Apparatus and method for electrophoretic separation of proteins

Abstract: A device and method for capillary zone electrophoresis is provided in which the inner surface of the capillary (205) is modified with an amphoteric coating to adjust the point of zero charge of the capillary surface to fall at any desired pH. The modified capillary is employed in CZE at the desired pH; moreover, the magnitude and direction of the electroosmotic flow is controlled by the application of an external electric field.

Description: Field of the Invention The present invention generally relates to the separation of proteins and particularly to capillary zone electrophoresis of proteins at a desired pH wherein the electroosmotic flow is controlled by an applied external electric field. Background of the Invention Capillary zone electrophoresis ("CZE") in small capillaries (less than or equal to 75µ) was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of small solutes. J. Chromatog ., 218 (1981), page 209; Anal. Chem ., 53 (1981), page 1298. The separation process relies upon an electroosmosis effect generally described as the flow of a liquid in contact with a solid surface under the influence of a tangentially applied electric field. Attractive factors for electrophoretic separations by capillary zone electrophoresis are the small sample sizes, little or no sample pretreatment, and the potential for quantification and recovery of biologically active samples. One of the single greatest disadvantages of capillary zone electrophoresis lies when attempts are made to separate macromolecules such as proteins. Separations of macromolecules by CZE leads to untoward interactions of the biopolymers with the silica capillary wall. One approach to the problem attendant to CZE separation of proteins was reported in U.S. Patent No. 4,931,328, inventor Swedberg, issued June 5, 1990, which described a modified capillary tube wherein an interfacial layer is covalently bonded to the inner wall of the capillary tube. The interfacial layer is effective to reduce interactions between the inner wall and protein solutes, and includes a hydratable amphoteric phase. This amphoteric phase has a determinable isoelectric point and permits electroosmotic flow control by selection of solution pH. Lee et al., Anal. Chem ., 62 (1990), pages 1550-1552, reported a CZE technique in which bare fused silica capillary electroosmosis was controlled by using an external electric field to modify the zeta potential at the aqueous/inner capillary interface. Summary of the Invention It is an object of the present invention to provide a CZE method and apparatus for protein separations in which the protein remains biologically active. It is another object of the present invention to provide a CZE method and apparatus in which protein is separated at or near the point of zero charge of the capillary inner surface, and in which the electroosmotic flow is controlled by regulating an external electric field across the capillary wall. Further objects and advantages of the invention will become apparent to those skilled in the art upon examination of the specification and appended claims, as well as in practice of the present invention. In one aspect of the invention capillary tubes with modified inner surfaces where the interaction phase includes amino groups were prepared. These amino-phase columns had a predetermined point of zero charge. These columns were employed in CZE wherein the pH is adjusted to fall at or close to the predetermined PZC while the magnitude and direction of the electroosmotic flow was controlled by an externally applied electric field. Brief Description of the Drawings Figure 1 is a schematic diagram of a CZE device wherein the electroosmotic flow is controlled by an applied external electric field. Figure 2 is a graph plotting the absolute value of the electroosmotic mobility versus the applied external potential gradient at pH 3.0. Figure 3 is a graph plotting the electroosmotic mobility versus the applied external potential gradient at pH 4.0. Figure 4 is a plot of electrophoretic mobility versus pH. Figure 5 is a plot of electrophoretic mobility versus external field at a constant pH of 4.0. Figure 6 is a plot of electrophoretic mobility versus applied external field at a pH of 4.0 with the addition of a surfactant. Figure 7a illustrates an electrotherogram of a protein mixture with no external control. Figure 7b illustrates an electrotherogram with the application of an external electric field. Detailed Description of the Preferred Embodiments The point of zero charge (PZC) is generally defined as the pH at which the net charge of a surface is zero. As described in U.S. Patent No. 4,931,328, a capillary tube can be modified so that an interfacial layer is covalently bonded to the inner wall of the capillary tube. The interfacial layer, which helps minimize interactions between the inner wall and proteins, includes a hydratable amphoteric phase. The amphoteric phase has a determinable isoelectric point. Thus, the amphoteric phase can provide a variety of surfaces with unique PZC characteristics. Lee et al., Anal. Chem ., 62 (1990), pages 1550-1552, demonstrated direct control of electroosmotic flow in capillary electrophoresis by using an additional electric field applied from outside the separation capillary. This technique vectorially couples the externally applied potential with the potential across the buffer solution inside the capillary. It is believed that this electric potential gradient across the capillary wall controls the polarity and magnitude of the zeta potential on the interior surface of the capillary wall. The