> CHIRAL TEMPERATURE GRADIENT FOCUSING

### CROSS-REFERENCE TO RELATED APPLICATIONS

1.  This application is a continuation‑in‑part of U.S. Patent
    Application No. 10/197,331 filed on July 18, 2002 which claims
    benefit of the filing date of both co‑pending Provisional Patent
    Application No. 60/307,691, filed on July 25, 2001, and 60/323,404,
    filed on September 19, 2001 and this application also claims benefit
    of the filing date of Provisional Patent Application No. 60/544,126
    filed on February 12, 2004.

### STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

2.  This invention was made by employees of the United States Government
    and may be manufactured and used by or for the Government for
    governmental purposes without the payment of any royalties.

### FIELD OF THE INVENTION

3.  The present invention relates to a method for electrokinetic
    focusing of materials in a fluid, and in particular, methods for
    electro-focusing materials in fluidic devices using electric field
    gradients.

### BACKGROUND OF THE INVENTION

4.  Over the past decade a great deal of research has been focused on
    the development of technology related to micro‑total‑analytical
    systems. This technology is based on the concept of a series of
    microfluidic channels also known as microchannels for the movement,
    separation, reaction, and/or detection of various materials, such as
    chemicals or biological compounds like amino acids, proteins, and
    DNA.

5.  One disadvantage with prior microfluidic devices is that there is
    frequently a mismatch between the extremely small quantities of
    sample material used for analysis and the often much larger
    quantities needed for loading the material into the microfluidic
    device and transporting it to the point of analysis. For example, a
    typical analysis sample may be around one nanoliter or less of a
    liquid containing one or more materials that is injected into a
    separation channel and then separated electrokinetically as it moves
    down the channel to a detection region. However, the channels used
    to transport the sample materials to the injection point are
    typically also filled with the sample, thus increasing the required
    amount of the sample by a factor of 100 or more. In addition, the
    sample is typically loaded onto the microfluidic device into a
    reservoir from a pipette so that in all, approximately 99.9% of the
    sample is discarded as waste.

6.  One way of addressing the problem of requiring a large amount of
    sample material for analysis due to the inefficiencies of
    conventional devices which result in wasted sample is to use one of
    any number of focusing techniques to concentrate the materials. In
    the context of this disclosure, focusing refers to methods for
    manipulating the velocity of a material and thereby causing it to
    move towards a position at which the velocity is zero. At the zero
    velocity position the material will accumulate and increase in
    concentration, i.e., it will become focused. In addition, the
    location of the zero velocity position is often dependent upon some
    characteristic of the material being focused, so that different
    materials are focused at different positions, and can thereby be
    separated from a mixture of materials.

7.  Prior focusing methods include isoelectric focusing; electromobility
    focusing; counteracting chromatographic electrophoresis; temperature
    gradient focusing, disclosed in U.S. Patent Application Publication
    No. 2003/0019752, herein incorporated by reference; and most
    recently, mixing reactions by temperature gradient focusing,
    disclosed in co‑pending U.S. Patent Application
    No. 10/**\[P07526US03/RFH\]**, herein incorporated by reference; and
    micellar affinity gradient focusing, disclosed in U.S. Patent
    Application Publication No. 2004/0206626, herein incorporated by
    reference. With the exception of the recently described micellar
    affinity gradient focusing method, the focusing methods all separate
    different materials based upon their electrophoretic properties,
    e.g., mobility in the case of electromobility focusing and
    temperature gradient focusing, and the isoelectric point in the case
    of isoelectric focusing.

8.  Electric field gradient focusing is one focusing technique which can
    be used to concentrate a material at a given position within a
    microfluidic device before the analysis or other step. Further, the
    electric field gradient can be used to concentrate all of a selected
    material at the beginning of the separation channel so that very
    little of the material would be wasted.

9.  Electric field gradient focusing is accomplished by the application
    of an electric field gradient within a microchannel. In response to
    the electric field gradient, there is a corresponding gradient in
    the electrophoretic velocity of any ionic material within the
    microchannel. The total velocity of the material is the sum of its
    electrophoretic velocity and the bulk fluid velocity. If these two
    components of the velocity are in opposite directions, they can be
    balanced so that the material will have zero total velocity.

10. When there is a gradient in the electrophoretic velocity, the
    balance between the bulk fluid velocity and the electrokinetic
    velocity can occur at a single position along the microchannel and
    therefore can result in focusing of the material at that position.
    Typically, the electric field gradient used in focusing is generated
    by the external manipulation of the electric field in the middle of
    the microchannel through the use of conducting wires, salt bridges,
    porous membranes, or other structures that will pass electric
    current but will restrict the flow of bulk fluid and the materials
    that are to be focused.

11. Several recent developments with regard to focusing methods in
    microfluidics, and in particular, the use of electric field
    gradients, have been made. A description of related methods of
    focusing can be found in C. F. Ivory, W. S. Koegler, R. L. Greenlee,
    and V. Surdigio, Abstracts of Papers of the American Chemical
    Society 207, 177-BTEC (1994); C. F. Ivory, Separation Science and
    Technology 35, 1777 (2000); Z. Huang and C. F. Ivory, Analytical
    Chemistry 71, 1628 (1999); W. S. Koegler and C. F. Ivory, Journal of
    Chromatography a 726, 229 (1996); and P. H. Ofarrell, Science 227,
    1586 (1985), all of which are hereby incorporated by reference.

12. To illustrate the basic principles disclosed in these publications,
    reference is made to Figure 1(a) which depicts a length of
    fluid-filled microchannel of constant cross-sectional area with an
    electrode, denoted 4, in the middle, and two further electrodes at
    each end, denoted 3 and 5, so that the voltages V~1~, V~3~ at the
    ends and the voltage V~2~ at the middle of the channel can be
    controlled. A single species of negatively charged ionic material is
    present in the fluid that is within the microchannel. The electrical
    connection, represented as electrode 4, can be accomplished with a
    simple metal wire as depicted in Figure 1(a), or through a more
    complicated structure consisting of additional fluid channels and
    porous membrane structures or salt bridges.

13. The electric field in the section 1, i.e., the channel between
    electrodes 3 and 4 is
    $E_{1} = (V_{2} - V_{1}\frac{)}{(}\frac{l}{2})$ and the electric
    field in section 2, i.e., between electrodes 4 and 5, is
    $E_{2} = (V_{3} - V_{2}\frac{)}{(}\frac{l}{2})$, where V~1~, V~2~,
    and V~3~ are the voltages applied to the three electrodes 3, 4, and
    5, and $l$ is the length of the microchannel. If *E~1~* differs from
    *E~2~* as shown in Figure 1(b), the electrophoretic velocity of the
    ionic material in the channel, $u_{\text{EP}}$, will be different in
    section 1 than in section 2. If an overall bulk fluid velocity,
    $u_{B}$ \<0, is applied, e.g., either electro-osmotic or
    pressure-driven, the bulk fluid velocity must be the same, due to
    continuity, in all parts of the microchannel. The total velocity of
    the ionic material, *u~T~* = *u~B~* + *u~EP~*, will then be the sum
    of the electrophoretic and bulk velocities, which can differ in
    section 1 from section 2.

14. The use of the microchannel device of Figure 1(a) for focusing of
    the ionic material is illustrated in Figure 2 where
    $u_{T,1} > 0 > u_{T,2}$, so that the ionic material moves into the
    middle from both directions and is thus focused in the middle of the
    channel near electrode 4.

15. One major drawback to electric field gradient focusing is that the
    device tends to be difficult to construct and that it requires the
    control of voltage on an additional electrode, e.g. 4 of Figure
    1(a), that is used to apply the electric field gradient. In
    addition, if electrodes are used to generate electric field
    gradients, unwanted chemical products will be generated
    electrochemically at the fluid-electrode interface. If the electric
    field gradient is produced through the use of a salt bridge or
    membrane, the electrochemical products can be avoided, however only
    materials or chemical species that cannot pass through the membrane
    or salt bridge can be focused.

16. Two additional methods for concentrating a sample include sample
    stacking and field amplified sample injection in which a sample is
    concentrated as the sample crosses a boundary between low and high
    conductivity fluids. These methods can achieve preconcentration
    factors of 100 to 1000-fold although these methods require multiple
    fluids. Sweeping is yet another concentration method which is
    capable of a very high degree of sample concentration (e.g., up to
    5000-fold), but is useful only for small molecular weight
    hydrophobic materials with a high affinity for a mobile micellar
    phase.

