MICELLAR GRADIENT FOCUSING 

FIELD OF THE INVENTION 

[0001] The present invention relates to a method and device for separating and or 

concentrating one or more analytes in a solution, and in particular, to a method and 

device which focuses and separates analytes using affinity gradient focusing. 

BACKGROUND OF THE INVENTION 

[0002] Over the past decade a great deal of research has been focused on the 

development of micro-total-analytical systems. This technology is based on the concept 

of integration of a series of microfluidic channels for the movement, separation, 

reaction, and/or detection of various chemicals, e.g., proteins, DNA, chemical 

compounds, etc. 

[0003] Prior methods for concentrating analytes include stacking and focusing. In 

the context of this disclosure, focusing refers to methods for manipulating the velocity of 

an analyte and thereby causing the analyte to move towards a point at which its velocity 

is zero and where the analyte will therefore accumulate and increase in concentration. 

In addition, the location of the zero velocity point is often dependent upon some 

characteristic of the analyte molecule being focused, so that different analyte molecules 

are focused at different points, and thereby separated. 

[0004] In this context, focusing is to be distinguished from stacking, which is a 

related class of methods in which analytes are moved through a velocity gradient (which 

is often transient) and the analyte peaks become narrower and more concentrated, but 

there is no point of zero analyte velocity. In stacking methods, the maximum degree to 

which analyte concentration can be increased is theoretically limited to the ratio of the 

velocities on the fast and slow sides of the velocity gradient. In contrast, for focusing at 

a zero velocity point, there is no theoretical limit to the concentration factor. 

[0005] Previously known focusing methods include isoelectric focusing (hereinafter 

“IEF”), electric field gradient focusing (hereinafter “EMF”), counteracting 

chromatographic electrophoresis, and temperature gradient focusing (hereinafter 

“TGF”). In general, all of these previously known methods work by creating a gradient 
in the electrophoretic velocity of the analyte. Therefore, they only work with analytes 

that have non-zero electrophoretic mobility. For example, in IEF, the analyte has zero 

electrophoretic mobility only at the zero velocity point. 

[0006] Micellar electrokinetic chromatography and related methods (hereinafter 

“EKC”) take advantage of an analyte’s affinity for a pseudostationary phase to facilitate 

separations using capillary electrophoresis. Traditional EKC separations differ from 

traditional focusing techniques in that analyte molecules move along a separation 

channel at an essentially constant velocity, whereas in focusing techniques, analytes 

migrate through the channel to a point where they have a zero velocity. Separation in 

EKC is achieved because different analytes migrate with different velocities dependent 

on their affinity for the pseudostationary phase. Often EKC separations are 

implemented in conjunction with stacking procedures to preconcentrate analytes and 

facilitate lower detection limits. 

[0007] In EKC (and in chromatography in general), the buffer is referred to as the 

mobile phase, and a second phase such as a micellar phase is referred to as the 

pseudostationary phase. The pseudostationary phase serves the same function – to 

provide selectivity – as a stationary phase in chromatography, but is not actually 

stationary, and can move with the buffer and/or with its own electrophoretic mobility. 

SUMMARY OF THE INVENTION 

[0008] The invention provides a method for the preconcentration and or separation 

of analytes in solution. In particular, it provides a focusing method for analytes that 

cannot be separated or focused based purely upon their electrophoretic mobilities 

(neutral species, or chiral species, for example). The separation can be used as a part 

of a chemical or biochemical analysis in a microfluidic chip or capillary system. 

[0009] The new method, described here, combines the two concepts of focusing 

and EKC to achieve something that could not be done with either of them separately: 

the focusing of analytes based upon their affinity for a pseudostationary 

chromatographic phase. The new method combines the characteristics and utility of 

EKC with those of focusing wherein the pseudostationary phase provides a medium for 

2
[0010] Paragraphs 10-16 will need to be rewritten once the claims are finalized. In 

one form of the invention, a method is provided for directing at least one analyte in a 

solution containing a pseudostationary phase and located within a conduit. The method 

includes establishing a gradient in the retention factor of the pseudostationary phase for 

the at least one analyte and moving the at least one analyte within the conduit, so that 

the concentration of the at least one analyte is caused to increase at one or more 

positions along the gradient. 