direction and flow rate of electroosmosis is dependent upon the polarity and magnitude of the zeta potential. The electroosmosis in a bare fused silica capillary electrophoresis at the solution condition of 1 mM phosphate buffer at pH 5 were directly controlled by simply varying the external electric potential. The present invention is based in part on utilizing an interfacial layer coated on the inner surface of a capillary tube to adjust the PZC of the capillary surface to fall at any desired pH. The layer can be chemically bonded or highly physisorbed to the capillary bore; the layer is stable under capillary separation conditions. The interfacial layer can comprise a hydratable amphoteric phase or an ionizable species having a basic or an acidic equilibrium. The modified capillary tube can be employed in CZE wherein the pH is adjusted to fall at or close to the predetermined PZC of the modified surface while the magnitude and direction of the electroosmotic flow ("EOF") is controlled by an externally applied electric field. The present invention is particularly suited for protein separations, since protein conformation (and function) is very sensitive to pH. For protein separations, it is desirable to choose specific pH conditions under which the separations will be done. In addition, control of the EOF is required for optimizing the resolution of components being separated. Experimental A capillary tube with a modified inner surface where the interaction phase includes amino groups (referred to herein as amino-phase columns and illustrating an ionizable species embodiment of the invention) was prepared as described in U.S. Patents 4,931,328 and 5,006,313, inventor Swedberg, issued June 5, 1990, and April 9, 1991, respectively, by pumping 0.5% solution of 2 amino propyltriethyoxysilane (pH 4.5) through capillaries for 15 minutes, followed by curing. The amino-phase column was used as the inner capillary 205 of the system as shown in Fig. 1, described herein. Referring to Fig. 1, a capillary 205 with 50 µm i.d., 150 µm o.d. and 25 cm in length was placed inside the bore of a larger capillary 206 200 µm i.d., 350 µm o.d. and 22 cm in length). The inner (sample) capillary was attached to reservoir 201 and reservoir 204, while the larger (outer) capillary was attached to reservoir 202 and reservoir 203. The reservoirs and outer capillary were filled with buffer. A bipolar high voltage power supply 207 was connected to the buffer in reservoir 201 via line 208 and electrode 209. Upon activation, power supply 207 generates an electric field inside the inner capillary between reservoir 201 and reservoir 204. Similarly, bipolar high voltage power supply 210 was connected to the buffer in reservoir 202 via line 211 and electrode 212. The buffer in reservoir 203 was connected via line 216 and electrode 217 to a selectable resistor ladder 215 that was in turn connected to a bipolar high voltage power supply 213 through line 214. Reservoir 204 was grounded. For instance, if reservoir 201 is at +10 kV and reservoir 204 is at 0 volts, an axial field of 10 kV is established. After an axial internal electric field is established in the inner (sample) capillary, a uniform radial electric field is created across the interstitial capillary space between the two coaxial capillaries by establishing a potential gradient between reservoirs 202 and 203. The external field can be "positive" or "negative." For example, a uniform axial positive radial external field is established if the potentials at reservoirs 202 and 203 are +15 kV and +5 kV, respectively. This establishes a +5 kV radial field across the interstitial capillary space. On the other hand, if the polarity is changed so that the potentials at reservoirs 202 and 203 are +5 kV and -5 kV, respectively, then a uniform negative radial external field of -5 kV is established. Fig. 2 is a graph plotting the absolute value of the electroosmotic mobility against the applied external potential gradient at pH 3.0 (50 mM citrate). As is apparent, a positive external field increases the EOF, i.e., faster anodic flow. Application of a negative external field slows the EOF, but did not reverse it. The above experiment was repeated at pH 4.0, one-half pH unit from the PZC of the amino-phase column which was experimentally calculated to be 4.5. Fig. 3 is a plot of the electroosmotic mobility against the applied external potential applied gradient. As is apparent, at a potential of +8 kV, the EOF increased generally, but the negative 8 kV offset reversed the flow direction. Fig. 4 is a plot of electrophoretic mobility against pH using the amino-phase column. Fig. 5 is a plot of electrophoretic mobility against external field at a constant pH of 4.0 (10 mM citrate buffer). Fig. 6 is a plot of electrophoretic mobility against applied external field at a constant pH of 4.0 with the addition of surfactant (10 mM OAC buffer, CHAPSO). Referring to Fig. 7a, a 50µ amino-phase column was utilized to separate a mixture of bacteriorhodopsin and bacterioopsin at pH 4.0 with no external control, i.e., no external electric field, (93 mm to detector, λ 210, 10 mM OAC, 18.0 mM CHAPSO, 200 V/cm). Fig. 7b refers to the same protein mixture separation under the same conditions but with the application of a +10 kV gradient. The increase in electroosmotic flow from 0.11 mm/sec (Fig. 7a) to 0.25 mm/sec (Fig. 7b) caused the elution times to be decreased in proportion to the increase in EOF. It is to be understood that while the invention has been described above in conjunction with preferred specific embodiments, the description and examples are intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.