17. An additional technique for concentrating an ionic material includes
    isoelectric focusing. Isoelectric focusing is commonly used for the
    concentration and separation of proteins and involves the focusing
    of materials at their respective isoelectric points along a pH
    gradient.

18. Two examples of recent isoelectric focusing techniques are provided
    by U.S. Patent Nos. 3,664,939 to Luner et al. and 5,759,370 to
    Pawliszyn. Both references relate to isoelectric focusing with pH
    gradients that are created by the application of a temperature
    gradient. Isoelectric focusing uses a pH gradient to focus materials
    and in particular proteins, at positions along the pH gradient where
    the local pH is equal to the isoelectric points of the materials.
    The isoelectric point is the pH at which the material has zero
    electrophoretic mobility, i.e., approximately zero charge. pH
    gradients for isoelectric focusing are typically generated using
    ampholyte mixtures or immobilized ampholytes in gels. The two above
    referenced patents are included here as examples of prior art uses
    of temperature gradients for focusing. It is, however, very unusual
    for isoelectric focusing to be done with a pH gradient generated
    using a temperature gradient.

19. One disadvantage of isoelectric focusing is that it is limited in
    application because it can only be used with materials having an
    accessible isoelectric point. Additionally, the concentration to
    which a protein can be focused with isoelectric focusing is severely
    limited due to the low solubility of most proteins at their
    isoelectric points.

20. In addition, further methods have been developed specifically to
    separate chiral species. Of the conventional methods of chiral
    separation, chiral capillary electrophoresis typically gives the
    best resolution, and is consequently widely used for analytical
    scale separations of chiral compounds and assays of enantiomeric
    purity. The major limitation of chiral capillary electrophoresis and
    of capillary electrophoresis in general is its poor detection
    limits, which are typically too high for the analysis of chiral
    compounds, e.g., drugs and drug metabolites in body fluids such as
    serum or urine. This major limitation is discussed throughout the
    scientific literature, including Scriba, G. K. E. *Electrophoresis*
    **2003**, *24*, 2409-2421; Bonato, P. S. *Electrophoresis* **2003**,
    *24*, 4078-4094; Hempel, G. *Electrophoresis* **2000**, *21*,
    691-698; Chen, S. H.; Chen, Y. H. *Electrophoresis* **1999**, *20*,
    3259-3268; and Altria, K. D.; Elder, D. *Journal of Chromatography
    A* **2004**, *1023*, 1-14.

21. Capillary electrophoresis is also commonly used to assess the
    enantiomeric purity of drugs. In order to detect and quantify a
    trace contaminant of one enantiomer against a large excess of the
    other, it is often necessary to reverse the order of peak migration
    so that the tail of the major enantiomer peak does not obscure the
    small impurity peak.

22. Isoelectric focusing and isoelectric trapping have also been used in
    conjunction with chirally selective additives for enantiomeric
    separations. However, these techniques are limited in their
    applicability because the chiral materials or molecules to be
    focused must be chemically derivatized so that 1) they have an
    isoelectric point, and 2) the isoelectric point is dependant on the
    degree of interaction with a chiral selector. The vast majority of
    pharmaceutical molecules do not have isoelectric points and so
    cannot be focused or separated with isoelectric focusing.
    Furthermore, most pharmaceutical molecules would not be amenable to
    the kind of chemical derivatization required for isoelectric
    focusing, see, e.g., Ogle, D.; Ho, A.; Gibson, T.; Rylatt, D.;
    Shave, E.; Lim, P.; Vigh, G. *Journal of Chromatography A* **2002**,
    *979*, 155-161; Rizzi, A. M.; Kremser, L. *Electrophoresis*
    **1999**, *20*, 3410-3416; Righetti, P. G.; Ettori, C.; Chafey, P.;
    and Wahrmann, J. P. *Electrophoresis* **1990**, *11*, 1-4 for a
    further discussion regarding the separation of chiral \[no
    pharmaceutical molecule has ever been focused by IEF\] molecules
    using isoelectric focusing.

BRIEF SUMMARY OF THE INVENTION

23. The present invention is directed to overcoming the limitations of
    prior chiral separation methods by combining the high resolution of
    chiral capillary electrophoresis with the high concentration
    enhancement and low detection limits of temperature gradient
    focusing. According to one aspect of the present invention, there is
    provided a method for focusing a material using temperature gradient
    focusing in conjunction with a fluid additive, such as a chirally
    selective additive which modifies the electrophoretic mobility of
    materials in the fluid. The method may be conducted in fluid
    conduits such as channels, separation columns, tubes, microchannels,
    and capillary tubes. The concentration is achieved by balancing the
    electrophoretic velocity of a material against the bulk flow of
    fluid in the presence of a temperature gradient. Using an
    appropriate fluid in conjunction with a fluid additive which
    interacts with the material of interest, the temperature gradient
    generates a corresponding gradient in the electrophoretic velocity
    of the material so that the electrophoretic and bulk velocities sum
    to zero at a given position along the fluid conduit and the material
    will be focused at that position. The types of fluids that would
    typically be used include ionic aqueous solutions, ionic non-aqueous
    solutions, aqueous buffer solutions, and mixtures of aqueous and
    non-aqueous solutions. Because the present invention does not use
    semi-permeable structures such as salt bridges or membranes to form
    a velocity gradient, it may be adapted for use with any charged
    material, including small ions, large ions, fluorescent dyes, amino
    acids, proteins, DNA, cells, and particles and may provide up to or,
    in some instances, exceed a 10,000‑fold concentration of a dilute
    material. Further, the present method can be used to separate
    stereoisomers of a material which have different affinities for the
    additive.

24. Since the present method combines temperature gradient focusing, in
    conjunction with a fluid additive that modifies the electrophoretic
    mobility of the material to be focused, the degree to which the
    electrophoretic mobility is modified will depend upon the strength
    of the interaction of the material with the additive. Consequently,
    two different materials that focus at the same position in
    temperature gradient focusing without the additive can potentially
    be focused at different positions in temperature gradient focusing
    with the additive if they differ in their interaction with the
    additive, and thus can be separated.

25. One aspect of the present invention concerns a method for directing
    materials contained in a fluid and which may include concentrating
    or separating materials present in the fluid. The method includes
    applying an electric field to a fluid containing one or more species
    of material to cause the materials to migrate electrophoretically. A
    temperature gradient is established in the fluid, thereby generating
    a gradient of the electrophoretic velocity of the materials. An
    additive is added to the fluid to modify the normal electrophoretic
    mobility of the materials based on the interaction between the
    materials and the additive. A flow is produced in the fluid such
    that the magnitudes and directions of the electric field,
    temperature gradient, and flow are such that at least one of the
    materials in the fluid will accumulate or be focused at a position
    along the gradient.

26. In various further alternative embodiments, the additive is a chiral
    selector, a non‑ionic additive and forms a pseudostationary phase.
    In an additional alternative embodiment, the material is first
    focused without the additive which focuses all stereoisomers of a
    material at the same point. Subsequently, the additive is added to
    the fluid and the focusing continues, separating the stereoisomers.

27. According to another aspect of the present invention, a fluidic
    device includes a fluid conduit, a fluid disposed in the conduit and
    a source of one or more materials to be focused in fluid connection
    with the conduit. An electric power supply or other means is used to
    provide an electric field to the conduit. Heated or cooled blocks
    thermally coupled to the conduit or other means are used to generate
    a temperature gradient within the fluid conduit. A source of bulk
    fluid flow is used to provide a flow of fluid in the conduit. An
    additive is added to the fluid to modify the normal electrophoretic
    mobility of the materials based on the materials' interaction with
    the additive. In alternate, further embodiments, the fluid has
    either a temperature dependent ionic strength or a temperature
    dependent pH such that when a temperature gradient is applied to the
    fluid conduit, a gradient of the materials' electrophoretic velocity
    is established in the fluid present in the fluid conduit.

28. One advantage or feature of the present invention is provided by a
    technique that allows for simultaneous concentration and separation
    in a manner similar to isoelectric focusing but which is adoptable
    for use with any charged material and is not limited to materials
    with a specific isoelectric point or range of isoelectric points.
    Further, the temperature gradient focusing of the present invention
    can be used to achieve higher degrees of sample concentration, e.g.,
    more than 10,000 fold concentration of a dilute material, when
    compared with any prior single sample preconcentration method.

29. A further feature of the present invention is that the
    electrophoretic velocity gradient is formed within the channel or
    capillary in response to the temperature gradient without the need
    for externally manipulated voltages or complicated and difficult to
    fabricate semi-permeable structures.

