CA3074958A1 - A method and apparatus for counterflow gradient focusing in free-flow electrophoresis - Google Patents

Abstract

The present work describes a free-flow electrophoresis apparatus that uses a novel counterflow gradient focusing method. The main use for this apparatus is the continuous separation and collection of analytes such as charged molecules and biomolecules. The counterflow gradient focusing mechanism will be used to improve resolution during the separation and offer versatility. The free-flow electrophoresis device consists of a flow chamber with an electric field applied perpendicular to the direction of fluid flow. The counterflow gradient will be generated by introducing an additional counterflow through the sidewalls of the flow chamber. The purpose of the counterflow gradient is to counter electrophoretic migration such that each analyte will reach a unique equilibrium point in the transverse direction that depends on their electrophoretic mobility.

Description

54 Description 55 Title 56 A Method and Apparatus for Counterflow Gradient Focusing in Free-Flow Electrophoresis.

58 Field of the Invention 59 Free-flow electrophoresis describes the continuous separation and collection of charged analytes such 60 as proteins, DNA, amino acids, and small molecules. Free-flow electrophoresis is often used for the 61 preparative-scale purification of proteins, but also has potential for real-time monitoring and two-62 dimensional separations. A typical free-flow electrophoresis device consists of a flow chamber with 63 an electric field applied perpendicular to the direction of fluid flow.
Then, analytes injected at the start 64 of the flow chamber will traverse the chamber with a velocity that depends on their electrophoretic 65 mobility.
66 In recent years, there have been several innovations in free-flow electrophoresis to improve 67 performance. For one, the miniaturization of the flow chamber has reduced band-broadening and Joule 68 heating. Moreover, the direct integration of electrodes within the device has enabled a higher voltage 69 efficiency while preventing electrolysis products from entering the separation region. Finally, there 70 have also been several operation modes proposed in free-flow electrophoresis to reduce band-71 broadening during migration for improved separation resolution. These methods include 72 isotachophoresis, field-step electrophoresis, and isoelectric focusing.
All of these techniques rely on 73 altering the buffer composition to generate a gradient across the chamber in either electrophoretic 74 mobility, conductivity, or pH. Then, analytes traversing the chamber will either concentrate and/or 75 focus at some point along the gradient for narrower separation peaks and better resolution.
76 Each of the aforementioned operation modes for free-flow electrophoresis have their drawbacks. Free-77 flow isotachophoresis uses a low mobility buffer and a high mobility buffer to generate a steep electric 78 field gradient at the buffer-buffer interface. Analytes will then stack in this region according to their 79 mobility at high concentration factors, but the issue is they cannot be resolved without the use of 80 suitable spacers. Free-flow field-step electrophoresis also uses a multi-buffer system to generate a 81 conductivity gradient that reduces electrophoretic migration as analytes traverse the chamber for 82 concentrated peaks. The issue with this technique is that analytes will stack at the buffer-buffer 83 interface, therefore limiting the peak capacity. Free-flow isoelectric focusing uses carrier ampholytes 84 to generate pH gradient that allows analytes to focus at an equilibrium point across the chamber where 85 their net charge is zero, also known as their isoelectric point (pI).
Free-flow isoelectric focusing 86 perhaps has more potential than the aforementioned techniques because of its ability to separate 87 several analytes at a time with a high-resolution. Free-flow isoelectric focusing has its own drawbacks, 88 though, as many analytes either lack a well-defined pI or have poor solubility at that point.
89 Furthermore, the required carrier ampholytes are expensive and have compatibility issues when used 90 in tandem mass spectroscopy.

91 The advantages of isoelectric focusing have inspired researchers and inventors to explore other 92 methods that rely on this equilibrium gradient focusing method. In capillary electrophoresis, there is 93 an equilibrium gradient focusing method known as counterflow gradient electrofocusing. In this 94 technique, a bulk flow of liquid solution is introduced in the capillary to oppose electrophoretic 95 migration. A gradient exists in one or both of these transport mechanisms such that analytes will focus 96 at a mobility-dependant equilibrium point where their net velocity is zero. In most cases, there is a 97 gradient in electrophoretic velocity while the counterflow is constant.
Several electric field parameters 98 can be varied to generate such as gradient, including conductivity, current density, cross-sectional 99 area, and temperature. This gradient can be varied overtime to produce a scanning mode that allows 100 the focused analytes to elute from the capillary sequentially. Bilinear gradients have also been 101 explored to improve the peak capacity and resolution of the separation simultaneously. The resulting 102 focusing mechanism is similar to that of isoelectric focusing, but does not rely on carrier ampholytes 103 or the pI of the analyte. While capillary electrophoresis is a useful analytical tool, the throughput for 104 collecting separated analytes is limited. Therefore, free-flow electrophoresis is a more suitable 105 platform when collection is required. The present work will describe a free-flow counterflow gradient 106 focusing method that will be produced by introducing a fluid flow from each sidewall in the flow 107 chamber.