[0011] The invention in another form thereof concerns a method for directing at 

least one analyte in a solution containing an ionic pseudostationary phase. The method 

includes establishing a gradient in the retention factor of the ionic pseudostationary 

phase for the at least one analyte. An electrical current is produced in the solution 

which has a significant component substantially aligned with the gradient and in a 

direction such that a resulting electrophoretic motion of the ionic pseudostationary 

phase is substantially aligned in a direction from a region of high retention factor to a 

region of low retention factor. A bulk flow is produced in the solution which has a 

significant component substantially aligned with a direction opposite the direction of the 

electrophoretic motion of the ionic pseudostationary phase so that the concentration of 

the at least one analyte is caused to increase at one or more positions along the 

gradient. 

[0012] The present invention in another form thereof, includes a method for 

directing at least one analyte in a solution containing a nonionic pseudostationary 

phase. The method includes establishing a gradient in the retention factor of the 

nonionic pseudostationary phase for the at least one analyte. An electrical current flow 

is produced in the solution which has a significant component substantially aligned with 

the gradient and in a direction such that a resulting electrophoretic motion of the at least 

one ionic analyte is substantially aligned in a direction from a region of low retention 

factor to a region of high retention factor. A bulk flow of the solution is produced which 

has a significant component substantially aligned in a direction opposite the direction of 

the electrophoretic motion of the at least one ionic analyte, so that the concentration of 

3
[0013] The present invention in another form thereof concerns a device for gradient 

focusing which includes a fluid conduit and a solution containing a pseudostationary 

phase located in the fluid conduit. At least one heat source or heat sink is thermally 

coupled to the fluid conduit for providing a temperature gradient which thereby 

establishes a gradient in the retention factor of the pseudostationary phase for at least 

one analyte. An electrical source is provided for establishing a flow of electric current in 

the solution which has a significant component substantially aligned along the gradient. 

A pump establishes a bulk flow in the solution which has a significant component 

substantially aligned along the gradient so that the concentration of the at least one 

analyte is caused to increase at one or more positions along the fluid conduit. 

[0014] The present invention in yet another form thereof concerns a device for 

gradient focusing which includes a fluid conduit which has two segments joined by a 

semi-permeable membrane. A solution containing a pseudostationary phase is 

disposed in disproportionate concentrations in each of the two segments of the fluid 

conduit thereby establishing a gradient in the retention factor of the pseudostationary 

phase for at least one analyte. An electrical current source is provided for establishing 

a flow of electric current in the solution having a significant component substantially 

aligned along the gradient. A pump provides for establishing a bulk flow in the solution 

having a significant component substantially aligned along the gradient so that the 

concentration of the at least one analyte is caused to increase at one or more positions 

along the fluid conduit. 

[0015] The present invention in another form thereof concerns a device for gradient 

focusing which includes a fluid conduit and a solution containing a pseudostationary 

phase disposed in the fluid conduit. The device further includes means for establishing 

a gradient in the retention factor of the pseudostationary phase for at least one analyte 

and means for causing one or more analytes to move within the conduit so that the 

concentration of the at least one analyte is caused to increase at one or more fixed 

positions along the fluid conduit. 

4
[0016] The present invention in yet another form thereof concerns a device for 

gradient focusing which includes a fluid conduit and a solution containing a 

pseudostationary phase disposed in the fluid conduit. The device further includes 

means for establishing a gradient in the retention factor of the pseudostationary phase 

for at least one analyte, means for establishing a flow of electric current in the solution 

having a significant component substantially aligned along the gradient, and means for 

establishing a bulk flow of the solution having a significant component substantially 

aligned along the gradient, so that the concentration of the at least one analyte is 

caused to increase at one or more positions along the fluid conduit. 