30. A further feature of the present invention is the ability thereof to
    focus and separate chiral stereoisomers. For example, with the
    addition of a chiral additive, the present focusing method allows
    for simultaneous separation and concentration of materials that
    cannot be separated using temperature gradient focusing based purely
    upon their electrophoretic mobilities. One benefit of being able to
    separate chiral stereoisomers is that many drugs and drug candidates
    are chiral and in most cases, one stereoisomer is more desired for
    drug use than the other. In some instances, one stereoisomer is a
    beneficial drug, whereas the other results in adverse side effects.

31. An additional advantage of the present invention for the separation
    of the stereoisomers of a chiral material is that it is very easy to
    reverse the positional order of the focused peaks corresponding to
    the different stereoisomers. This can be done simply by reversing
    the direction of the temperature gradient, the sign of the applied
    electric field, and the direction of the bulk fluid flow. As
    mentioned above, this can be beneficial for the analysis of the
    purity of chiral materials that are desired to contain only one
    stereoisomer.

32. 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 

33. The invention will now be described in detail with respect to
    preferred embodiments with reference to the accompanying drawings,
    wherein:

34. Figure 1(a) is a schematic depicting a prior art microchannel device
    which provides for electric field gradient and Figure 1(b) is a plot
    of the electric field versus distance (x) along the microchannel of
    Figure 1(a);

35. Figure 2 is a plot of velocity versus distance along the
    microchannel of Figure 1(a);

36. Figure 3(a) is a schematic illustration of temperature gradient
    focusing and fluid conduit in the form of a microchannel in
    accordance with the present invention, Figure 3(b) depicts
    temperature distribution along the microchannel of Figure 3(a), and
    Figure 3(c) is a plot of the function
    $f(T) = \frac{\sigma(\text{20}) \cdot \eta(\text{20})}{\sigma(T) \cdot \eta(T)}$
    plotted as a function of the distance along the microchannel of
    Figure 3(a), where σ(T) is the temperature dependent conductivity,
    σ~0~ is a constant, and η(T) is the temperature dependent viscosity,
    and Figure 3(d) is a plot depicting velocity as a function of
    distance along the microchannel;

37. Figure 4(a) is a schematic illustration of a microchannel for
    temperature gradient focusing created by Joule heating according to
    another embodiment of the present invention, Figure 4(b) depicts the
    temperature profile along a length of the microchannel of Figure
    4(a), Figure 4(c) is a plot of the function
    $f(T) = \frac{\sigma(\text{20}) \cdot \eta(\text{20})}{\sigma(T) \cdot \eta(T)}$
    plotted as a function of the distance along the microchannel of
    Figure 4(a), and Figure 4(d) is a plot showing electrophoretic
    velocity, bulk velocity, and total velocity vs. distance along the
    microchannel of Figure 4(a);

38. Figure 5 is a schematic drawing of a fluidic device according to
    further embodiment of the present invention;

39. Figure 6 is a schematic drawing of a capillary fluidic device
    according to an alternate embodiment of the present invention;

40. Figure 7 is a schematic drawing of a fluidic device according to a
    further embodiment of the present invention; and

41. 

42. Figure 8 is a plot depicting the concentration of two stereoisomers
    of a material versus distance along a microchannel in accordance
    with the present invention.

### DETAILED DESCRIPTION OF THE INVENTION 

43. A material can be focused in a fluid conduit such as a channel or
    capillary when its total velocity is in opposite directions in
    different portions of the conduit, so that the material will move
    into the middle of the conduit and stop at a position where its
    velocity is zero. One way of accomplishing this is to cause the
    material to move with an electrophoretic velocity that varies along
    the length of the conduit. This can be accomplished by producing an
    electrophoretic velocity gradient along the conduit. If an
    electrophoretic velocity gradient is established, but the
    electrophoretic velocity is still in the same direction along the
    entire length of the conduit, a counter‑balancing bulk flow can be
    applied to the fluid in the conduit, driven either by
    electroosmosis, pressure gradients, or both, so that the total
    velocity of the material, i.e., the vector sum of the bulk flow
    velocity and electrophoretic velocity of the material is equal to
    zero at some position along the conduit.

44. Temperature gradient focusing is a method for focusing materials
    that produces the required material electrophoretic velocity
    gradient through the application of a temperature gradient. In
    temperature gradient focusing, a fluid with a temperature dependent
    ionic strength is typically used to give an electrophoretic velocity
    that is temperature dependent. Then the electrophoretic velocity of
    the material can be altered in different portions of the conduit or
    capillary simply by heating or cooling different portions of the
    conduit. The application of a temperature gradient then results in
    an electrophoretic velocity gradient, which, with a counteracting
    bulk fluid flow, can be used for focusing. With temperature gradient
    focusing, different materials are focused at different positions and
    thereby separated based upon differences in their electrophoretic
    mobilities.

45. The present method includes temperature gradient focusing of a
    material with the addition of an additive that interacts with the
    materials to be focused, and thereby modifies their normal
    electrophoretic mobilities, thus changing the positions at which
    they focus. If two materials have the same or very nearly the same
    electrophoretic mobilities (as is the case for stereoisomers of a
    pharmaceutical molecule, for example), they can not normally be
    separated by temperature gradient focusing without the addition of
    an additive. If the additive interacts more strongly with one of the
    materials than the other however, they can be simultaneously
    concentrated and separated using the present method of temperature
    gradient focusing with an additive.

46. Further description of the present invention will now be made with
    reference to the drawings, and in particular to Figure 3(a), where a
    fluid-filled microchannel 10 includes electrode connections 12, 14
    at each end, one of which is connected to a high voltage source such
    as an electrical power supply and the other of which is electrically
    grounded. The velocity of a material in the microchannel 10 is given
    by the sum of its electrophoretic velocity, u~EP~, and the bulk
    velocity, u~B~, of the fluid:

$u_{T} = u_{\text{EP}} + u_{B}$.

47. If there is a gradient in the electrophoretic velocity, the bulk
    velocity can be adjusted so that the total velocity is equal to zero
    at a single point along the channel, and the material will be
    focused at a position around that point. The electrophoretic
    velocity of a material in the microchannel 10 is given by the
    product of the electric field, E, and the electrophoretic mobility
    of the material: $u_{\text{EP}} = E \cdot \mu_{\text{EP}}$.

48. A temperature gradient is applied along the length of the channel as
    shown in Figure 3(b). This results in corresponding gradients in
    both the electric field E and the electrophoretic mobility μ~EP~.

49. The electric field in the microchannel 10 is given by:

$E = \frac{I}{A \cdot \sigma}$,

where *I* is the electric current running through the microchannel 10,
*A* is the channel cross-sectional area of the microchannel 10, and *σ*
is the conductivity of the fluid. Since the conductivity of the fluid is
almost always temperature-dependent, the electric field is also almost
always temperature-dependent. Here, constant current is presumed because
the current running through any given section of the microchannel 10
will be the same for all parts of the microchannel, whereas the voltage
drop across a portion of the microchannel 10 and the electric field in
the microchannel 10 will depend on the temperature of that portion. One
skilled in the art will readily appreciate that the present temperature
gradient focusing differs from electric field gradient focusing in that
in electric field gradient focusing, the velocity gradient that is used
for focusing results from a gradient in the electric field imposed by
the addition or subtraction of current from a point or points within the
microchannel.

50. Using microchannel 10, it is possible to manipulate the conductivity
    of the fluid by changing the temperature. Consequently, it is
    possible to produce electric field gradients in microfluidic
    devices, such as microchannel 10, through the application of a
    temperature gradient.

51. At fixed current density, the electric field in microchannel 10 is
    inversely proportional to the conductivity of the fluid in the
    microchannel. Most often, the primary temperature dependence of the
    conductivity is due to the variation of the fluid viscosity with
    temperature, so it can be written as
    *σ* = *σ*~0~·*η*(20)/(*η*(*T*)·*f*(*T*)), where σ is the
    conductivity, σ~0~ is a constant, η(T) is the temperature dependent
    viscosity, and $f(T)$ is a dimensionless function that accounts for
    any other temperature dependence. Similarly, the temperature
    dependence of the electric field is given by *E* =
    *E*~0~·*η*(*T*)·*f*(*T*)/*η*(20), where E is the electric field and
    E~0~ is a constant.

52. For the types of fluids most commonly used microfluidic analysis
    (aqueous buffers), the function $f(T)$ is constant or only weakly
    dependent on temperature. However, it can be non-constant, i.e.,
    variable, if, for example, the ionic strength of the fluid is
    temperature dependent. Advantageously, the fluids of the present
    invention are characterized by a non-constant $f(T)$.