109 References Cited 111 US. Pat. No. 3,412,008 to Strickler (Nov. 19, 1968) 112 US. Pat. No. 4,310,408 to Rose and Richman (Jan. 12, 1982) 113 US. Pat. No. 4,309,268 to Richman (Jan. 5, 1982) 114 US. Pat. No. 5,080,770 to Culkin (Jan. 14, 1992) 115 US. Pat. No. 5,336,387 to Egan et al. (Aug. 9, 1994) 116 US. Pat. No. 6,478,942 to Rhodes and Snyder (Nov. 12, 2002) 117 US. Pat. No. 6,767,443 to Rhodes and Snyder (Jul. 27, 2004) 119 Novo and Janasek, "Current Advances and Challenges in Microfluidic Free-Flow Electrophoresis - A
120 Critical Review", Analytica Chimica Acta, vol. 991, 2017, pp. 9-29.
121 Ivory, "A Brief Review of Alternative Electrofocusing Techniques", Separation Science and 122 Technology, vol. 35, No. 11, 2000, pp. 9-29.

123 Shackman and Ross, "Counter-flow Gradient Electrofocusing", Electrophoresis, vol. 28, 2007, pp.
124 556-571.
125 Courtney and Ren, "Counter-flow Gradient Electrophoresis for Focusing and Elution", 126 Electrophoresis, vol. 40, 2019, pp. 643-658.

128 Description of Prior Art 129 Free-flow electrophoresis has been a technique used to continuously separate and collect charged 130 analytes since the 1960s. In a traditional device, a flow chamber is sandwiched between two parallel 131 plates. Additional chambers containing electrodes connected to a voltage source are located on each 132 side of the flow chamber, such that a uniform electric field can be applied across the flow chamber.
133 The side chambers are separated by an ionically conductive membrane, and continuously purged with 134 an electrode buffer to ensure no electrolysis products enter the flow chamber. Analytes are injected at 135 the start of the flow chamber, and as they are transported by the carrier fluid to the end of the flow 136 chamber, the charged analytes will simultaneously migrate across chamber due to the electric field.
137 The distance traveled by the analytes across the flow chamber is determined by its electrophoretic 138 mobility. Several outlets are located at the end of the flow chamber to collect the separated fractions 139 of analytes.
140 Since its invention, there have been several inventions in free-flow electrophoresis to improve stability 141 and resolution through efficient electrode integration, reduced band-broadening, and improved 142 cooling methods for less Joule heating. This prior art description will only focus on previous 143 inventions that are relevant to the present method and its introduction.of a crossflow through the flow 144 chamber.
145 US. Pat. No. 4,309,268 to David W. Richman (Jan. 5, 1982) incorporated at lateral outflow through 146 the sidewalls of the separation chamber to minimize band-broadening due to crescent distortion. At 147 that point, crescent distortion had been well-documented as a contributor to poor resolution. Crescent 148 distortion is caused by hydrodynamic dispersion as well as electrodynamic dispersion, which is due 149 to the pressure-driven backflow in the lateral direction generated due to electroosmotic flow. The 150 purpose of the outflow through the sidewalls is to produce an opposing pressure-driven flow in the 151 lateral direction to "cancel out" the crescent distortion.
152 US. Pat. No. 5,080,770 to Joseph B. Culkin (Jan. 14, 1992) used a flow chamber containing 153 subcompartments that were separated by porous membranes. The pump used to circulate the buffer 154 through the subcompartments is biased such that a crossflow is produced through the porous 155 membranes. This crossflow counters electrophoretic migration such that analytes will focus in one of 156 the subcompartments where the two transport mechanisms are counterbalanced before exiting through 157 the outlet. In a similar invention, US. Pat. No. 5,336,387 to Ned B.
Egan, Garland E. Twitty, and 158 David W. Sammons (Aug. 9, 1994) also used a flow chamber containing subcompartments separated 159 by porous membranes. By withdrawing a controlled fraction of fluid flow from each subcompartment, 160 a crossflow gradient was generated to counter electrophoretic migration. Analytes would then focus 161 in the compartment where the crossflow and their electrophoretic velocity were approximately equal 162 in magnitude, before exiting through the subcompartment outlets.
163 US. Pat. No. 6,478,942 to Percy H. Rhodes and Robert S. Snyder (Nov.
12, 2002) used a free-flow 164 electrophoresis chamber with porous sidewalls. Fluid flow is introduced through one sidewall and 165 exits through the other sidewall to produce a uniform crossflow across the separation chamber to focus 166 an analyte that is counterbalanced by an equivalent electrophoretic velocity. The electric field can be 167 applied in a scanning mode to focus and collect analytes sequentially.
A method for generating an 168 electric field gradient across the chamber was also suggested to focus several analytes at a time. US.
169 Pat. No. 6,767,443 to Percy H. Rhodes and Robert S. Snyder (Jul. 27, 2004) was then introduced as a 170 variation to the aforementioned invention, where electrode chambers, crossflow chambers, and the 171 separation chamber were stacked on top of each other. This version could therefore sequentially focus 172 analytes in the separation chamber with a higher voltage gradient, less Joule heating effects, and 173 reduced electrodynamic dispersion.

