WO2017205985A1

WO2017205985A1 - System and method for the transfer of fluid from one flow to another

Info

Publication number: WO2017205985A1
Application number: PCT/CA2017/050671
Authority: WO
Inventors: Cameron Skinner; John D. CHIN
Original assignee: Valorbec Societe En Commandite
Priority date: 2016-06-03
Filing date: 2017-06-02
Publication date: 2017-12-07

Abstract

A system and method that uses non-turbulent fluid flow for transferring a fluid from one flow to another. The system and method allows for the manipulation of small amounts of fluids, for example sub-microliter volumes. Also, the system and method allows for the analysis of complex / multicomponent samples such as biological samples. Moreover, the system and method results in analytical data that are aligned.

Description

SYSTEM AND METHOD FOR THE TRANSFER OF FLUID FROM ONE FLOW TO ANOTHER

FIELD OF THE INVENTION

[0001] The present invention relates generally to systems and methods for the transfer of fluid from one flow to another. More specifically, the invention relates to a system and method that uses non-turbulent fluid flow for transferring a fluid from one flow to another. The system and method of the invention allows for the manipulation of small amounts of fluids, for example sub-microliter volumes. Also, the system and method of the invention allows for the analysis of complex / multicomponent samples such as biological samples. Moreover, the system and method of the invention yields analytical data that are aligned.

BACKGROUND OF THE INVENTION

[0002] One of the key problems in biological analysis is the complexity of the sample. A typical person has about 50000 different proteins distributed throughout the body. Each of these proteins performs a function, and many can be key indicators of health status (e.g. cancer, stress, infection etc.). A serum sample can contain many thousands of proteins and other metabolites. Biological researchers need to understand the interplay of many of these proteins to have a chance at mapping and establishing how important biological processes, such as gestation, cancer, response to drugs, stress etc. work in the body.

[0003] The traditional approach to surveying the proteins in a biological sample is via two-dimensional poly acrylamide gel electrophoresis (2-D PAGE). The 2D-PAGE technique separates the sample by combining two different separation mechanisms. This is the "classic" multidimensional separation. The first dimension is typically iso-electric focusing and the second dimension is gel electrophoresis. Each of these techniques is capable of separating the sample into, roughly, 80 distinct peaks. Therefore each separation technique has a peak capacity of 80 and, in the most optimistic consideration, can separate a sample with up to 80 different analytes by itself. A more realistic assessment is that about 35-40% of the peak capacity is the maximum number of components that can be successfully separated. Therefore, single separation techniques are capable of analyzing simple samples but are wholly inadequate in the face of a sample that has thousands of proteins. [0004] The power of multidimensional separations comes from the combination of orthogonal separations where each technique operates on a distinct aspect of the sample. When the separations are brought together, as in 2-D PAGE, the combined peak capacity is the product of the individual separations. Wth 2-D PAGE this equates to 80 x 80 = 6400 which means that thousands of proteins can be surveyed. This still is not quite enough to comfortably tackle biological samples, although it is much better than just a single separation alone. 2-D PAGE is used extensively, but has a few drawbacks associated thereto. 2D-PAGE is generally slow (about 8 hours). The analytes (proteins) must not precipitate during focusing or electrophoresis and more importantly for comparative analyses, 2D-PAGE is generally not quite a reproducible method.

[0005] Due to these limitations, and also a desire to increase the separation power of separation processes in general, researchers have interfaced two liquid chromatographic systems together. Each system operates in a different orthogonal mode and the interface is via a rotary valve similar to what is used for standard sample injection on a typical liquid chromatograph. Briefly, the first dimension separation effluent leaves the column and is collected in the interface valve's sampling loop. Typical volumes would be on the order of 10 μΙ_. Once the loop is full, this sample is injected onto the second dimension where it is quickly separated. During this separation time, the loop refills by a next sample which is then injected and separated etc. In order to synchronize the two instruments, the first dimension separation is usually operated at a very slow rate so that there is sufficient time for the second dimension separation to complete, with reasonable efficiency, between the periodic sample injections. Drawbacks associated to this technique are that both separation dimensions operate far away from their maximum efficiency and the total separation time is the product of the number of second dimension separations (hundreds) and the second dimension separation time (minutes) and can therefore be many hours long. These multidimensional separations can have combined peak capacities in the low thousands and are, to a first approximation, competitive with 2-D PAGE. They have the additional advantages of being fully automated, have better reproducibility and keep analytes in an all liquid-phase.

[0006] Systems for multidimensional separations currently known in the art involve a mechanical valve interface that allows for only a limited number (usually one) of second dimension separations to operate. This, as outlined above, forces the first dimension separation to operate very slowly and the second dimension to operate very quickly, which significantly reduces separation efficiency (peak capacity) and time efficiency (many hours long). Also, the mechanical valve interface involved in these systems requires the use of relatively large volume / flowrate chromatographic techniques.