53. The electrophoretic mobility of an ionic (e.g., material) species in
    the fluid is also dependent on the viscosity, and so can be written
    as *μ~EP~* = *μ*~0~·*η*(20)/(*η*(*T*)·*f~EP~*(*T*)), where $\mu_{0}$
    and *f~EP~*(*T*) are defined in analogy to σ~0~ and $f(T)$ where,
    for most materials, *f~EP~*(*T*) will be constant. The
    electrophoretic velocity of the material can then be written as
    *u~EP~* = *E*~0~·*μ*~0~·*f*(*T*)/*f~EP~*(*T*). It should be noted
    that if $f(T)$ and *f~EP~*(*T*) have the same temperature
    dependence, e.g., they are both constant, then *u~EP~* will not be
    temperature dependent, and an electric field gradient produced in
    this way can not be used for focusing.

54. If, on the other hand, $f(T)$ and *f~EP~*(*T*) do not have the same
    temperature dependence, then temperature gradients will result in
    gradients in the electrophoretic velocity, which can be used for
    focusing as described above.

55. One skilled in the art will readily appreciate a major advantage of
    this present method over some other methods of preconcentration is
    that the concentrations of the background ions (those ions that are
    included in the fluid to provide a temperature-dependent ionic
    strength) are completely unaffected by the focusing. This results
    from the fact that if the background ions are considered as
    materials to be focused, then, by definition, *f~EP~*(*T*) = $f(T)$
    and there is no gradient in the electrophoretic velocities of the
    background ions.

56. Most commonly this technique would be implemented with a fluid
    characterized by a strongly temperature dependent $f(T)$ and with
    materials characterized by a constant or nearly constant
    *f~EP~*(*T*). However, the present temperature gradient focusing can
    also be implemented in a system in which $f(T)$ is constant and
    *f~EP~*(*T*) is not, or in which both $f(T)$ and *f~EP~*(*T*) are
    non-constant, but differ in their temperature dependence. One type
    of fluid that would have a temperature dependent *f(T)*, and could
    therefore be used for temperature gradient focusing, would be a
    fluid with a temperature dependent ionic strength.

57. One preferred fluid system is composed of 0.9 mol/L
    Tris(hydroxymethyl)aminomethane and 0.9 mol/L boric acid in water
    (0.9 M Tris-borate buffer), with an expected pH of about 8.7 (at
    room temperature). From measurements of the conductivity of the 0.9
    M Tris-borate buffer and the known viscosity of water, the function
    $f(T)$ for 0.9 M Tris-borate buffer was determined to vary from 1 at
    20ºC to 0.77 at 70ºC.

58. The counterbalancing bulk flow can be applied electroosmotically if
    the electro-osmotic mobility does not differ too much from the
    electrophoretic mobility of the material to be focused. If the
    electro-osmotic mobility is written as
    *μ~EO~* = *μ~EO~*^0^·*η*(20)/*η*(*T*), then by adjusting the ratio
    of the lengths of the hot and cold channels, (assuming
    *f~EP~*(*T*) = constant) focusing can be achieved if
    *f*(*cold*)/*f*(*hot*) \< ‑*μ*~0~/*μ~EO~*^0^ \< *f*(*hot*)/*f*(*cold*),
    where $f(\text{hot}) > f(\text{cold})$
    (If$f(\text{hot}) < f(\text{cold})$, then the inequalities have the
    opposite sign). If x is the fraction of the total channel length
    that is hot, then focusing will occur if:
    *x*·*f*(*hot*)/*f*(*cold*) + (1 ‑ *x*) \< ‑*μ*~0~/*μ~EO~*^0^ \<
    *x* + (1 ‑ *x*)·*f*(*cold*)/*f*(*hot*), where
    $f(\text{hot}) > f(\text{cold})$. By adjusting x, it is then
    possible to tune the range of material mobilities that are focused.
    In many cases, the electrophoretic and electroosmotic mobilities
    will not be so well balanced, and therefore although the bulk
    velocity will be largely due to electroosmosis, it must also be
    adjusted using an externally applied pressure gradient.

59. It should be noted that temperature gradient focusing can also be
    implemented in microchannels or other fluid conduits of non-constant
    cross-section. The final results are essentially unchanged, since in
    most instances, the dependence on the cross-sectional area of the
    channel cancels out in the equations. As a result, it is possible to
    generate the temperature gradient using Joule heating within the
    microchannel. This would serve to simplify the design and operation
    of a microfluidic device using this technique even further, since
    the focusing and the temperature gradient could be produced using
    the same voltage source and pair of electrodes as illustrated in
    Figure 4(a).

60. Microchannel 20 shown schematically in Figure 4(a), has electrodes,
    22, 24, and two sections, sections 26, 28, of different
    cross-sectional area. Section 26 has a cross-sectional area of 27
    and section 28 has a cross-sectional area of 29. The electrical
    resistance per unit length of each section is given by:
    $R_{i} = \frac{1}{(}\sigma \cdot A_{i})$, where σ is the
    conductivity of the fluid in the microchannel 20. When a current,
    *I*, is passed through the microchannel 20, the power per unit
    length dissipated through Joule heating in each section will be:
    $P_{i} = I^{2} \cdot R_{i} = \frac{I^{2}}{(}\sigma \cdot A_{i})$. In
    general, the resulting temperature in section 28 will be higher than
    that in section 26, as shown in Figure 4(b): $T_{2} > T_{1}$. The
    electric field in each section of the microchannel 20 is given by
    the current multiplied by the resistance per unit length:
    *E~i~* = *I*·*R~i~* = *I*/(*σ*·*A~i~*) = *I*·*η*(*T~i~*)·*f*(*T~i~*)/(*σ*~0~·*h*(20)·*A~i~*).

61. The electrophoretic velocity of a material in each section of the
    channel is: *u~EP~^i^* =
    *μ*~0~·*f*(*T~i~*)·*I*/(*σ*~0~·*f~EP~*(*T*)·*A~i~*). If a bulk flow
    velocity is applied along the channel, it will not be the same in
    each section, but will instead be given by *u~B~^i^* =
    *u~B~*^0^/*A~i~*, where, *u~B~*^0^ is a constant. The ratio of the
    electrophoretic velocity to the bulk velocity is then given by:
    *u~i~^ratio^* = *μ*~0~·*f*(*T~i~*)·*I*/(*σ*~0~·*f~EP~*(*T*)·*u~B~*^0^) ≡ *u~0~^ratio^*·*f*(*T~i~*)/*f~EP~*(*T*)
    via adjusting *u~B~*^0^ so that
    $\left| u_{1}^{\text{ratio}} \right| > 1 > \left| u_{2}^{\text{ratio}} \right|$
    as shown in Figure 4(d), which can result in focusing. Because the
    ratio of the electrophoretic velocity to the bulk velocity does not
    depend on the cross‑sectional areas of the two sections, the same
    considerations as above apply if bulk flow is applied
    electroosmotically.

62. Joule heating may be used to generate the temperature gradient in
    the microchannel device of Figure 4(a). The following is a
    non-limiting example demonstrating Joule heating of a microchannel
    of the type shown in Figure 4 (a).

63. The microchannel used for this demonstration was similar to the one
    shown schematically in Figure 4(a). The width, i.e., cross sectional
    area 29, of the narrow channel, i.e., section 28, was about 70 μm,
    and the width of the wide section, i.e., section 26, cross sectional
    area 27 was about 350 μm. The length of the tapered portion of the
    channel was about 500 μm. The depth of all portions of the channel
    was about 30 μm. The total length of the microchannel was about 2
    cm, with the length of the section 28 divided by the total length, x
    ≅ 0.8. Access to each end of the microchannel was provided by a 3 mm
    hole through the lid piece of the microchannel.

64. 0.9 M Tris-borate buffer was used as the fluid for temperature
    gradient focusing. An 8 μmol/L solution of carboxyfluorescein in 0.9
    M Tris-borate buffer was prepared. The material to be concentrated
    was the carboxyfluorescein. Detection of the material was performed
    using a fluorescence microscope and CCD cameras. Simultaneous color
    and grayscale images were obtained.

65. To demonstrate gradient focusing using Joule heating, the
    microchannel was filled with the caboxyfluorescein solution and 1900
    V was applied along its length using a high voltage power supply and
    platinum electrodes, with the positive voltage V~2~ applied to the
    narrow end via electrode 22, and the wide end held at ground at
    electrode 24.

66. After 6 min., the carboxyfluorescein was highly concentrated at the
    junction between sections 26 and 28 of the microchannel 20. The
    concentration factor achieved by using this example was typically
    about 100-fold per minute.