175 Summary of the Invention 176 The present work highlights a free-flow electrophoresis apparatus that introduces a counterflow 177 through the permeable sidewalls of the separation chamber. The purpose of the counterflow is to 178 produce a velocity gradient in the transverse direction. This velocity gradient serves to counter 179 electrophoretic migration such that analytes focus according to their electrophoretic mobility before 180 they are collected at different outlets at the end of the separation chamber. The focusing mechanism 181 is similar to that of free-flow isoelectric focusing, except the equilibrium point is not pI-dependant 182 and no carrier ampholytes are required. Compared to existing methods for free-flow counterflow 183 gradient focusing techniques, no subcompartments within the separation chamber are required, and 184 several analytes can be focused at a time.

186 List of figures 187 FIG. 1 is a schematic of the present method with a plot of relevant velocity fields in the chamber.
188 FIG. 2 shows the focusing mechanism for different analytes for the present method.
189 FIG. 3 is an alternative embodiment of the present method which uses an asymmetric gradient.
190 FIG. 4 is another alternative embodiment of the present method which uses a bilinear gradient.
191 FIG. 5 shows the crescent distortion of the separation peaks for the present method.
192 FIG. 6 shows the crescent distortion of the separation peaks for the present method when there is 193 electroosmotic flow.

194 FIG. 7 shows the crescent distortion of the separation peaks for the present method when there is flow 195 from the top and bottom of the separation chamber.
196 FIG. 8 shows the crescent distortion of the separation peaks for the present method when the 197 separation chamber is a porous matrix.
198 FIG. 9 is a schematic of the top view of an apparatus illustrative of the present invention.
199 FIG. 10 is a schematic of the front view of the apparatus shown in FIG.
9.
200 FIG. 11 is a schematic of the top view of an apparatus illustrative of an alternative embodiment of the 201 present invention.
202 FIG. 12 is a schematic of the front view of the apparatus shown in FIG.
10.
203 FIG. 13 is a schematic of the top view of an apparatus illustrative of another alternative embodiment 204 of the present invention when there is flow from the top and bottom of the separation chamber.
205 FIG. 14 is a schematic of the front view of the apparatus shown in FIG.
13.
206 FIG. 15 is a schematic of the top view of an apparatus illustrative of another alternative embodiment 207 of the present invention when the separation chamber is a porous matrix.
208 FIG. 16 is a schematic of the front view of the apparatus shown in FIG.
15.
209 FIG. 17 is an image of various small molecules being separated and focused at different applied 210 currents using the method presented in FIG. 1 and the apparatus presented in FIG. 9.
211 FIG. 18 is an image of various small molecules being separated and focused at different applied 212 currents using the method presented in FIG. 3 and the apparatus presented in FIG. 9.
213 FIG. 19 is an image of various proteins being separated and focused at different applied currents using 214 the method presented in FIG. 1 and the apparatus presented in FIG. 9.