[0017] In its implementation, the present method functions to focus or separate 

analytes using either a neutral or charged pseudostationary phase. For example, the 

pseudostationary phase may be charged micelles such as negatively charged micelles, 

although the same principles discussed apply for positively charged micelles. A voltage 

is applied so that the charged micelles move from the region of high retention to the 

region of low retention. The background buffer is made to flow in the opposite direction, 

from the region of low retention to the region of high retention. In the region of high 

retention, the analyte is predominantly located within the micelles and so the analyte 

moves with the micelles. In the region of low retention, the analyte is predominantly 

located outside the micelles, and so moves with the buffer, in the opposite direction. 

Somewhere between the regions of high and low retention, the net velocity of the 

analyte is zero, and the analyte is focused at that point. 

[0018] An advantage of this method over all of the previous focusing techniques is 

that it can be used with neutral analytes, which, in their native state, have zero 

electrophoretic mobility. An additional advantage over other focusing techniques is that 

it provides for focusing based upon properties of the analyte molecules other than the 

electrophoretic mobility of the molecule. 

[0019] An advantage of this method over the previous EKC techniques is that the 

present method is a focusing technique and as such has no theoretical upper limit to the 

concentration factor. An additional advantage over EKC stacking techniques is that the 

position of the focused analyte can be manipulated, e.g., held stationary or moved, as 

5
[0020] Further features and advantages of the present invention will be set forth in, 

or apparent from, the detailed description of preferred embodiments thereof which 

follows. 

BRIEF DESCRIPTION OF THE DRAWINGS 

[0021] Figure 1 is a schematic of micellar gradient focusing in a microchannel or 

capillary; 

[0022] Figure 2 is a schematic drawing of a fluidic device according to one 

embodiment of the present invention; 

[0023] Figures 3a-f depict fluorescence microscopy images of focusing of 

rhodamine B where the images of Figures 3a-f were taken just before initiating the 

focusing, i.e., 0 seconds, and at 10, 20, 30, 40, and 50 seconds, respectively, after the 

voltage was turned on; 

[0024] Figure 4 is a plot of peak concentration vs. time for focusing of rhodamine B 

in a capillary; 

[0025] Figure 5 depicts micellar gradient focusing and separation of two neutral 

fluorescent dyes, rhodamine B (left) and rhodamine 110 (right); and 

[0026] Figure 6 is a schematic of micellar focusing in a microchannel or capillary 

according to another embodiment of the present invention. 

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 

[0027] The present method provides focusing, i.e., concentrations and/or 

separation, based upon affinity of an analyte for a pseudostationary phase such as a 

micellar phase. The method works by creating a gradient in the retention factor of the 

solute of interest to the pseudostationary phase (i.e., affinity of solute for the micelles) in 

a channel such as in capillary systems or in a microfluidic chip. The solute has an 

inherent electrophoretic mobility when free in solution. When interacting with the 

pseudostationary phase, the solute assumes the electrophoretic mobility of the 

pseudostationary phase. On one side of the gradient, the solutes strongly interact with 

6
[0028] Referring now to Figure 1, on the left side of channel 10, the retention factor 

of an analyte in the micellar phase, or other pseudostationary phase as represented by 

micelles 12, is high, so that the analyte is predominantly found in the micelles 12. On 

the right side of channel 10, the retention factor is low, and the analyte is predominantly 

found in the mobile phase, i.e., buffer 14. The mobile phase is pumped via pump 

system 16, either electroosmotically, with pressure gradients, or by a combination of the 

two, so that the buffer 14 moves from the region of low retention factor to the region of 

high retention factor as indicated by arrow 17. 

[0029] In the embodiment of Figure 1, the micelles 12 are negatively charged, and a 

voltage is applied via power supply 18 to the channel 10 so that the micelles 12 

electrophoretically migrate from the region of high retention factor to the region of low 

retention factor as indicated by arrow 19. In the region of high retention factor, the 

analyte moves primarily with the micelles 12. In the region of low retention factor, the 

analyte moves primarily with the mobile phase, i.e., buffer 14. The result is that the 

analyte moves toward the center from both ends of the channel and is focused at some 

point F along the retention factor gradient at which the net velocity of the analyte is zero. 