[0007] Another problem in many fields of research and industry is the manipulation of small volumes of fluid, for example sub-microliter volumes. Traditional mechanical actuators, valves, tubing etc. are capable of operating efficiently with volumes larger than a few microliters. However, when volumes smaller than a few microliters are considered it is difficult to transfer, switch, and otherwise manipulate them. These limitations are often due to physical limitations such as the ability to machine valve components at the sub millimeter scale, the increased surface area to volume ratio that results in adsorption losses in micro vials, the difficulty in measuring volumes precisely when the sample occupies less than a cubic millimeter, and increased dispersion due to dead volumes within interconnected tubing.

[0008] Some of these problems can be addressed by utilizing tubing, connectors and fittings specifically designed to operate with small volumes. Generally, many of the successful macroscale "plumbing" processes can be scaled down to the microscale. The salient features are small internal cross sectional areas (small diameters of a few to a few hundred μηι as encountered with capillary tubing) and low sorption surfaces such as silica and polyether ether ketone (PEEK). Fittings can be designed with very small dead volumes to minimize dispersion. However, one remaining difficulty is precision flow switching (valving) where flow can be selectively directed into another flow. This control or manipulation of flow is an essential element for many types of micro analyses. This manipulation is essential for selective operations such as enzymatic digestion of a fraction of a sample for proteomic analysis, following time course processes and selecting sub-samples from a flowing stream for subsequent separation(s).

[0009] A further problem in electro-driven separations, such as capillary electrophoresis, is the variability in the apparent velocity of the analyte. This variability complicates comparison of data between separations on the same capillary and in-between capillaries. In multidimensional separations that use parallel separations, it may prevent alignment of the data across the parallel separations, which is critical to producing a multidimensional plot of the data and by extension prevent comparison of multidimensional data sets.

[0010] Typically, in electro-driven separations, the velocity of the analyte is largely the result of the summation of two separate velocities: o Apparent velocity = electrophoretic velocity + electroosmotic velocity o Apparent velocity = (electrophoretic mobility + electroosmotic mobility) x applied electric field.

[0011] The electrophoretic mobility is an intrinsic property of the analyte that depends on its charge state, volume and solvent viscosity. The electrophoretic mobility should, to a first approximation, be constant with a set of experimental conditions. However, the magnitude of the electroosmotic mobility is extremely sensitive to even small changes in the experimental conditions. Factors that affect the electroosmotic flow (EOF) include pH, ionic strength, temperature, solvent viscosity, surface condition of the wall etc. Observed variations in apparent velocity data are largely attributed to uncontrolled variations in the electroosmotic mobility.

[0012] Several experimental procedures can be used to minimize the variations such as using a background electrolyte with fixed pH, elevated buffer capacity and ionic strength, careful control of the capillary temperature, frequent rejuvenation of the capillary wall through appropriate washing procedures etc. Even with such precautions there will often remain significant variability, much of it due to intrinsic variations in-between capillaries and some due the influence of the sample components on the EOF and sample carry-over. In the context of separations carried-out on an array of capillaries, there may also be additional variations across the array due to non-uniform electric fields in the inlet and outlet cuvettes and the parabolic flow profiles generating differing hydrodynamic pressures at the entrance and exit of each capillary.

[0013] Internal standardization is an approach to addressing inter-experiment variations where an internal standard (IS) or non-analyte species is introduced into the sample. These species, under ideal conditions, produce peaks that can be readily distinguished from the analyte peaks. Depending on where, in the analytical process, the internal standards or non-analyte species are introduced, they may be used to control experiment-to-experiment and/or separation-to-separation variations. When used properly, the internal standards or non-analyte species may act as reference signals that allow variations in the separation conditions to be detected. Once the variation in the separation has been identified, it can be corrected, resulting in data that are aligned. Unfortunately, most species that can be used as IS possess mobilities that are similar to the analytes, which results in the IS peak(s) appearing within the analyte separation window. This can obscure the analyte(s), reduce the effective peak capacity and produce indistinct IS peaks that are useless for data alignment.

[0014] Applicant is aware of the prior art documents listed herein below at the section entitled "References." The content of these documents is herein incorporated by reference in their entirety.

[0015] There is still a need for systems and methods for the transfer of fluid from one flow to another. There is a need for such systems and methods that also allow for multidimensional separations. There is a need for such systems and methods that are efficient, that require less time for operation, that allow for the manipulation of small amounts, for example sub-microliter volumes of a sample, that allow for the analysis of complex / multicomponent samples, and that may be automated.