216 Detailed Description 217 FIG. 1 shows schematics of the free-flow counterflow gradient focusing method. In the preferred 218 embodiment, there will be a buffer flowing through a separation chamber 101 with electrodes 102 on 219 each side. There will be an electric field E across the separation chamber 101 when there is a potential 220 difference applied at the electrodes 102. A sample inlet 103 will be located at the top of the separation 221 chamber 101 to allow an inflow of different analytes through the separation chamber 101. The analytes 222 will migrate and focus across the separation chamber 101 according to their electrophoretic velocity 223 in the electric field E, before the outflow ports 104 at the bottom of the separation chamber 101 224 transport them to different collection reservoirs 105.
225 The novelty of this free-flow counterflow gradient focusing method is the introduction of buffer flow 226 through the sidewalls 106 of the separation chamber 101. In the preferred embodiment, the buffer 227 flow will be equal in magnitude through the sidewalls 106 and will be uniform along the axial 228 direction. As the buffer flow through the sidewalls 106 moves incrementally towards the centre of the 229 separation chamber 101, a fraction will begin to move in the axial direction, towards the outflow ports 230 104. As a result, a counterflow velocity gradient will be produced in the transverse direction that 231 opposes electrophoretic migration in the separation chamber 101. FIG 1a shows a schematic of the 232 transverse velocity field for the counterflow velocity gradient Ux. The continuous addition of buffer 233 flow through the sidewalls 106 also produces a velocity gradient Uz in the axial direction, and this 234 velocity field is shown in FIG. lb.
235 In the counterflow velocity gradient seen in FIG. la, there exists a position in the transverse direction 236 where the electrophoretic velocity of a given analyte is equal to the counterflow velocity in magnitude 237 but opposite in direction. Therefore, the analyte will focus at this unique equilibrium position 238 according to its electrophoretic velocity. In the example presented in FIG. 1, the sample contains but 239 is not limited to two analytes. The first analyte will have an electrophoretic velocity Uel as shown in 240 the velocity field in FIG. lc, and its resulting transport in the separation chamber 101 is represented 241 by a streamline 107. The second analyte will have an electrophoretic velocity Ue2 as shown in the 242 velocity field in FIG. id, and its resulting transport in the separation chamber 101 is represented by a 243 streamline 108. Because Ue2 is greater than Uel, its focal point is at a position in the transverse 244 direction where the opposing counterflow velocity Ux is greater. As the analytes are focused, they are 245 simultaneously transported by the axial fluid velocity Uz, towards the different outlets 104 and into 246 the collection reservoirs 105.
247 FIG. 2 describes the free-flow counterflow gradient focusing mechanism shown in FIG. 1 with more 248 detail. FIG. 2a is a close-up view of the separation chamber 101 where the analytes are focused. The 249 analyte represented by streamline 107 is focused at a transverse position where its electrophoretic 250 velocity Uel is counterbalanced by a transverse bulk fluid velocity Uxl. The analyte represented by 251 streamline 108 is focused at a transverse position where its electrophoretic velocity Ue2 is 252 counterbalanced by a transverse bulk fluid velocity Ux2. FIG. 2b is a graphical representation of the 253 relevant transverse velocities in the separation chamber 101. The electrophoretic velocities Uel and 254 Ue2 for each analyte are constant across the separation chamber 101 while the bulk fluid velocity Ux 255 is a linear gradient. The total transverse velocity of each analyte is therefore Utl = Uel + Ux and Ut2 256 = Ue2 + Ux. The resulting focal point xl and x2 of each analyte is where Utl and Ut2 cross the x-257 axis, respectively. FIG. 2a demonstrates the power of this counterflow gradient focusing mechanism, 258 as analytes will always migrate towards their focal point.