Although the embodiment of Figure 1 has the micelles being negatively charged, one 

skilled in the art will readily appreciate that the same focusing technique can easily be 

modified for positively charged micelles. 

[0030] Delete paragraph 30. 

7
[0031] Although the present micellar gradient focusing bears similarities in its 

implementation to temperature gradient focusing, micellar gradient focusing focuses 

analytes via a different mechanism and is functionally unique from temperature gradient 

focusing. Further, it is important to note that whereas temperature gradient focusing 

requires a buffer whose ionic strength is a function of temperature, micellar gradient 

focusing can be accomplished in any buffer capable of supporting a pseudostationary 

phase. 

[0032] Micellar gradient focusing requires the production of a spatial gradient in the 

analyte velocity. For the motion of an analyte in a system such as that illustrated in 

Figure 1, the total analyte velocity is given by: 

  uuu      kkuk   EPBT  1/1   MC  1/  , 

[0033] where uB is the mobile phase velocity, uEP is the electrophoretic velocity of 

the analyte through the mobile phase (equal to zero for neutral analytes), uMC is the 

(total) velocity of the micelles, and k is the retention factor (a factor indicating the 

relative amount of time the analyte spends in the pseudostationary phase). This 

description describes methods to produce the required velocity gradient by producing a 

gradient in the retention factor k. 

[0034] The retention factor is equal to k = K β, where K is the distribution coefficient 

which quantifies the affinity of the analyte for the pseudostationary phase, and β is the 

phase ratio defined as the ratio of the volume of pseudostationary phase to the volume 

of mobile phase. The production of a retention factor gradient can then be 

accomplished either through a gradient in the distribution coefficient or through a 

gradient in the phase ratio or a combination of the two. There are several different ways 

that this can be accomplished, some of which are listed below. 

[0035] A solution of surfactant will form micelles if and only if the concentration of 

surfactant is greater than the critical micelle concentration (hereinafter “CMC”). For 

surfactant concentration greater than the CMC, the volume of micellar pseudostationary 

phase is proportional to the difference between the concentration and the CMC. For 

most surfactants, the CMC is a function of temperature. Therefore, a gradient in the 

phase ratio can then be obtained by applying a temperature gradient at a fixed 

surfactant concentration. 

8
[0036] Implementation of this embodiment is described with reference to a 

microfluidic chip exemplar having a microchannel 20 as shown in Figure 2 which is one 

possible apparatus for controlling the temperature of different parts of the microchannel. 

The method of creating a temperature gradient is the same as described in U.S. Patent 

Application Serial No. 10/197,331, herein incorporated by reference. However, the 

apparatus of U.S. Patent Application Serial No. 10/197,331 provides for just one of 

many different ways to manipulate the temperature in a microchannel which are known 

to one of ordinary skill in the art. 

[0037] In the embodiment, the buffer is 5 mM carbonate buffer, pH 9.4 in 

approximately 5% by weight ethanol, 95% water, with a 5 mM concentration of sodium 

dodecyl sulfate (SDS). For an analyte, the neutral fluorescent dye rhodamine B is 

added to the solution at a concentration of about 2.5 μM. For this type of SDS solution 

the CMC is an increasing function of temperature, so that the phase ratio is a 

decreasing function of temperature. 

[0038] A microchannel 20 with dimensions 30 μm deep, 50 μm wide, and 2 cm long 

is used for the separation. The microchannel and fluid reservoirs 27 are initially filled 

with the uniform sample solution. The temperatures of the cold and hot portions of the 

apparatus are set to 10°C and 80°C using heat sinks such as cooling copper 

blocks 23a, 23b covering much of the ends of the microchannel 20, and one hot zone 

provided by a heat source such as heated copper block 22. Thermal contact between 

the poly(carbonate) and the copper blocks is insured using a thermally conductive 

adhesive 26. The copper blocks 22, 23a, 23b are arranged so that there was a 1 mm 

gap 28 between the heated copper block 22 and the cooling copper block 23a and a 

2 mm gap 29 between heated copper block 22 and the cooling copper block 23b. 