67. Referring now to Figure 5, in order to have better control of the
    temperature gradient, experiments were done using three temperature
    zones, two cold zones provided by cooling copper blocks 53a, 53b
    covering much of the ends of the microchannel 50, and one hot zone
    provided by heated copper block 52. The microchannel 50 was made out
    of thin (125 μm) sheets of poly(carbonate) substrate 51, which were
    pressed onto the copper blocks 52, 53a, 53b. Thermal contact between
    the poly(carbonate) and the copper blocks was insured using a
    thermally conductive adhesive 56. The copper blocks 52, 53a, 53b
    were arranged so that there was a 1 mm gap 58 between the heated
    copper block 52 and the cooling copper block 53a and a 2 mm gap 59
    between heated copper block 52 and the cooling copper block 53b.

68. Microchannel 50 also includes electrodes 55, 54, fluid
    reservoirs 57, and a narrow hot zone 50a near the middle of the
    microchannel 50. The heated copper block 52 was heated using a small
    high-power resistor embedded into the copper and its temperature was
    regulated using a PID temperature controller (Omega Engineering Inc,
    Stamford, CT). To regulate the temperature of the cold zones, ¼ inch
    diameter holes were drilled through the cooling copper blocks 53a,
    53b and cold water from a thermostatted bath (Neslab, Portsmouth,
    NH) was passed through them.

69. Thin polycarbonate microchannel chips, i.e. substrate 51 was
    attached to the copper blocks 52, 53a, 53b using thermally
    conductive adhesive 56 in the form of transfer tape (3M). The
    substrate 51 was pressed against the copper blocks 52, 53a, 53b from
    above with 3 mm thick PDMS (Sylgard 184, Dow Corning, Midland, MI)
    gaskets 60 and a 2 mm thick acrylic (Acrylite OP-4, Cyro Industries,
    Mt. Arlington, NJ)) top plate 61, which was secured to the outer
    copper clocks using nylon screws (not shown).

70. During temperature gradient focusing, a voltage potential is applied
    to electrode 55 and electrode 54 is set to ground using a high
    voltage power supply. In most cases, the electrophoretic and
    electroosmotic mobilities are not sufficiently balanced, so that an
    externally applied pressure gradient must be applied to microchannel
    50 to allow microchannel 50 to provide focusing and separation of
    different types of materials: small dye molecules, amino acids,
    proteins, DNA, colloidal particles, and cells. There are many
    possible means for applying the necessary pressure gradient,
    including the use of pneumatic pressure controllers, and adjusting
    the volume of fluid contained in the reservoirs 57 to take advantage
    of gravitational and/or capillary (surface tension-driven) forces.

71. The microchannel 50 may be formed by imprinting with a micro
    machined silicon template and then sealed with a similar material
    according to the method disclosed in Ross, D.; Gaitan, M.; Locascio,
    L.E., *Analytical Chemistry* 2001, 73, 4117-23, herein incorporated
    by reference.

72. The copper block arrangement was also used to determine the degree
    of focusing that could ultimately be reached with temperature
    gradient focusing. Beginning with a 8 nM solution of Oregon Green
    488 carboxylic acid in 0.9 M Tris-borate, 100 min of focusing
    resulted in a focused plug of Oregon Green 488 carboxylic acid with
    a peak concentration over 80 μM -- a greater than 10000-fold
    increase in concentration.

73. It will become readily apparent to one of ordinary skill in the art
    that the present method provides for use in numerous applications.
    For example, temperature gradient focusing could be used as a
    preconcentration step before an analysis or separation or as a
    simultaneous concentration and separation technique. In addition,
    temperature gradient focusing could be used for preparative scale
    separation of different materials or different enantiomers if the
    focused material or materials are collected after focusing.

74. In addition, temperature gradient focusing may be used with any
    charged material and not just small molecules. For example, the
    materials may include larger molecules such as proteins and DNA, or
    even particles and cells. Further, the present method can be used
    with particles to create packed beds of particles or cells for use
    in other analysis steps. In addition, the present method can be
    adapted for use to sort particles or cells by electrophoretic
    mobility.

75. In one separation mode, the bulk velocity could be ramped over time
    to scan focused sample peaks past a fixed detector, e.g. the
    detector shown in Figure 5. This would produce results similar to
    capillary electrophoresis but the widths of the sample peaks would
    be determined by the applied gradients and the peak heights would be
    determined by how long a given peak was in the focusing "window". If
    the ramp speed were halved, the peak heights would all be doubled,
    so that the ramp rate could be chosen dependent on the concentration
    limit of detection necessary. Alternatively, the focusing window
    could remain fixed and a scanning or imaging detector could be used
    to locate the separate peaks.

76. In a further embodiment, the method may be adapted for a system
    where temperature dependence is due to something other than the
    ionic strength. An example is a system having *f(T)* constant but
    *f~EP~(T)* not constant, or variable. One way to accomplish this
    would be to use a fluid with a temperature dependent pH. In such a
    system, this embodiment of the present invention is similar to
    isoelectric focusing schemes. However, the present invention differs
    from isoelectric focusing in that, in the present system, an
    opposing fluid flow is applied so that materials are focused at a pH
    other than their isoelectric points.

77. When using any of the embodiments of the present method, operating
    parameters which include voltage, bulk flow rate, and temperature of
    the different zones may be held constant with time or varied with
    time to affect the position and width of focused sample peaks.
    Varying of parameters may be accomplished using any of a number of
    methods which include the methods previously described above in
    which the focused sample peaks are scanned past a fixed detector.

78. Advantageously, in order to achieve the fastest accumulation of
    material in the focused peak, the highest possible voltage should be
    used. However, a higher applied voltage requires a faster bulk flow
    which can result in greater dispersion, i.e., wider focused peaks,
    which is disadvantageous for separation and for achieving
    pre-concentration of a sample to a high concentration in a very
    narrow peak. Therefore, a high voltage and fast bulk flow could be
    used for the initial accumulation of material into a relatively
    broad peak, and the voltage flow and flow rate could then be reduced
    to the values which give the narrowest peaks. Further, temperature
    gradients could be turned on and off to first concentrate the sample
    and then release the focused peak and allow it to flow on down the
    channel. Further, the temperature gradient can be adjusted to be
    linear or nonlinear, and the temperature gradient may be monotonic
    or non-monotonic. Thus, operating parameters may be adjusted to
    achieve the desired results.

79. While the previously disclosed embodiments are directed to a
    microchannel or microfluidic device, the present method may be
    adapted for incorporation for use with substantially larger channels
    which may include millimeter and centimeter if not larger in
    dimension which should now be apparent to one of ordinary skill in
    the art. Because temperature gradient focusing uses low conductivity
    fluids, one can adapt the present method for use in much larger
    scale geometries than the micron-sized channels and capillaries
    described in detail herein.

80. Further, the previously described method can be adapted for use in
    modified capillary fluidic systems known to one of ordinary skill in
    the art. Figure 6 depicts a capillary fluidic system having a
    capillary tube 70 spanning between two fluid reservoirs 77. Two
    temperature blocks, denoted as heated block 72 and cooling block 73
    are located along the length of the capillary tube 70 to provide a
    desired temperature gradient in the capillary tube 70.
    Alternatively, temperature blocks being both cooling, both heated,
    both at ambient temperature, or any combination, thereof, may be
    substituted to provide the desired temperature gradient.

81. The fluid reservoirs 77 contain a fluid with temperature dependent
    ionic strength. Electrodes 74, 75 are connected at one end to a
    power supply and on the other end, are in contact with the fluid in
    the reservoirs 77. The power supply applies a driving voltage
    through the capillary tube 70. A source of bulk flow is driven
    either by electro-osmosis with the applied driving voltage, by a
    pressure gradient applied, e.g. by a pump, or a combination of the
    two. Detector 80 is used to detect materials present in the fluid.

82. In a specific chiral temperature gradient method of the present
    invention, one or more additives are added to the fluid to modify
    the normal electrophoretic mobility of the materials based on the
    interaction between the material and the additive. In one
    implementation, the additive is a chiral selector such as a
    cyclodextrin, in which case the method can be used for the focusing
    and separation of stereoisomers with respect to the chirality of the
    material. This method can be employed in the pharmaceutical industry
    as drug compounds are often chiral where one enantiomer may have a
    therapeutic effect while the other enantiomer is not efficacious,
    and is sometimes harmful. The chiral selectors that could be used
    with this new method include all of the many chiral selectors
    commonly used in chiral capillary electrophoresis such as
    alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin,
    heptakis-O-methyl beta-cyclodextrin, heptakis(2,6-di-O-methyl)
    beta-cyclodextrin, heptakis (2,3,6-tri-O-methyl) beta-cyclodextrin,
    hydroxyethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin,
    sulfated beta-cyclodextrin, 6-O-alpha-D-glucosyl-alpha-cyclodextrin,
    6-O-alpha-D-glucosyl-beta-cyclodextrin,
    2-hydroxy-3-trimethylammoniopropyl-beta-cyclodextrin, carboxymethyl
    beta-cyclodextrin, carboxyethyl beta-cyclodextrin, sulfobutyl
    beta-cyclodextrin, vancomycin, heparin, maltooligosaccharides,
    dextrin, teicoplanin, deoxy Big CHAP, and others.

