Patent Application
20070051628
The present invention generally relates to surface treatment, films and coatings, and more particularly coatings for chromatography and electrophoresis in capillaries and microchannels. Specifically, the invention is directed to wall coatings in capillary electrophoresis to reduce electroosmotic flow and adsorption of analytes on the wall.
Capillary electrophoresis has achieved a remarkably rapid development from its introduction in the early 1980s. This technique miniaturizes the electrophoretic process and presents significant advantages over traditional slab gel electrophoretic techniques. Most of materials used to prepare separation channels or capillaries for capillary electrophoresis (CE) contain ionizable groups on their surface that are responsible for so-called electrokinetic potential or ξ-potential. This potential is a cause of electroosmotic flow (EOF). The presence of EOF and especially its uneven distribution along the electrophoretic capillary or channel causes disturbances called eddy migration and a loss of resolution during electrophoretic separation. To eliminate EOF, a good wall coating eliminates ξ potential at the wall and/or increases viscosity inside the electric double layer. To reduce ξ-potential, it may react with charged groups incorporated in the wall (silanol groups in the case of fused silica capillary). To some extent, compounds with an opposite charge to the wall can be also used to titrate ξ-potential.
A number of various wall coatings have been proposed and developed to eliminate EOF and adsorption of analytes in fused silica capillaries. A vast majority of them merely reduced EOF and did not eliminate it completely. Frequently a dynamic wall coating was formed by simply adding an active ingredient to the background electrolyte. It adsorbs on the wall and reduces capillary surface charge and/or viscosity of solution in the electric double layer. Dynamic wall coatings are popular because of the simplicity of their preparation. However, they do not eliminate electroosmotic flow completely. Among many dynamic coatings, a guaran dynamic coating has been developed (Liu, Q., Lin, F., Hartwick, R. A. Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier. Chromatographia 1998, 47, 219-224).
To eliminate EOF completely, static wall coatings have to be applied. Typically, a static wall coating is made of two layers: an intermediate layer and a hydrophilic polymer layer. A bifunctional reagent that reacts with both the capillary surface and functional groups of the polymer molecule usually forms the intermediate layer. The first polymer used for the preparation of a static wall coating was a linear polyacrylamide attached to the fused silica capillary wall by y-methacryloxypropyltrimethoxysilane (Hjertén, S., Coating for electrophoresis tube. U.S. Pat. No. 4,680,201). More hydrolytically stable coating was obtained when polyacrylamide was attached to the silica wall by using a Grignard reagent with an olephinic moiety, e.g., vinylmagnesium bromide after activating silanol groups by a reaction with thionyl chloride (Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,074,982; Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,143,753). Polyacrylamide is, however, hydrolytically unstable at high pH and hydrolyzes forming poly(acrylic acid). The presence of carboxylic groups leads to generation of ξ-potential on the wall and to an increase of EOF. A more stable wall coating is usually obtained if acrylamide is replaced with its derivative having some substituents on nitrogen (Dolnik, V. and Chiari, M., Compounds for molecular separations. U.S. Pat. No. 6,074,542).
Thermal immobilization of a polymer on a capillary wall is another way how to anchor a polymer on the capillary wall. Schomburg and coworkers proposed a poly(vinyl alcohol) (PVA) coating fixed thermally to the wall by heating the capillary at 140.degree.C. They assumed that formation of a permanent PVA coating was based on PVA becoming water insoluble by thermal treatment and expected PVA to form semicrystalline highly associated structures, which were not covalently bound to the fused silica capillary. PVA molecules became more strongly associated by hydrogen bridges and water molecules could not penetrate microcrystalline domains. The authors expressed their opinion that this was a unique property of PVA. In the pH range of 5-9, the PVA coating did not, however, completely eliminate EOF and the coated capillaries exhibited a pH-independent electroosmotic mobility of 1.2×10−9 m2V−1s−1 as measured in 20 mM sodium phosphate (Schomburg, G. and Gilges, M., Deactivation of the inner surfaces of capillaries. U.S. Pat. No. 5,502,169). The procedure was further modified to make a PVA wall coating on a glass microchip. A newly introduced crosslinking of PVA with glutaraldehyde should improve the stability of the coating. No heating is necessary, just drying is sufficient to provide a stable wall coating (Belder, D., Deege, A., Husmann, H., Kohler, F., and Ludwig, M. Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis. Electrophoresis. 2001; 22, 3813-3818).