[0039] Microchannel 20 also includes electrodes 25, 24, input wells, i.e., buffer 

reservoirs 27 for the mobile phase buffer, and a narrow hot zone 20a near the middle of 

the microchannel 20. The heated copper block 22 is heated using a small high-power 

resistor embedded into the copper and its temperature is regulated using a PID 

temperature controller (Omega Engineering Inc., Stamford, Conn.). To regulate the 

temperature of the cold zones, 1/4 inch diameter holes are drilled through the cooling 

copper blocks 23a, 23b and cold water from a thermostatted bath (Neslab, Portsmouth, 

9
[0040] A voltage of +125 V is applied, and the distribution of rhodamine B dye in the 

channel is monitored using a fluorescence microscope and CCD camera 32. A series 

of the resulting images taken at 10 second intervals from 0 to 50 seconds as is shown in 

Figures 3a-3f, respectively with the rhodamine B identified as B. After 50 seconds, a 

bright band of focused rhodamine was clearly visible near the hot end (the right side in 

the figure) of the temperature gradient (Figure 3f). 

[0041] The same apparatus as described above can be used to produce the 

temperature gradient in a capillary. The solution used is similar to that used for the 

device in a microchannel, and consists of 20 mM SDS, and 5 mM carbonate buffer in 

25% ethanol, 75% water. An initial concentration of rhodamine B of 0.2 μM is used. 

[0042] Figure 4 shows a plot of the focused peak concentration, inferred from the 

measured fluorescence intensities, as a function of time. After the voltage is switched 

on around 5 seconds, the peak concentration increased with time, reaching a value 

greater than 7 μM in about 3 minutes. About a 35-fold increase in concentration is 

achieved in three minutes. 

[0043] Referring now to Figure 5, in a further embodiment, the present method can 

be used for focusing and separating more than one species in a capillary. Building on 

the embodiment of Figures 2 and 3 from above for focusing rhodamine B, after the 

rhodamine B is focused for several minutes, identified as B, a solution of approximately 

3 μM rhodamine 110 is introduced at the entrance to the capillary. After a few minutes 

of additional focusing, the rhodamine 110 was clearly visible as an additional focused 

peak as shown in Figure 5 identified as 110. Note that in normal operation, a separation 

would be accomplished by injecting mixed sample into the separation channel rather 

than by sequential injection of different samples as was done for this demonstration. 

[0044] In most cases the distribution coefficient is temperature-dependent, so that a 

retention factor gradient could be made by applying a temperature gradient, even if the 

CMC and the phase ratio were temperature-independent. In the above example, there 

10
[0045] There have also been pseudostationary phases specifically developed to 

provide a temperature-dependent affinity for various analytes. These are used in 

“temperature-programming” EKC, where the temperature of the separation is changed 

over time to achieve improved separations. Use of these types of pseudostationary 

phases for focusing with a temperature gradient according to the methods described 

herein would be relatively straight forward and readily implemented by one skilled in the 

art. Examples include the use of a thermoresponsive polymer 

poly(N-isopropyl-acrylamide), which can be used to make pseudostationary phases that 

can be switched on and off at a transition temperature. 

[0046] Both the distribution coefficient and phase ratio can be dependant upon the 

composition of the solution used, and so a retention factor gradient can be produced 

using a gradient in the solution composition. 

Referring now to Figure 6, in another embodiment of a microchannel or capillary for use 

in gradient focusing, where like elements to the embodiment of Figure 1 are identified 

as reference numbers increased by 50, microchannel 60 is divided into two adjacent 

channels, 60a, 60b. The two channels over a certain portion of their length, the gradient 

region, are connected by a selectively-permeable membrane or other selectively 

permeable structure 61. The analyte and the pseudostationary phase are contained in 

the separation channel 60a. As in the embodiment of figure 1, the solution in the 

separation channel is caused to flow from the region of low retention factor to the region 

of high retention factor. The control channel 60b and the membrane 61 are used to form 

a gradient in one or more components of the buffer in the separation channel and to 

thereby form the retention factor gradient. The control channel contains a solution of a 