83. One preferred embodiment uses a nonionic additive that does not form
    a pseudostationary phase such as micelles, microemulsion droplets,
    liposomes, particles, or dendrimers. An illustrative example of
    temperature gradient focusing with a nonionic additive system would
    be the use of aqueous tris‑borate buffer as the fluid with
    gamma‑cyclodextrin which is a neutral chiral selector as the
    additive.

84. Referring to Figure 7, microchannel 710 is filled with a fluid 716
    which contains a non‑ionic chiral additive. The ends of microchannel
    710 are connected to fluid reservoirs 722 and 724, which are at
    least partially filled with fluid. Reservoir 724 can serve as a
    source of materials to be focused, in which case those materials are
    dissolved in the fluid that is in the reservoir 724. Alternately,
    the materials to be focused can be dissolved in the fluid in the
    microchannel 710. An electrical power supply 726 is used to apply an
    electric field to microchannel 710 via electrodes 712 and 714. A
    heated block 720 and a cooling block 718 are thermally connected to
    microchannel 710 and provide a temperature gradient in microchannel
    710 thereby generating a gradient in the electrophoretic velocity of
    the materials in the fluid. The counterbalancing bulk fluid flow is
    provided largely by the electroosmosis generated by the application
    of the electric field and is adjusted using a pressure controller
    728 which is connected via reservoir 722 to one end of
    microchannel 710. The chiral additive interacts with the material or
    materials to be separated or focused. This interaction modifies the
    normal electrophoretic mobility of the material based on the
    interaction of the additive with the material.

85. As with the prior embodiments, the gradient of electrophoretic
    velocity of the materials can be established by the fluid having an
    ionic strength or pH which is temperature dependent. The velocity of
    the bulk fluid flow through the microchannel 710 is adjusted using
    the pressure controller 728 so that the bulk flow velocity has a
    significant component substantially aligned in the direction
    opposite the direction of the electrophoretic migration of one or
    more materials and so that the total velocity of one or more of the
    materials is equal to zero at region F, the focus region in the
    microchannel 710.

86. In a second chiral temperature gradient focusing embodiment, the
    additive used with the temperature gradient focusing forms a
    pseudostationary phase. The pseudostationary phase includes but is
    not limited to micelles, microemulsion droplets, liposomes,
    particles, or dendrimers, which can modify the electrophoretic
    mobility of the material based upon hydrophobic interactions between
    the material to be focused and the pseudostationary phase.

87. Although this method is similar to micellar affinity gradient
    focusing of U.S. Patent Application No. 10/864,485, herein
    incorporated by reference, this method is distinguishable in that in
    micellar affinity gradient focusing the electrophoretic velocity
    variation is due to a variation in the interaction with the additive
    whereas in the present method, the interaction is more typically
    constant and the velocity variation results primarily from a
    variation in the ionic strength or other property of the fluid.

88. It should be noted, because the fluid ionic strength is typically a
    critical parameter in temperature gradient focusing, preferably, the
    additive should not affect the ionic strength and advantageously,
    the additive is neutral or comprises zwitterionic molecules. If the
    additive is ionic, it is preferable to use a sufficiently low
    concentration of the additive so as to not significantly affect the
    conductivity of the fluid.

89. However, if the additive is ionic it could be used in conjunction
    with temperature gradient focusing to focus nonionic materials that
    would not be focusable without the additive. The nonionic material
    would effectively become ionic through its interaction with the
    ionic additive.

90. In a third chiral temperature gradient focusing form of the present
    invention, the materials to be focused include chiral species which
    are first focused without the addition of a chirally selective
    additive. If the materials include two or more stereoisomers of a
    given molecular species those stereoisomers will be focused at the
    same position along the fluid conduit. Once the stereoisomers are
    focused to a desired concentration, a chiral selector is introduced
    into the fluid conduit such as microchannel 710, where the additive
    will interact with the chiral species resulting in a shift in the
    focusing position of the stereoisomers and separation of the
    stereoisomers.

91. One advantage of this third chiral temperature gradient focusing
    embodiment is that it can provide additional information about the
    interaction between the additive and the materials through
    monitoring of the focused peaks after the introduction of the
    additive.

92. In order to provide further understanding of the present chiral
    temperature gradient focusing, the following example is provided
    which in no way limits the scope of the present invention in any
    way. In this example, the temperature gradient focusing was
    performed in a 30 μm I.D. fused silica capillary, 3 cm in length.
    The capillary was mechanically and thermally anchored to two copper
    blocks at different temperatures to create a temperature gradient
    along a 2 mm section approximately midway along the length of the
    capillary. A temperature gradient from 20°C to 60°C was applied.

93. The fluid was 1 mol/L Tris(hydroxymethyl)aminomethane, 1 mol/L boric
    acid, with 20 mmol/L gamma‑cyclodextrin as the chirally selective
    additive. A voltage of ‑3000 V was applied to the 20°C end of the
    capillary and the 60°C end was electrically grounded. The pressure
    applied to the 60°C end of the capillary was adjusted to control the
    bulk flow rate so that the D and L enantiomers of glutamic acid were
    simultaneously focused in the 2 mm gradient section of the
    capillary, which was imaged using a fluorescence microscope. A plot
    of the measured intensity as a function of distance along the
    capillary is shown in Figure 8. The two peaks are the D and
    L enantiomers of dansyl‑glutamic acid. The fine structure near the
    top of the left peak is due to scratches in the plastic cladding
    surrounding the capillary.

94. An additional experiment was conducted for the enantiomeric
    separation of dansyl‑DL‑aspartic acid using 10 mm β‑cyclodextrin as
    the chirally selective additive.

95. A similar procedure was also used to achieve a baseline resolved
    separation of two different isomers of an achiral fluorescent dye
    molecule, Oregon green 488 carboxylic acid (available from Molecular
    Probes). The separated isomers in this case are comparable to the 5‑
    and 6‑ isomers of carboxyfluorescein.

96. A similar procedure was also used to achieve a baseline resolved
    separation of the fluorescently-labeled R and S enantiomers of the
    drug baclofen.

97. One of ordinary skill in the art now will readily appreciate that
    the present temperature gradient focusing differs from prior art
    methods such as sample stacking and isotachophoresis. In both cases,
    samples are focused or concentrated as a result of gradients in
    their electrophoretic velocities. In sample stacking and
    isotachophoresis, the velocity gradients are generated at the
    interfaces between solutions of different composition, and the point
    at which the concentration or focusing occurs is not stationary, but
    moves along with the bulk flow in the channel or capillary. In
    contrast to both sample stacking and isotachophoresis, the velocity
    gradients that produce material focusing in the present temperature
    gradient focusing result from applied temperature gradients and are
    stable for as long as the temperature gradient is maintained.

98. Further, one skilled in the art will recognized that the present
    temperature gradient focusing differs from isoelectric focusing
    techniques such as those disclosed in U.S. Patent Nos. 3,664,939 and
    5,759,370. Unlike isoelectric focusing techniques in which the pH
    gradient is established by using a fluid system that has a
    temperature dependent pH, the present temperature gradient focusing
    typically utilizes a fluid that has a temperature dependent ionic
    strength. When a temperature gradient and a voltage are applied to a
    microchannel, the ionic strength gradient of the fluid gives rise to
    a velocity gradient, which is used for focusing. As a result, a
    material present in the fluid is focused at a point where the
    material\'s total velocity, i.e., the sum of the electrophoretic
    velocity of the material and the bulk velocity of the fluid is zero.
    Therefore, in the present temperature gradient focusing, the pH and
    the isoelectric point of the material are typically not critical. In
    the embodiment of the present invention that utilizes fluids with a
    temperature-dependent pH, and thereby uses a thermally generated pH
    gradient for focusing, a bulk fluid flow is applied so that rather
    than focusing at its isoelectric point as in isoelectric focusing, a
    material will focus at a position along the pH gradient where the
    local pH is unequal to the isoelectric point of the material.