259 FIG. 3 presents an alternative embodiment of the free-flow counterflow gradient focusing method.
260 For this alternative method, the buffer flow may not be equal in magnitude through opposite sidewalls 261 106a and 106b, though it will remain uniform along the axial direction.
For example, the transverse 262 flow through sidewall 106b is much greater than the transverse flow through sidewall 106a. The 263 resulting transverse velocity field across the separation chamber 101 is shown in FIG. 3a, where the 264 counterflow velocity gradient Uxa is now asymmetric. Therefore, when no electric field E is present 265 across the separation chamber 101, the sample streamline will be skewed to the left. When an electric 266 field E is applied across the separation chamber 101 as shown in FIG.
3, positively charged analytes 267 presented in FIG. 1 now focus near the centre of the chamber. In general, this method is particularly 268 useful when all of the analytes within the sample have the same charge, as this effectively doubles the 269 width of the separation chamber 101 as well as the amount of collection reservoirs 105. Furthermore, 270 the electric field E or the flow through sidewalls 106a and 106b can be varied to enable a scanning 271 mode that can shift the location of each focal point overtime. This scanning mode is useful for a 272 scenario where a detector or mass spectrometer is positioned inline with the one of the outlets 104, as 273 analytes can sequentially pass through this location for further analysis.
274 FIG. 4 presents another alternative embodiment of the free-flow counterflow gradient focusing 275 method. For this alternative method, the buffer flow may or may not be equal in magnitude through 276 opposite sidewalls 106, and will not be uniform along the axial direction. For example, if the flow 277 velocity through the sidewalls 106c in the upper half of separation chamber 101c is greater than the 278 flow velocity through the sidewalls 106d in the lower half of separation chamber 101d, then the 279 transverse velocity gradient Uxc in the upper half will be steeper than the transverse velocity gradient 280 Uxd in the lower half. As a result, a bilinear transverse velocity gradient will be created in the 281 separation chamber 101. Bilinear gradients have been used in counterflow gradient focusing 282 techniques because it solves an inherent disadvantage of linear gradients, being that they cannot 283 improve peak capacity and resolution simultaneously. Therefore, in the upper half of the separation 284 chamber 101c, the steep transverse velocity gradient Uxc will produce narrow streamlines 107c and 285 108c that are stacked together. Then, as analytes are transported to the lower half of the separation 286 chamber 101d, the shallower transverse velocity gradient Uxd will resolve the stacked streamlines to 287 produce streamlines 107d and 108d. Such a bilinear gradient can be produced using separate flow 288 sources for the upper sidewalls 106c and lower sidewalls 106d, or by tuning the dimensions of the 289 upper sidewalls 106c and lower sidewalls 106d. As an alternative to creating a bilinear gradient using 290 fluid flow, it is also possible to vary the electric field E in the upper half of the separation chamber 291 101c and the lower half of the separation chamber 101d by tuning either the conductivity or current 292 density in the buffer.
293 FIG. 5 and FIG. 6 demonstrate the resolution of the separation peaks in this free-flow counterflow 294 gradient focusing method. Understanding the flow profile in the height direction of the separation 295 chamber 101 is critical for predicting the resolution of this free-flow counterflow gradient focusing 296 method. In traditional counterflow gradient focusing techniques, Taylor dispersion is often a factor 297 because of the parabolic flow profile of the counterflow Ux. This parabolic flow profile is 298 demonstrated in FIG. 5a, and it decreases from the chamber sidewalls 106 to the middle of the 299 separation chamber 101 in the transverse direction. The parabolic profile will cause the focal point of 300 an analyte to vary along the height direction of the separation chamber 101. As a result, the separation 301 peak for a given analyte is crescent shaped, and its width and orientation depend on its position in the 302 transverse velocity gradient Ux, as shown in FIG. 5b. This result would indicate that band-broadening 303 will be higher closer to the sidewalls 106, and this distortion will affect resolution.
304 In reality, there is electroosmotic flow Ueof to consider in any free-flow electrophoresis device, and 305 this produces a flat velocity profile across the separation 101 chamber in the transverse direction as 306 shown in FIG. 6a. Because of the partial blockage of electroosmotic flow Ueof at the sidewalls 106, 307 a pressure-driven backflow Ub will be generated in the transverse direction with a parabolic flow 308 profile, as shown in FIG. 6a. By adding the velocity profiles for transverse counterflow Ux, the 309 electroosmotic flow Ueof, and the pressure-driven backflow Ub, a depiction of the resulting 310 separation peaks at a given position in the transverse counterflow gradient is shown in FIG. 6b. The 311 resulting crescent-shaped separation peaks are also dependant on the transverse position in the 312 separation chamber 101. In this case, however, the size of the crescent-shaped peaks are reduced when 313 the transverse counterflow Ux and the pressure-driven backflow Ub are in opposite directions, while 314 the size is increased when they are in the same direction. In practice, the electroosmotic flow Ueof 315 can tuned to reduce the band-broadening and therefore improve resolution for a given set of analytes.
316 FIG. 7 and FIG. 8 demonstrate potential methods for reducing Taylor dispersion in free-flow 317 counterflow gradient focusing method. In FIG. 7a, an additional buffer flow is introduced through the 318 top wall 109 and bottom wall 110 of the separation chamber, to produce an additional velocity profile 319 Uy in the height direction. This buffer flow Uy is equal in magnitude from the top wall 109 and bottom 320 wall 110, and is uniform along the transverse and axial direction. The result is that analytes will now 321 be focused in the centre of the separation chamber 101 in the height direction, where the portion of 322 the transverse counterflow parabolic profile Ux is relatively flat.
Therefore, the Taylor dispersion for 323 a given separation peak will be significantly reduced as shown in FIG.
7b, and this will ultimately 324 improve the resolution. In FIG. 8a, the separation chamber 112 is made from a porous matrix, which 325 produces more of a flat transverse counterflow velocity profile Ux when compared to an open chamber 326 101. The resulting separation peaks shown in FIG. 8b will therefore have reduced band-broadening 327 and improved resolution. Note that the methods presented in FIG. 7 and FIG. 8 could also reduce 328 dispersion due to other sources in traditional free-flow electrophoresis devices. For example, they 329 could reduce hydrodynamic dispersion, which is caused by the parabolic velocity profile Uz of the 330 flow in the axial direction. They could also reduce electrodynamic dispersion, which is caused by the 331 parabolic velocity profile Ub of the pressure-driven backflow in the transverse direction.
332 FIG. 9 and FIG. 10 show schematics of the free-flow counterflow gradient focusing apparatus in the 333 preferred embodiment of the invention. Most notably, there is a separation chamber 1 with additional 334 counterflow chambers 2 on each side. Between the separation chamber 1 and each counterflow 335 chamber 2 is a barrier 4 that is permeable to fluid flow and ionic current. On the other side of each 336 counterflow chamber 2 is an electrode chamber 3. Between each counterflow chamber 2 and electrode 337 chamber 3 is an ionically conductive barrier 5.