composition differing from that in the separation channel. The difference between the 

two solutions will most often be in the concentration of one or more of the solution 

components. The membrane is chosen to be permeable to one or more of the 

components that are present in differing concentrations in the two solutions. The 

passage of the solution components through the membrane will cause the composition 

of the solution in the separation channel to change. Because the solution in the 

11
separation channel is flowing from the region of low retention factor to the region of high 

retention factor, this change will be greater at the high retention factor end of the 

gradient region than at the low retention factor end. Thus, a steady-state gradient in the 

composition of the solution in the separation channel will be established. The 

composition gradient can be, for example, a gradient in the surfactant concentration in a 

micellar system, or a gradient in the concentration of an organic modifier, or a gradient 

in the concentration of a salt dissolved in the solution. Because the retention factor is a 

function of the composition of the solution used, this will result in a steady-state gradient 

of the retention factor in the separation channel. This retention factor gradient can then 

be used for focusing as in the previously described embodiments. Note that the 

geometrical arrangement of the separation channel and the control channel need not be 

side-by-side as drawn in figure 6. They could also be arranged coaxially, annularly, or in 

any other arrangement that accomplished the desired solution composition gradient. In 

addition, the relative sizes of the two channels need not be equal as is shown in Figure 

6. The control channel could in fact be replaced with a much larger container of solution. 

For examples of devices using similar semi-permeable structures to separate to 

channels see Z. Huang and C.F. Ivory, Analytical Chemistry 71, 1628 (1999) and 

Z. Huang and C.F. Ivory, Abstracts of Papers of the American Chemical Society 219, 

208-BIOT (2000), herein incorporated by reference. 

[0047] As previously noted, materials and strategies that have been developed for 

EKC methods could be modified and applied to the present method as would be now 

readily apparent to one of ordinary skill in the art. For example, it is common in EKC to 

vary the solution composition (e.g., salt concentration, fraction of alcohol used, 

surfactant concentration, etc.) as a function of time to produce a time variation of the 

retention factor. These strategies – referred to as “solvent programming” – could be 

combined with a spatial gradient of the solution composition to produce a spatial 

gradient of the retention factor that could be used for focusing. 

[0048] A neutral micellar or other pseudostationary phase would always move with 

the bulk buffer. In this case, Equation (1) would become: 

  kuuu  EPBT 1/1  . 

12
[0049] Because the electrophoretic mobility of the analyte changes when it moves 

from the mobile phase to the pseudostationary phase, a gradient in the retention factor 

will still produce a gradient in velocity (if uEP ≠ 0), and so a neutral micellar or other 

pseudostationary phase can be used for focusing. If the analyte is neutral, however, the 

pseudostationary phase must be charged. 

[0050] The present micellar gradient focusing can also be implemented through the 

superposition of multiple separation modalities to yield highly selective chiral 

separations. Traditionally, microchannel-based separations of chiral analytes are 

conducted by adding a chiral selector to the medium in which the separation is 

performed. Commonly these chiral selectors have a chiral handedness associated with 

their structure and thereby interact more strongly with either the D or L enantiomer of a 

racemic analyte mixture. This interaction either will lead to a retardation of the 

electrophoretic velocity of one enantiomer over the other and or differentially effect the 

partitioning into the pseudostationary phase, and thereby provide their separation. 

[0051] Implementation of chiral selectors in the present method can be done by 

adding the desired chiral selector (in addition to the pseudostationary phase) to the 

buffer medium. Thus, the addition of the chiral selector would effect the velocities (and 

therefore the focusing points) of the two enantiomers differently in one or both of two 

ways: The chiral selector could either differentially modify the electrophoretic mobilities 

of the two enantiomers and/or it could differentially effect their partitioning into the 

pseudostationary phase. Thus micellar gradient focusing, where a spatial gradient in 

pseudostationary phase affinity for the analyte exists, allows for focusing of enantiomers 

at differing spatial points in the channel when combined with a chiral selector. Note that 

chiral separations could also be accomplished within this new method by using a chiral 

pseudostationary phase, such as micelles formed with chiral surfactants, either with or 

without an additional chiral selector additive. 