99. It will now be apparent to one of ordinary skill in the art that the
    present microfluidic device and temperature gradient focusing method
    provide numerous advantages over prior devices and methods. The
    present device and method are simpler to implement as no imbedded
    electrodes or salt bridges are necessary. In addition, like
    isoelectric focusing, temperature gradient focusing can be used to
    both concentrate and separate materials, but without the
    disadvantages associated with isoelectric focusing.

100. It will now also be apparent to one of ordinary skill in the art
     that the present invention provides advantages over prior methods
     for separating enantomers which include chiral crystallization,
     chiral HPLC, chiral capillary electrophoresis and chiral
     isoelectric focusing. The advantages of the present method are
     especially applicable to the pharmaceutical industry where
     low‑level enantomeric contaminants have an adverse effect.

101. An example of a composition containing such contaminants is
     thalidomide, where one enantiomer (the R) is an effective treatment
     for morning sickness, sleep disorders, and nausea whereas the other
     stereoisomer (the S) leads to phocomelia (a prenatal developmental
     abnormality in which the limbs of fetuses do not develop). A large
     number of other drugs have optical isomers, with one isomer being
     beneficial and the other being non‑beneficial and often harmful.
     Thus, chiral temperature gradient focusing would be excellently
     suited for the detection and quantification of low‑level
     enantiomeric impurities in pharmaceutical compounds as is critical
     for the prediction of the efficacy of the final preparation.

102. An additional advantage of the present chiral temperature gradient
     focusing over prior chiral capillary electrophoresis is that it is
     much easier to reverse the peak order. In temperature gradient
     focusing, the peak order can be reversed simply by changing the
     sign (direction) of the applied temperature gradient, the applied
     electric potential, and the bulk flow.

103. A further advantage of the present chiral temperature gradient
     focusing method over prior isoelectric focusing and isoelectric
     trapping techniques which use chirally selective additives for
     enantiomeric separations is provided by the use of temperature
     gradient focusing for focusing in that temperature gradient
     focusing has a number of advantages over any isoelectric
     focusing‑based approach. For example, temperature gradient focusing
     can provide greater degrees of concentration enhancement than
     isoelectric focusing. Temperature gradient focusing is applicable
     to all types of ionic materials, where isoelectric focusing is
     applicable only to materials with a well‑defined isoelectric point
     in the range from about 3 to about 11. In practice, this limits
     isoelectric focusing to use with proteins, peptides, and some
     chemically-modified amino acids. In addition, because the
     temperature gradient for temperature gradient focusing is much
     simpler and easier to create than the pH gradient required for
     isoelectric focusing, temperature gradient focusing‑based chiral
     separations are much simpler to control and implement.

104. A further advantage of the present invention is provided in that
     only a single, continuous fluid system is required. Solid phase
     extraction and related preconcentration methods of the prior art
     require multiple fluids where one fluid is used to carry the
     material to the preconcentrator and a second fluid is used to
     release the material from the preconcentrator. Further examples of
     multiple fluid systems include sample stacking, field amplified
     injection, iosotachophoresis, and sweeping.

105. Further, the present temperature gradient focusing provides
     enhanced concentration when compared with the prior art of other
     single preconcentration methods.

106. 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.

What is claimed is:

1\. A method for directing one or more materials in a fluid, said method
comprising the steps of:

applying an electric field to a fluid thereby causing one or more
materials to move electrophoretically;

establishing a temperature gradient in said fluid thereby generating a
gradient of the electrophoretic velocity of said one or more materials;

producing a flow of said fluid thereby changing the total velocity of
said one or more materials; and

adding an additive to said fluid thereby modifying the normal
electrophoretic velocity of said one or more materials based on an
interaction of said one or more materials with said additive.

2\. The method of claim 1, wherein:

said temperature gradient has a significant component substantially
aligned with the electrophoretic motion of said one or more materials,

said flow of said fluid has a significant component substantially
aligned in a direction opposite a direction of said electrophoretic
motion of said one or more materials, and

adjusting magnitudes of said electric field, said temperature gradient,
and said flow so that at least one of said one or more materials will
accumulate or be focused at at least one position along said temperature
gradient, the pH at said at least one position being unequal to the
isoelectric point of said at least one of said one or more materials
that are focused at said at least one position;

whereby said step of adding an additive causes a change in the focusing
position of at least one of said one or more materials.

3\. The method of claim 2, wherein the pH of said fluid is temperature
dependent and said temperature gradient establishes a gradient in the pH
of said fluid.

4\. The method of claim 2, wherein the ionic strength of said fluid is
temperature dependent and said temperature gradient establishes a
gradient in the ionic strength of said fluid.

5\. The method of claim 4, wherein at least two of said one or more
materials being initially spatially mixed within said fluid are
separated.

6\. The method of claim 4, wherein said one or more materials are
selected from the group consisting of fluorescent dyes, ions, amino
acids, peptides, proteins, nucleic acids, cells, and colloidal
particles.

7\. The method of claim 4, wherein said fluid is selected from the group
consisting of ionic aqueous solutions, ionic non-aqueous solutions,
aqueous buffer solutions, and mixtures of aqueous and non-aqueous
solutions.

8\. The method of claim 4, wherein said electric field is applied using
a set of components comprising an electrical power supply and two or
more electrodes contacting said fluid.

9\. The method of claim 4, wherein the temperature gradient is one of
linear and non-linear.

10\. The method of claim 4, wherein the temperature gradient is one of
monotonic and non-monotonic.

11\. The method of claim 4, wherein said step of establishing a
temperature gradient comprises applying an electric current to the fluid
to produce the temperature gradient by Joule heating.

12\. The method of claim 4, wherein said step of establishing a
temperature gradient comprises supplying thermal energy to said fluid
via a heated block.

13\. The method of claim 4, wherein said step of establishing a
temperature gradient comprises removing thermal energy from said fluid
via a cooled block.

14\. The method of claim 4, wherein said flow is generated by
electroosmosis.

15\. The method of claim 4, wherein said flow is generated by pressure
gradients.

16\. The method of claim 4, wherein said flow is generated by a
combination of electroosmosis and pressure gradients.

17\. The method of claim 4, wherein said additive comprises a chiral
selector.

18\. The method of claim 17, wherein said chiral selector is selected
from the group consisting of alpha-cyclodextrin, beta-cyclodextrin,
gamma-cyclodextrin, heptakis-O-methyl beta-cyclodextrin,
heptakis(2,6-di-O-methyl) beta-cyclodextrin, heptakis
(2,3,6-tri-O-methyl) beta-cyclodextrin, hydroxyethyl beta-cyclodextrin,
hydroxypropyl beta-cyclodextrin, sulfated beta-cyclodextrin,
6-O-alpha-D-glucosyl-alpha-cyclodextrin,
6-O-alpha-D-glucosyl-beta-cyclodextrin,
2-hydroxy-3-trimethylammoniopropyl-beta-cyclodextrin, carboxymethyl
beta-cyclodextrin, carboxyethyl beta-cyclodextrin, sulfobutyl
beta-cyclodextrin, vancomycin, heparin, maltooligosaccharides, dextrin,
teicoplanin, and deoxy Big CHAP.

19\. The method of claim 4, wherein said additive comprises a nonionic
additive.

20\. The method of claim 4, wherein said additive does not form a
pseudostationary phase.

21\. The method of claim 4, wherein said additive forms a
pseudostationary phase.

22\. The method of claim 21, wherein said pseudostationary phase is
selected from the group consisting of micelles, microemulsion droplets,
liposomes, particles and dendrimers.

23\. The method of claim 21, wherein said additive is selected from the
group consisting of anionic surfactants, cationic surfactants, and
nonionic surfactants.

24\. The method of claim 4, wherein the interaction of a first
stereoisomer of one of said one or more materials with said additive is
stronger than the interaction of a second stereoisomer of said one of
said one or more materials with said additive, thereby allowing the
separation of said first stereoisomer from said second stereoisomer.

25\. The method of claim 4, wherein said step of applying an electric
field, said step of establishing a temperature gradient, and said step
of producing a bulk flow comprise using an electrical power supply to
apply a voltage to said fluid, and wherein the electric field provided
by said electrical power supply causes the electrophoretic motion of
said one or more materials, a flow of electric current in said fluid
thereby generating said temperature gradient by Joule heating, and
electroosmosis of said fluid thereby producing said flow of said fluid.