338 The features 1, 2, 3, 4, and 5 will be enclosed by a housing 17 that is made using soft lithography, 339 micromachining, additive manufacturing, or injection molding processes.
The housing 17 can be made 340 from glass, acrylic, polycarbonate, or any other electrically insulating material. If multiple layers are 341 used in the assembly of the housing 17 then it can be held together using chemical bonding, double-342 sided adhesives, or gaskets. The permeable barrier 4 can consist of several parallel microchannels or 343 a porous matrix. The permeable barrier 5 can consist of an ion exchange membrane, several parallel 344 microchannels, or a porous matrix.
345 The counterflow is pumped into the inlet ports 8 which are connected to the counterflow chambers 2 346 by a fluidic channel 13. There are no outlet ports for the counterflow chambers, as all of the incoming 347 fluid is transported through the permeable barriers 4 and into the separation chamber 1. The 348 hydrodynamic resistance of the permeable barriers 4 must be relatively large to ensure that the 349 counterflow in the transverse direction is uniform along the axial direction as it enters the separation 350 chamber 1 from the barriers 4. As the counterflow moves incrementally towards the center of the 351 separation chamber 1, a fraction will begin to move in the axial direction, towards the outlet ports 11 352 located at the end of the separation chamber 1. As a result, a counterflow velocity gradient will be 353 created in the transverse direction, such that the counterflow is maximum at the sides of the separation 354 chamber 1 and zero at the center of the separation chamber 1.
355 Within each electrode chamber 3 is a platinum electrode 6 that is connected to a voltage source. The 356 barriers 4 and 5 must be ionically conductive to ensure that the electric potential applied at the 357 electrodes 6 results in a uniform electric field through the separation chamber 1. The electrode 358 chambers 3 may be purged with a well-buffered solution to clear electrolysis products from the 359 chamber. The inlet ports 9 for the electrode buffer solution are connected to the electrode chambers 3 360 by fluidic channels 14. The outlet ports 10 for the electrode buffer solution are on the opposite end of 361 the electrode chambers 3 and are connected by fluidic channels 15. The ionically conductive 362 membrane 5 should be impermeable to fluid flow to ensure the electrode buffer does not enter the 363 separation chamber 1.
364 To operate the apparatus, a sample containing analytes with different electrophoretic mobilities will 365 be pumped through the inlet port 7 that is connected to the start of the separation chamber 1 by a 366 fluidic channel 12. While the analytes are transported by the carrier fluid in the axial direction, they 367 will simultaneously migrate across the separation chamber 1 in the transverse direction due to the 368 electric potential applied at the electrodes 6. The counterflow gradient in the separation chamber 1 369 will counter the electrophoretic migration such that each analyte will reach a unique mobility-370 dependant equilibrium point where their net velocity in the transverse direction is zero. The focusing 371 analytes will continue to travel in the axial direction until they reach the outlets 11 at the end of the 372 separation chamber 1. The outlet channels 16 that connect the outlet ports 11 to the separation chamber 373 1 are used to collect the separated analytes without disrupting the fluid flow profile. Any number of 374 outlet channels 16 and outlets 11 can be included at the end of the separation region 1, depending on 375 the number of analytes that must be collected as well as the manufacturing limitations.