[0052] Delete paragraph 53 and 54. 

[0053] Delete paragraph 53 and 54.

[0054] It will now be apparent to one skilled in the art that the present method 

combines focusing with EKC and thereby incorporates the advantages of both. 

Advantages inherent to focusing include the high degree of analyte concentration that 

13
[0055] The combination of focusing with micellar chromatography adds the ability to 

focus and separate analytes that cannot be focused or separated with any other prior 

focusing technique. In particular, neutral species now can be focused, and different 

analytes with identical electrophoretic mobilities, such as stereo-isomers, can be 

separated. 

[0056] Further, the pseudostationary-phase-based focusing of the present invention 

also allows for the tuning of the property of the analyte molecules that is the basis for 

the focusing and separation. For example, different pseudostationary phases can be 

used to separate analytes based upon their hydrophobicity, chirality, or specific affinity 

for a ligand. Any of the pseudostationary phases that have been developed for EKC 

can potentially be used in a focusing mode with the present novel method. 

[0057] In addition, the present micellar gradient focusing technique, which 

separates molecules on the basis of properties other than electrophoretic mobility, can 

be used in conjunction with one of the previously developed focusing techniques or in 

combination with a different variation or embodiment of this present micellar gradient 

focusing technique to implement a 2D separation scheme in which both separation 

dimensions result from focusing techniques. 

[0058] Since the retention factor gradient can be imposed using a temperature 

gradient, the present method potentially shares the advantage of the simplicity of 

implementation of temperature gradient focusing. 

[0059] Although the invention has been described above in relation to preferred 

embodiments thereof, it will be understood by those skilled in the art that variations and 

modifications can be effected in these preferred embodiments without departing from 

the scope and spirit of the invention. 

14
CLAIMS: 

1. A method for directing at least one analyte in a solution containing a 

pseudostationary phase, the pseudostationary phase having a retention factor for {one 

or more of?} said at least one analyte, said method comprising the steps of: 

establishing a steady-state spatial gradient in the retention factor in the 

pseudostationary phase for the at least one analyte; and 

moving {one or more of?} the at least one analyte in the solution, so that the 

concentration of {one or more of?} the at least one analyte is caused to increase at one 

or more positions along said gradient. 

2. The method of claim 1, wherein the step of moving the at least one analyte is 

accomplished using a combination of an applied electric field and an applied bulk 

solution flow. 

3. The method of claim 2, wherein the pseudostationary phase is charged, the 

analyte is either neutral or charged; 

and the applied electric field has a significant component substantially aligned 

with said gradient and in a direction such that the resulting electrophoretic motion of the 

ionic pseudostationary phase is substantially aligned in a direction from a region of high 

retention factor to a region of low retention factor; 

and the applied bulk solution flow has a significant component substantially 

aligned in a direction opposite the direction of the electrophoretic motion of the ionic 

pseudostationary phase. 

4. The method of claim 3, wherein said step of establishing a gradient in the 

retention factor comprises producing a gradient in the phase ratio of the ionic 

pseudostationary phase. 

5. The method of claim 4, wherein producing a gradient in the phase ratio 

comprises producing a temperature gradient along the ionic pseudostationary phase. 

15
6. The method of claim 4, wherein producing a gradient in the phase ratio 

comprises producing a gradient in the solution composition. 

7. The method of claim 3, wherein said step of establishing a gradient in the 

retention factor comprises producing a gradient in the distribution coefficient of the ionic 

pseudostationary phase. 

8. The method of claim 7, wherein the distribution coefficient of the ionic 

pseudostationary phase is temperature dependent and producing a gradient in the 

distribution coefficient comprises producing a temperature gradient along the ionic 

pseudostationary phase. 

9. The method of claim 7, wherein producing a gradient in the distribution coefficient 

comprises producing a gradient in the solution composition. 

10. The method of claim 3, further comprising adding a chiral selector to the solution 

in the conduit. 