Claims: An electrophoretic apparatus useful for electrophoretic separations of proteins in solution at a desired pH comprising: a capillary tube (205) defining an outer capillary wall, an inner capillary wall and a bore, wherein at the inner capillary wall surface there is a chemically bonded or highly physisorbed interfacial layer that is effective to reduce interactions between the inner wall and protein solutes; means for applying an internal electric field along the bore axis; and means for applying an external electric field across the outer wall and inner wall. The electrophoretic apparatus as defined in claim 1 wherein the interfacial layer comprises a hydratable amphoteric phase that comprises a protein, a peptide or an ampholyte. The electrophoretic apparatus as defined in claim 1 wherein the interfacial layer includes an ionizable species having an acidic equilibrium or a basic equilibrium. The electrophoretic apparatus as defined in claim 3 wherein the ionizable species is an amino group. The electrophoretic apparatus as defined in claim 3 wherein the ionizable species is a carboxyl group. The electrophoretic apparatus as defined in claim 1 wherein the inner capillary wall surface comprises a highly physisorbed interfacial layer that comprises an amphoteric or zwitterionic surfactant. The electrophoretic apparatus as defined in either claims 2, 3, 4, 5, or 6 wherein the interfacial layer surface has a point of zero charge that is at or near the desired pH of the solution. The electrophoretic apparatus as defined in claim 7 wherein the external electric field permits electroosmotic flow control. An electrophoretic method useful for separating proteins in solution at a desired pH which comprises the steps of: positioning one end of a capillary tube into a buffer reservoir, wherein the capillary tube defines an outer capillary wall, an inner capillary wall, and a bore, and wherein at the inner capillary wall surface there is a chemically bonded or highly physisorbed interfacial layer that is effective to reduce interactions between the inner wall and protein solutes; establishing an internal electric field between two points along the capillary bore; and applying an external electric field across the outer wall and inner wall. The electrophoretic method as defined in claim 9 wherein the interfacial layer comprises a hydratable amphoteric phase that comprises a protein, a peptide or an ampholyte. The electrophoretic method as defined in claim 9 wherein the interfacial layer includes an ionizable species having an acidic equilibrium or a basic equilibrium. The electrophoretic method as defined in claim 11 wherein the ionizable species is an amino group. The electrophoretic method as defined in claim 11 wherein the ionizable species is a carboxyl group. The electrophoretic method as defined in claim 9 wherein the inner capillary wall surface comprises a highly physisorbed interfacial layer that comprises an amphoteric or zwitterionic surfactant. The electrophoretic method as defined in either claims 10, 11, 12, 13, or 14 wherein the interfacial layer surface has a point of zero charge that is at or near the desired pH of the solution. The electrophoretic method as defined in claim 15 further comprising the step of controlling electroosmotic flow by adjusting the external electric field.

Inventors: MCMANIGILL DOUGLAS, SWEDBERG SALLY, MCMANIGILL, DOUGLAS, SWEDBERG, SALLY
Applicants: HEWLETT PACKARD CO, HEWLETT-PACKARD COMPANY
Application year: 1992
Main classification: G01N27
IPC codes: C07K1, G01N27