376 A potential drawback of the preferred embodiment of the present invention is that the large 377 hydrodynamic resistance requirements for the barrier 4 will also result in a large electrical resistance 378 across the barrier 4. The resulting voltage drop across the barrier 4 will therefore be high, and this will 379 limit the electric field that can be applied through the separation chamber 1. Consequently, the 380 separation resolution will be limited. To address this issue, an alternative embodiment of the present 381 invention has been proposed and shown in FIG. 11 and FIG. 12. The barriers 4 and 5 in this alternative 382 embodiment are now located on separate planes in the height direction, such that the fluidic and ionic 383 transport into the membrane are decoupled. Therefore, the high hydrodynamic resistance requirements 384 of the barrier 4 have no impact on the electric field through the separation chamber 1.
385 FIG. 11 has been divided into parts a, b, and c to highlight the relevant planes in the height direction 386 for this embodiment. FIG. 12 has also been divided into parts a, b, and c to provide a reference for the 387 corresponding plane being shown in FIG. 11a, b, and c, respectively.
FIG. lla shows the plane in the 388 height direction where the separation chamber 1 is located. FIG. llb shows the plane in the height 389 direction above that of FIG. lla where the counterflow chambers 2 are located. The permeable barriers 390 4 are located on a height plane that is between the separation chamber 1 and the counterflow chambers 391 2 as shown in FIG 12. FIG. 11c shows the plane in the height direction below that of FIG. ha where 392 the electrode chambers 3 are located. The ionically conductive barriers 5 are located on a height plane 393 that is between the separation chamber 1 and the electrode chambers 2 as shown in FIG 12.
394 A fluid pumped into a counterflow chamber 2 from the corresponding inlet port 8 will then flow 395 through the corresponding permeable barrier 4 and into the separation chamber 1. The counterflow 396 chambers 2 and corresponding permeable barriers 4 are positioned on each side of the separation 397 chamber 1, such that a counterflow gradient in the transverse direction is produced in the separation 398 chamber 1. There may be channels 25 at the sides of the separation chamber 1 that are part of the 399 housing 17 and ensure that the counterflow from the permeable barriers 4 enters the separation 400 chamber in the transverse direction. At the same time, an electric potential will be applied at the 401 platinum electrodes 6 in the electrode chambers 3, which will then allow an electric field to travel 402 through the ionically conductive barriers 5 and across the separation chamber 1. A well-buffered 403 solution should be pumped into the inlet port 9, through the electrode chamber 3, and out of the outlet 404 port 10 to ensure that all electrolysis products are cleared from the electrode chamber 3. From there, 405 the separation and collection of analytes will occur in the separation chamber 1 using the same 406 counterflow gradient focusing mechanism discussed in the preferred embodiment.
407 Another potential drawback of the preferred embodiment in the present invention is that Taylor 408 dispersion caused by the parabolic flow-profile of the counterflow could limit the separation 409 resolution. A similar issue in free-flow electrophoresis known as crescent distortion has been well 410 documented in the prior art, and is mainly caused by hydrodynamic and electrohydrodynamic 411 dispersion. Therefore, a schematic of the proposed apparatus that will limit Taylor dispersion is shown 412 in FIG. 13 and FIG. 14. There are three relevant planes shown in the height direction that are labeled 413 as a, b, and c for FIG. 13 and FIG. 14. The alternative embodiment of the present invention shown in 414 FIG. 13 and FIG. 14 is a slight variation from the alternative embodiment presented in FIG. 11 and 415 FIG. 12. The only difference is that there is now an additional flow introduced from the top and bottom 416 walls of the separation chamber 1. As a result, the analytes will be hydrodynamically focused at the 417 center of the separation chamber in the height direction, such that they are located at the center of the 418 parabolic flow-profile where less distortion occurs.
419 FIG. 13 and FIG. 14 show additional chambers 18 and 21 located above and below the separation 420 chamber 1 that is shown in FIG. 13a and FIG. 14a. The chamber 18 is shown in FIG. 13b and FIG.
421 14b, where fluid is pumped into this chamber 18 from inlet port 19 through the fluidic channel 20. A