11. The method of claim 3, wherein the pseudostationary phase is selected from the 

group comprising micelles, microemulsions, lyposomes, and dendrimers. 

12. The method of claim 2, wherein the pseudostationary phase is neutral, the 

analyte is charged; 

and the applied electric field has a significant component substantially aligned 

with said gradient and in a direction such that the resulting electrophoretic motion of the 

at least one ionic analyte is substantially aligned in a direction from a region of low 

retention factor to a region of high retention factor; 

and the applied bulk solution flow has a significant component substantially aligned in a 

direction opposite the direction of the electrophoretic motion of the at least one ionic 

analyte. 

16
Claim 12 should have the same set of dependent claims as claim 3. 

13. A device for equilibrium gradient focusing, said device comprising: 

a separation channel {we’d like separation channel to be taken as generally as 

possible – including things like microchannels and capillaries. Would it be ok to insert 

our own definition of separation channel in the body of the application or should we 

leave it as is, assuming that it is more general like that, or should we use a different 

word at this point in the claims? Or should we write two sets of claims – one for 

capillaries and one for channels as we did with the TGF apatent application?} ; 

a solution containing a pseudostationary phase disposed in said separation 

channel, said pseudostationary phase having a retention factor for at least one analyte; 

a first means for producing a steady-state gradient in said retention factor; 

a second means for moving said at least one analyte within said separation 

channel; 

whereby the concentration of {one or more of?} said at least one analyte is 

caused to increase at one or more positions along said separation channel. 

13. The device of claim 12, wherein said first means is comprised of 

 a third means for applying a temperature gradient along the length of said 

separation channel. 

14. The device of claim 13, wherein said second means is comprised of 

 a power supply for applying an electric field along said separation channel; 

 a fourth means for applying a bulk flow of said solution through said separation 

channel. 

15. The device of claim 14, wherein said fourth means is comprised of a pump. 

16. The device of claim 14, wherein said fourth means is comprised of said power 

supply and whereby said bulk flow is driven by electroosmosis. 

17
17. The device of claim 14, wherein said fourth means is comprised of the 

combination of a pump and said power supply and whereby said bulk flow is driven by a 

combination of pressure gradients and electroosmosis. 

18. The device of claim 12, wherein said first means is comprised of 

 a third means for creating a steady-state gradient in the composition of said 

solution. 

19. The device of claim 18, wherein said second means is comprised of 

 a power supply for applying an electric field along said separation channel; 

 a fourth means for applying a bulk flow of said solution through said separation 

channel. 

20. The device of claim 19, wherein said fourth means is comprised of a pump. 

21. The device of claim 19, wherein said fourth means is comprised of said power 

supply and whereby said bulk flow is driven by electroosmosis. 

22. The device of claim 19, wherein said fourth means is comprised of the 

combination of a pump and said power supply and whereby said bulk flow is driven by a 

combination of pressure gradients and electroosmosis. 

23. The device of claim 18, wherein said third means is comprised of 

 a fluid chamber containing a second solution and being largely disconnected 

from said separation channel; 

a semi-permeable structure connecting said fluid chamber to said separation 

channel and allowing the passage of one or more components of said solution and said 

second solution. 

Should we repeat some of the dependent claims? 

18
ABSTRACT OF THE DISCLOSURE 

A method and device are provided for affinity gradient focusing for directing at 

least one analyte in a solution containing a pseudostationary phase and located in a 

conduit or microchannel. The method includes establishing a gradient in a retention 

factor of the pseudostationary phase for the at least one analyte. The analyte is caused 

to be moved within the conduit whereby the concentration of the at least one analyte 

increases at one or more positions along the gradient. The pseudostationary phase is 

charged and the analyte is either neutral or charged or alternatively, the 

pseudostationary phase is neutral and the analyte is charged. The device includes a 

fluid conduit, a pseudostationary phase having a retention factor gradient, an electrical 

current source and a pump system for establishing the bulk flow in the solution in the 

conduit. 

Figure 6

19

Separation c

60a 

hannel, 

Control c

60b 

hannel, 

high r

factor 

etention etention 

Gradient region 

low r

factor 

61