Thermal immobilization was also applied to hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC) (Shen, Y. and Smith, R. D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcol. Sep. 2000; 12, 135-141). The authors found that the wall coating was stable if the silica capillaries were heated at 140° C. for 20 min rather than just being dried at room temperature for 4 days. From this observation they concluded that a chemical reaction must have occurred between cellulose derivatives and fused silica capillary inner wall.
There are several types of galactomannans, a class of linear polysaccharides with 1,4 linked β-D-mannopyranosyl units and 1,6-linked α-D-galactopyranosyl side groups (Dolnik, V., Gurske, W. A. and Padua, A.: Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 2001, 22, 707-719). The four most important galactomannans are locust bean gum, tara gum, guar gum (guaran), and fenugreek gum, which differ by the frequency of galactosyl side group attachment to the polymannose. The ratio of D-mannosyl to D-galactosyl units is approximately 3.8:1 for locust bean gum, 3:1 for tara gum, 1.8:1 for guar gum, and about 1:1 for fenugreek gum. Locust bean gum, guar gum, and tara gum are commercially produced and have various applications in the food industry and as an additive to fracturing fluids in the petroleum industry. Guar gum is obtained from the endosperm portion of the legume seed (Cyamopsis tetragonoloba) that grows mainly on the Indian subcontinent and in some parts of Texas and Oklahoma. Typical guar gum contains 75-85% of galactomannan, 8-14% water, 5-6% proteins, 2-3% fiber, and 0.5-1% ash. Guar gum shows an excellent resistance to shear degradation (Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226).
The present invention is useful as a capillary coating that satisfies the above objectives. The polysaccharide coating described here eliminates electroosmotic flow and reduces adsorption on the capillary wall. The coating is stable for at least 48 hours at pH 10.3.
The present invention is also a method of preparing said coating and using this coating to cover the interior of a capillary tube.
Preparation of thermally immobilized guaran wall coating
3 m piece of fused silica capillary 75 μm ID, 360 μm OD is flushed with 0.1 mL thionyl chloride under pressure of 500 psi to clean the inner surface of the capillary. Then the capillary is filled with 1 g/L guaran at pressure of 1000 psi. Guaran (
Similarly other polysaccharides can be used to make a hydrophilic neutral wall coating including locust bean gum (
Electroosmotic mobility of guaran wall coating and its dependence on electrolyte concentration
Electroosmotic mobility (μEEO) of the prepared wall coating is measured in a 335 mm long capillary with effective length of 250 mm using equimolar solution of tris(hydroxymethyl)aminomethane (Tris) and N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) as a background electrolyte (BGE) and 100 mM nicotinamide or 1 g/L hydroxyethyl methacrylate as a neutral marker. First a band of the marker is injected as a 2 s pulse at 50 mbar to the capillary inlet and is moved forward by pumping BGE electrolyte for 35 s at 50 mbar into capillary. Then another 2 s pulse of the neutral marker is injected at 50 mbar and moved forward by pumping BGE for 35 s at 50 mbar into capillary. The −10 kV voltage is applied for 180 s and a 3rd pulse of the neutral marker (2 s at 50 mbar) is introduced into the capillary Then the all three bands of the neutral marker are pumped through the capillary by a pressure of 50 mbar, measuring absorption at 214 nm. Migration times of these three peaks are measured and used to calculate electroosmotic mobility (Williams, B. A. and Vigh, G., Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis. Anal. Chem. 1997, 69, 4445-4451).
