**MICELLAR GRADIENT FOCUSING**

FIELD OF THE INVENTION

1.  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

2.  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.

3.  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.

4.  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.

5.  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.

6.  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.

7.  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

8.  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.

9.  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
    analytes to move at differing velocities along the separation
    channel, and hence facilitate a focusing separation modality.

10. 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.

11. 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.

12. 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 the at least one ionic analyte is caused to increase at one or
    more positions along the gradient.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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 needed to maneuver the sample for detection or further analysis
    while maintaining the sample in a narrow focused plug.

20. 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

21. Figure 1 is a schematic of micellar gradient focusing in a
    microchannel or capillary;

22. Figure 2 is a schematic drawing of a fluidic device according to one
    embodiment of the present invention;

23. 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;

24. Figure 4 is a plot of peak concentration vs. time for focusing of
    rhodamine B in a capillary;

25. Figure 5 depicts micellar gradient focusing and separation of two
    neutral fluorescent dyes, rhodamine B (left) and rhodamine 110
    (right); and

26. 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

27. 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 the pseudostationary phase and have a net mobility dominated by
    that of the pseudostationary phase. On the other side of the
    gradient, the retention factor is low and the solute assumes its
    native electrophoretic mobility. If the pseudostationary phase is
    charged, a combination of electrokinetic and pressure‑driven flow
    can be applied so that the pseudostationary phase and the mobile
    phase move in opposite directions. Conversely, focusing can be
    performed with a neutral pseudostationary phase if the analyte is
    charged and made to migrate in the opposite direction of the mobile
    phase. Under these conditions, the analyte can be made to focus at a
    point along the retention factor gradient. Different analytes with
    different affinities for the pseudostationary phase (or different
    electrophoretic mobilities) will focus at different points. The
    present method thereby provides a focusing equivalent of EKC.

28. 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.

29. 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.

30. Delete paragraph 30.

31. 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.

32. 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:

$u_{T} = \left( u_{B} + u_{\text{EP}} \right) \cdot \left\lbrack \frac{1}{(1 + k)} \right\rbrack + u_{\text{MC}} \cdot \left\lbrack \frac{k}{(1 + k)} \right\rbrack$,

33. where *u~B~* is the mobile phase velocity, *u~EP~* is the
    electrophoretic velocity of the analyte through the mobile phase
    (equal to zero for neutral analytes), *u~MC~* 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*.

34. 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.

35. 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.

36. 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.

37. In the embodiment, the buffer is 5 m[m]{.smallcaps} carbonate
    buffer, pH 9.4 in approximately 5% by weight ethanol, 95% water,
    with a 5 m[m]{.smallcaps} 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 µ[m]{.smallcaps}. 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.

38. 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.

39. 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, N.H.) is passed
    through them. The amount of solution in the input wells at each end
    of the channel 20 is adjusted to produce pressure‑driven flow so
    that when a voltage is applied, the micelles would move from cold to
    hot above the 1 mm gap while the mobile phase would move from hot to
    cold above the gap.

40. 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).

41. 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 m[m]{.smallcaps} SDS, and 5 m[m]{.smallcaps} carbonate buffer in
    25% ethanol, 75% water. An initial concentration of rhodamine B of
    0.2 µ[m]{.smallcaps} is used.

42. 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 µ[m]{.smallcaps} in about 3 minutes. About a 35‑fold increase in
    concentration is achieved in three minutes.

43. 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 µ[m]{.smallcaps} 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.

44. 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 is probably a
    combination of these two temperature‑dependent effects that
    contribute to the focusing.

45. 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.

46. 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 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.

47. 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.

48. A neutral micellar or other pseudostationary phase would always move
    with the bulk buffer. In this case, Equation (1) would become:

$u_{T} = u_{B} + u_{\text{EP}}\left\lbrack \frac{1}{(1 + k)} \right\rbrack$.

49. 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 *u~EP~* ≠ 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.

50. 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.

51. 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.

52. Delete paragraph 53 and 54.

53. Delete paragraph 53 and 54.

54. 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 can be achieved which
    translates to lower concentration limits of detection, simultaneous
    separation and concentration of one or more analytes of interest,
    and the ability to manipulate the sample peak while maintaining
    focusing.

55. 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.

56. 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.

57. 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.

58. 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.

59. 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.

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.

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.

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\. 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?

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.