26\. The method of claim 4, wherein said step of applying an electric
field and said step of producing a bulk flow comprise using an
electrical power supply to apply a voltage to said fluid, and wherein
the electric field provided by said electrical power supply causes the
electrophoretic motion of said one or more materials, and electroosmosis
of said fluid thereby producing said flow of said fluid.

27\. The method of claim 4, wherein said steps of applying an electric
field, establishing a temperature gradient, and producing a flow are
implemented first, thereby focusing said one or more materials into at
least one group, said at least one group each containing all of the
extant stereoisomers of each of said one or more materials,
respectively;

after which, said step of adding an additive is implemented, whereby
said stereoisomers of one or more of said at least one group are
refocused at different positions along said temperature gradient and are
thereby separated.

28\. The method of claim 4, wherein said fluid is supplied as a
continuous single fluid flow.

29\. The method of claim 4, further comprising the step of collecting
said at least one of said one or more materials after it has been
focused.

30\. A fluidic device, comprising:

a fluid conduit;

a fluid disposed in said fluid conduit;

a source of one or more materials in fluid communication with said fluid
conduit;

a first means for providing an electric field within said fluid conduit,
thereby causing said one or more materials to move electrophoretically;

a second means for providing for a temperature gradient within said
fluid conduit, thereby generating a gradient of the electrophoretic
velocity of said one or more materials;

a third means for providing a flow of said fluid in said fluid conduit;
and

an additive, which when added to said fluid, modifies the normal
electrophoretic mobility of said one or more materials within said fluid
based on the affinity of said one or more materials for said additive.

31\. The fluidic device of claim 30, wherein said second means provides
for a temperature gradient which has a significant component
substantially aligned with the electrophoretic motion of said one or
more materials, and wherein when said third means produces flow of said
fluid, the flow has a significant component substantially aligned in a
direction opposite a direction of said electrophoretic motion of said
one or more materials, and wherein said first means, said second means
and said third means are adjustable to adjust magnitudes of said
electric field, said temperature gradient, and said flow, respectively,
such that at least one of said one or more materials will accumulate or
be focused at at least one position along said temperature gradient, the
pH at said at least one position being unequal to the isoelectric point
of said at least one of said one or more materials that are focused at
said at least one position.

32\. The fluidic device of claim 31, wherein the pH of said fluid is
temperature dependent and said second means provides for establishing a
gradient in the pH of said fluid.

33\. The fluidic device of claim 31, wherein the ionic strength of said
fluid is temperature dependent and said second means provides for
establishing a gradient in the ionic strength of said fluid.

34\. The fluidic device of claim 33, wherein said fluid conduit
comprises a microchannel formed in a substrate, having a geometry with
at least one spatial dimension on the order of micrometers, and said
second means provides for a temperature gradient being applied to said
substrate.

35\. The fluidic device of claim 33, wherein said at fluid conduit
comprises a channel formed in a substrate and having a geometry with at
least one spatial dimension on the order of at least one millimeter, and
said second means provides for a temperature gradient being applied to
said substrate.

36\. The fluidic device of claim 33, wherein said fluid conduit
comprises a capillary tube.

37\. The device of claim 33, wherein said fluid is selected from the
group consisting of ionic aqueous solutions, ionic non-aqueous
solutions, aqueous buffer solutions, and mixtures of aqueous and
non-aqueous solutions.

38\. The fluid device of claim 33, wherein said fluid has a temperature
dependent pH.

39\. The method of claim 33, wherein said one or more materials are
selected from the group consisting of fluorescent dyes, ions, amino
acids, peptides, proteins, nucleic acids, cells, and colloidal
particles.

40\. The device of claim 33, wherein said first means comprises an
electrical power supply.

41\. The device of claim 33, wherein said first means comprises an
electrical power supply and two or more electrodes in contact with said
fluid.

42\. The device of claim 33, wherein said second means comprises at
least one element selected from the group consisting of a heat source
and a heat sink, thermally coupled to said fluid conduit, for providing
a temperature gradient having a significant component substantially
aligned with an electric field produceable from said first means.

43\. The fluidic device of claim 42, wherein said heat source comprises
a heated block thermally coupled to said fluid conduit for providing
thermal energy to said fluid conduit.

44\. The fluidic device of claim 42, wherein said heat sink comprises a
cooling block thermally coupled to said fluid conduit for removing
thermal energy from said fluid conduit.

45\. The fluidic device of claim 33, wherein said second means comprises
a heated block thermally coupled to said fluid conduit for providing
thermal energy to said fluid conduit and a cooling block spaced from
said heated block and thermally coupled to said fluid conduit for
removing thermal energy from said fluid conduit.

46\. The device of claim 33, wherein said first and second means
comprises an electrical power supply, and wherein the electric field
provided by said electrical power supply causes both the electrophoretic
motion of said one or more materials and a flow of electric current in
said fluid conduit thereby generating said temperature gradient by Joule
heating.

47\. The device of claim 33, wherein said third means provides for a
fluid flow substantially in opposition to the electrophoretic migration
of said one or more materials.

48\. The device of claim 33, wherein said third means comprises a
pressure control means for providing a pressure driven flow in said
fluid conduit.

49\. The device of claim 48, wherein said pressure control means is
selected from the group consisting of a pneumatic pressure controller, a
fluid pump, and a reservoir filled to a predetermined height with fluid,
said height being adjustable and, in conjunction with gravity,
determining the pressure applied to said fluid conduit.

50\. The device of claim 33, wherein said third means comprises an
electrical power supply for providing electroosmotic flow of said fluid
in said fluid conduit.

51\. The device of claim 33, wherein said third means comprises a
pressure control means for providing a pressure driven flow in said
fluid conduit and an electrical power supply for providing
electroosmotic flow of said fluid in said fluid conduit.

52\. The device of claim 33, wherein said first and third means
comprises an electrical power supply, and wherein an electric field
provided by said electrical power supply provides for the
electrophoretic motion of said one or more materials, and electroosmotic
flow of said fluid in said fluid conduit.

53\. The device of claim 33, wherein said first, second, and third means
comprise an electrical power supply, and wherein an electric field
provided by said electrical power supply provides for electrophoretic
motion of said one or more materials, a flow of electric current in said
fluid conduit thereby generating said temperature gradient by Joule
heating, and electroosmotic flow of said fluid in said fluid conduit.

54\. The fluidic device of claim 33, wherein said additive means
comprises a chiral selector.

55\. The fluidic device of claim 54, wherein said chiral selector is
selected from the group consisting of alpha-cyclodextrin,
beta-cyclodextrin, gamma-cyclodextrin, heptakis-O-methyl
beta-cyclodextrin, heptakis(2,6-di-O-methyl) beta-cyclodextrin, heptakis
(2,3,6-tri-O-methyl) beta-cyclodextrin, hydroxyethyl beta-cyclodextrin,
hydroxypropyl beta-cyclodextrin, sulfated beta-cyclodextrin,
6-O-alpha-D-glucosyl-alpha-cyclodextrin,
6-O-alpha-D-glucosyl-beta-cyclodextrin,
2-hydroxy-3-trimethylammoniopropyl-beta-cyclodextrin, carboxymethyl
beta-cyclodextrin, carboxyethyl beta-cyclodextrin, sulfobutyl
beta-cyclodextrin, vancomycin, heparin, maltooligosaccharides, dextrin,
teicoplanin, and deoxy Big CHAP.

56\. The fluidic device of claim 33, wherein said additive means
comprises a nonionic additive.

57\. The fluidic device of claim 33, wherein said additive means does
not form a pseudostationary phase.

58\. The fluidic device of claim 33, wherein said additive means forms a
pseudostationary phase.

59\. The fluidic device of claim 58, wherein said pseudostationary phase
is selected from the group consisting of micelles, microemulsion
droplets, liposomes, particles and dendrimers.

60\. The fluidic device of claim 58, wherein said additive means is
selected from the group consisting of anionic surfactants, cationic
surfactants, and nonionic surfactants.

## ABSTRACT OF THE DISCLOSURE

A method and device are provided for concentrating and separating
materials in fluids within a fluidic device having a fluid conduit such
as a channel or capillary. The concentration is achieved by balancing
the electrophoretic velocity of a material against the bulk flow of
fluid in the presence of a temperature gradient. An additive is added to
the fluid which interacts with the material and which modifies the
normal electrophoretic mobility of the material. Using an appropriate
fluid, the temperature gradient can generate a corresponding gradient in
the electrophoretic velocity so that the electrophoretic and bulk
velocities sum to zero at a unique position along the conduit and the
material will be focused at that position. The method and device may be
adapted for use with a variety of materials including fluorescent dyes,
amino acids, proteins, DNA and to concentrate a dilute material.