422 barrier 26 that is permeable to fluid flow is located between chamber 18 and the separation chamber 423 1 in the height direction. The fluid pumped into chamber 18 flows through the permeable barrier 26 424 to produce a flow from the top of the separation chamber 1. Similarly, the chamber 21 is shown in 425 FIG. 13c and FIG. 14c, where fluid is pumped into this chamber 21 from inlet port 22 through the 426 fluidic channel 23. A barrier 27 that is permeable to fluid flow is located between chamber 21 and the 427 separation chamber 1 in the height direction. The fluid pumped into chamber 21 flows through the 428 permeable barrier 27 to produce a flow from the bottom of the separation chamber 1. The top and 429 bottom flow in the separation chamber 1 must be approximately equal in magnitude to ensure that 430 analytes are focused at the center of the parabolic profile and Taylor dispersion is minimized.
431 Another alternative embodiment of the present invention is shown in FIG. 15 and FIG. 16. The 432 purpose of this embodiment is also to reduce Taylor dispersion due to the counterflow. Unlike the 433 embodiment shown in FIG. 13 and FIG. 14, the fabrication and operation of this apparatus is less 434 complicated. The embodiment shown in FIG. 15 and FIG. 16 is a slight variation to that of the 435 preferred embodiment shown in FIG. 9 and FIG. 10. The only difference is that there are no longer 436 permeable barriers 4, and the separation chamber 1 has been replaced with a porous matrix 24. The 437 purpose for using a porous matrix 24 as the separation region is that flow through a porous matrix 438 produces more of a flat flow-profile, and therefore Taylor dispersion is less of an issue. The porous 439 matrix 24 can be made from cellulose, glass, a ceramic material, a polymer, or a gel material. The 440 hydrodynamic resistance of the porous matrix 24 must be large enough to ensure that the counterflow 441 is uniform along the axial direction.
442 To reduce the concept of the free-flow counterflow gradient focusing method to practice, an apparatus 443 was developed according to the preferred embodiment shown in FIG. 9.
The apparatus was made from 444 a polydimethylsiloxane (PDMS) housing 17 according to standard soft lithography techniques. The 445 barrier 4 consisted of microchannels which had a height that was approximately ten times smaller than 446 that of the separation chamber 1. The barrier 5 consisted of a Nafion ion-exchange membrane while 447 the electrode chamber 3 consisted of a glass reservoir. The apparatus was used to separate a sample 448 containing small molecules of varying electrophoretic mobilities as well as a sample containing 449 proteins of varying electrophoretic mobilities.
450 FIG. 17 shows the apparatus separating and focusing small molecules according to the method 451 presented in FIG. 1. From left to right, the small molecules are xylene cyanol FF, bromophenol blue, 452 orange G, and ponceau S. FIG. 17a shows images of the separation chamber at various applied 453 currents. The streamlines of the small molecules follow a similar trajectory to the streamlines 107 and 454 108 presented in FIG. 1. FIG. 17b shows the corresponding separation profiles of the small molecules 455 at their focal point across the separation chamber.
456 FIG. 18 shows the apparatus separating and focusing small molecules according to the asymmetric 457 gradient method presented in FIG. 3. From left to right, the small molecules are xylene cyanol FF, 458 bromophenol blue, orange G, and ponceau S. FIG. 18a shows images of the separation chamber at 459 various applied currents. The streamlines of the small molecules appear to follow a similar trajectory 460 to the streamlines 107 and 108 presented in FIG. 3. FIG. 18b shows the corresponding separation 461 profiles of the small molecules at their focal point across the separation chamber.
462 FIG. 19 shows the apparatus separating and focusing proteins according to the method presented in 463 FIG. 1. From left to right, the proteins are hemoglobin and bovine serum albumin. The bovine serum 464 albumin was labeled with bromophenol blue dye to appear visible. FIG.
19a shows an image of the 465 separation chamber at various applied currents. The streamlines of the proteins appear to follow a 466 similar trajectory to the streamlines 107 and 108 presented in FIG. 1.
Note that some of the unbound 467 bromophenol blue dye appears to the right of the bovine serum albumin streamline. FIG. 19b shows 468 the corresponding separation profiles of the analytes at their focal point across the separation chamber.
469 It should also be noted that proteins are relatively large compared to the small molecules and therefore 470 the corresponding separation peaks were expected to be larger due to Taylor dispersion. However, 471 this does not appear to be the case, and therefore it is assumed that electroosmotic flow is reducing 472 the effects of Taylor dispersion, as described in FIG. 6.