By using this method we measured electroosmotic mobility of the prepared capillaries. For high-quality coating that suppresses EOF significantly (μEEO below 10−9 m2V−1s−1), it is necessary to apply high voltage during measurement for extended period of time to keep reproducibility in an acceptable range.
The resulting electroosmotic mobility of the prepared coating depends on the ionic strength. The higher concentration of Tris-HEPES, the lower value of electroosmotic mobility was obtained (
Capillary zone electrophoresis of acidic protein mixture
Quality of the prepared guaran wall coating was tested by CZE of model proteins in the guaran-coated capillary. The total length of the capillary was 335 mm, the effective length of the capillary was 250 mm. The capillary had ID 50 μm and OD 360 μm. For a CE separation of acid proteins, background electrolyte containing 100 mM Tris and 100 mM HEPES, pH 8.1 was used and a constant voltage of −10 kV was applied. The sample containing 10 g/L polyGlu, 4 g/L trypsin inhibitor, 8 g/L .beta.-lactoglobulin, 1 g/L .alpha.-lactalbumin in water was injected hydrodynamically applying pressure of 30 mbar for 3 s. The peaks were detected by measuring UV absorption at 214 nm. All five model proteins were separated in less than 25 minutes.
Capillary zone electrophoresis of basic protein mixture
Quality of the prepared guaran wall coating was tested by CZE of model proteins in the guaran-coated capillary. The total length of the capillary was 335 mm, the effective length of the capillary was 250 mm. The capillary had ID 75 μm and OD 360 μm. For a CE separation of acid proteins, background electrolyte containing 100 mM .beta.-alanine and 100 mM citric acid, pH 3.3 was used and a constant voltage of −10 kV was applied. The sample containing 2 g/L polylysine, lysozyme, cytochrome c, trypsinogen, and .alpha.-lactalbumin in water was injected hydrodynamically applying pressure of 30 mbar for 3 s. The peaks were detected by measuring UV absorption at 214 nm.
Capillary isoelectric focusing of pi standards in guaran-coated capillary
Utility of guaran-coated capillary for capillary isoelectric focusing was tested by isoelectric focusing of colored synthetic pl markers (
tm=7.50+0.51 pl
where tm is migration time and pl isoelectric point.
Hjertén, S., Coating for electrophoresis tube. U.S. Pat. No. 4,680,201, 1987.
Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,074,982, 1990.
Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,143,753, 1991.
Dolnik, V. and Chiari, M., Compounds for molecular separations. U.S. Pat. No. 6,074,542, 2000.
Schomburg, G. and Gilges, M., Deactivation of the inner surfaces of capillaries. U.S. Pat. No. 5,502,169, 1996.
V. Dolnik, W. A. Gurske, and A. Padua: Solution of galactomannans as a sieving matrix in capillary electrophoresis. U.S. Patent Application 20020049184, Sep. 5, 2001.
Whistler R., Conversion of Guar Gum to Gel-Forming Polysaccharides by the Action of Alpha-Galactosidase. U.S. Pat. No. 4,332,894, 1982.
Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226.
Dolnik, V., Gurske, W. A. and Padua, A.: Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 2001, 22, 707-719.
Williams, B. A. and Vigh, G. Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis. Anal. Chem. 1997. 69, 4445-4451.
Liu, Q., Lin, F., Hartwick, R. A. Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier. Chromatographia 1998, 47, 219-224.
Belder, D. Deege, A., Husmann, H., Kohler, F., and Ludwig, M. Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis. Electrophoresis. 2001; 22, 3813-3818.
Shen, Y. and Smith, R. D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcol Sep. 2000; 12, 135-141.
Slais, K., Friedl, Z. Low-Molecular-Mass pl Markers for Isoelectric Focusing. J. Chromatogr. A 1994, 661, 249-256.
