of silica and said hydrophobic polymeric functionality is covalently bound to said interior surface by a Si-O-Si bond. 3. The capillary column of claim 1 wherein said hydrophobic polymeric functionality is polybutadiene. 4. The capillary column of claim 1 wherein said polyvinylalcohol is a modified polyvinylalcohol. 5. The capillary column of claim 4 wherein said modified polyvinylalcohol is a polyvinylether. 6. A capillary column for electrophoretic separations of sample components, said capillary column comprising: a length of tubing having an interior surface, said interior surface having an interconnected polymeric coating, said interconnected polymeric coating comprising a polybutadiene functionality covalently bound to said interior surface and a modified polyvinylalcohol, comprising a polyvinylether, copolymerized with said polybutadiene functionality. 7. The capillary column of claim 6 wherein said capillary tubing is formed of silica and said polybutadiene functionality is covalently bound to said interior surface through a Si-O-Si bond. 8. The capillary column of claim 7 wherein said polybutadiene functionality is covalently bound to said interior surface by reacting polybutadieneyl triethoxy silane with Si-OH of said silica. 9. A process for preparing a capillary column, said process comprising the steps: providing a length of silica tubing having an interior surface and Si-OH functionalities on said interior surface; causing a Si-OH reactive compound to react with said Si-OH functionalities, said Si-OH reactive compound having a hydrophobic polymeric functionality; causing vinylacetate to polymerize in contact with said hydrophobic polymeric functionality, thereby forming polyvinylacetate; and hydrolyzing said polyvinylacetate to form polyvinylalcohol, thereby forming an interconnecting polymeric network of polyvinylalcohol and hydrophobic polymers. 10. The process of claim 9 wherein said Si-OH reactive compound is polybutadienenyl triethoxy silane. 11. The process of claim 9 further including the step of crosslinking said polyvinylalcohol. 12. The process of claim 9 wherein causing said vinylacetate to polymerize is accomplished by reacting vinyl acetate in an organic solvent in the presence of polymerization initiators. 13. The process of claim 12 wherein said organic solvent is ethylacetate. 14. The process of claim 12 wherein said initiator is benzoyl peroxide. 15. The process of claim 9 further including the step of reacting said polyvinyl alcohol with a hydroxyl reactive reagent to form a modified polyvinylalcohol. 16. The process of claim 9 further including the step of reacting said polyvinylalcohol with an ether halide to form a modified polyvinylalcohol comprising polyvinylether. 17. The process of claim 16 wherein said ether halide is 2-methoxy ethoxy-methyl chloride. 18. A process for analyzing a sample composition for sample constituents by capillary electrophoresis, said process comprising the steps: providing a capillary electrophoresis column comprising: a length of tubing having an interior surface, said interior surface having an interconnected polymeric coating, said interconnected polymeric coating comprising a hydrophobic polymeric functionality covalently bound to said interior surface and polyvinylalcohol copolymerized with said hydrophobic polymeric functionality; immersing said first end in an anodic reservoir and immersing said second end in a cathodic reservoir; introducing said sample composition into said length of capillary tubing at said first end or said second end; and applying an electric field across said reservoirs, said electric field capable of causing said sample constituents to migrate at different rates with respect to each sample constituent within said capillary column. 19. The process of claim 18 wherein said hydrophobic polymeric functionality is polybutadiene. 20. The process of claim 18 wherein said polyvinylalcohol is a modified polyvinylalcohol. 21. The process of claim 20 wherein said modified polyvinylalcohol is polyvinylether. 22. The process of claim 18 wherein said tubing is fabricated of silica and said hydrophobic polymeric functionality is covalently bound to said interior surface by a Si-O-Si bond. 23. A capillary column for electrophoretic separations of sample components, said capillary column comprising: a length of tubing having an interior surface, said interior surface having an interconnected polymeric coating, said interconnected polymeric coating comprising a hydrophobic polymeric functionality covalently bound to said interior surface and polyvinylether copolymerized with said hydrophobic polymeric functionality.

Inventors: GOETZINGER WOLFGANG, KARGER BARRY L, MUTH JOCHEN, SHIEH CHIA-HUI, GOETZINGER, WOLFGANG, KARGER, BARRY, L., MUTH, JOCHEN, SHIEH, CHIA-HUI
Applicants: BECKMAN INSTRUMENTS INC, GOETZINGER WOLFGANG, KARGER BARRY L, MUTH JOCHEN, SHIEH CHIA HUI, UNIV NORTHEASTERN, BECKMAN INSTRUMENTS, INC., GOETZINGER, WOLFGANG, KARGER, BARRY, L., MUTH, JOCHEN, NORTHEASTERN UNIVERSITY, SHIEH, CHIA-HUI
Application year: 1996
Main classification: G01N27
IPC codes: G01N27