[0016] Also, there is a need, in multidimensional separations, to obtain data that are properly aligned.

SUMMARY OF THE INVENTION

[0017] The inventors have designed and constructed a system for transferring a fluid sample from one flow to another using a non-turbulent flow of a fluid running in a sheath flow interface. The system according to the invention allows for multidimensional separation of a sample. Also, the system according to the invention and associated method allows for alignment and normalization of the analytical data obtained. The source of the sample / fluid sample may be from an organism, a bulk solution or a sub-sample thereof held in a container such as a vial.

[0018] The invention thus provides the following according to aspects thereof: (1) System for transferring a fluid sample from one flow to another, wherein transfer of the fluid sample is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

(2) System for transferring a fluid sample from one flow to another, wherein the fluid sample is fed to the system through at least one first tube and is transferred to at least one second tube using a non-turbulent flow of a fluid running in a sheath flow interface.

(3) System according to (1) or (2) above, wherein the fluid sample is fed to the system from a source which is an organism, a bulk solution or a sub-sample thereof.

(4) System according to (2) above, wherein an outlet of the at least one first tube and an inlet of the at least one second tube extend in the sheath flow interface, in overlooked alignment, the fluid running in a direction which is first tube towards second tube.

(5) System according to (2) above, wherein the first tubes are in parallel arrangement and the second tubes are in parallel arrangement; optionally, the second tubes are arranged in a plurality of sets of tubes, each set of tubes being supplied with a different fluid.

(6) System according to (2) above, wherein the tubes are in vertical arrangement in the interface, the first tubes being positioned above the second tubes.

(7) System according to (2) above, wherein the tubes are in vertical arrangement in the interface, the first tubes being positioned below the second tubes.

(8) System according to (2) above, wherein the at least one first tube and the at least one second tube are capillaries.

(9) System according to (2) above, wherein the at least one first tube and the at least one second tube each independently comprises one or more of: capillary electrophoresis (CE), liquid chromatography (LC), capillary electrochromatography (CEC), means for effecting a chemical modification such as enzymatic digestor (ED), flow injection analysis (FIA) etc.

(10) System according to (1) or (2) above, wherein the non-turbulent flow is selected from laminar flow and uniform flow. (1 1) System according to (2) above, wherein the interface comprises an inlet for introducing the fluid and an outlet for removing the fluid.

(12) System according to (2) above, wherein the direction of the running fluid comprises one or more contours.

(13) System according to (2) above, wherein the fluid running in the interface comprises at least one agent selected from analyte labeling agents.

(14) System according to (2) above, further comprising at least one motor for moving the tubes for sequentially positioning an inlet of one of the second tubes in overlooked alignment with an outlet of one of the first tubes.

(15) System according to (2) above, further comprising at least one of: sensors, detectors, cameras and computer programs.

(16) System according to (1) above, wherein at least one internal standard (IS) is introduced into said one flow and/or said other flow.

(17) System according to (2) above, wherein at least one internal standard (IS) is introduced into said at least one first tube and/or said at least one second tube before and/or after the transfer of the fluid sample.

(18) Method for transferring a fluid sample from one flow to another, comprising using a system as defined in any one of (1)-(17) above.

(19) Method for transferring a fluid sample from one flow to another, comprising: feeding the fluid sample to at least one first tube; and transferring the fluid sample to at least one second tube, wherein the transfer of the fluid sample is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

(20) Method for transferring a fluid sample from one flow to another, comprising:

(a) providing a source of the fluid sample, a sheath flow interface, at least one first tube having an outlet extending in the interface, and at least one second tube having an inlet extending in the interface and in overlooked alignment with the outlet of the at least one first tube; (b) running a non-turbulent flow of a fluid in the interface, in a direction which is first tube towards second tube; and

(c) causing the fluid sample to circulate from the source to the at least one first tube, wherein the fluid sample exiting the outlet of the at least one first tube is transferred to the inlet of the at least one second tube, the transfer being effected using the non-turbulent flow running in the interface.

(21) Method according to (19) or (20) above, wherein at least one internal standard (IS) is introduced into said at least one first tube and/or said at least one second tube before and/or after the transfer of the fluid sample.

(22) Method according to (19) or (20) above, wherein the source of the fluid sample is an organism, a bulk solution or a sub-sample thereof.

(23) System for multidimensional separation of a sample, comprising at least two separation instruments, wherein transfer of the sample between the instruments is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

(24) System for multidimensional separation of a sample, comprising first and second separation instruments each comprising at least one separation capillary, wherein a separated sample exiting an outlet of the at least one separation capillary of the first separation instrument is transferred to the second instrument through an inlet of the at least one separation capillary thereof, and wherein the transfer is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

(25) System for multidimensional separation of a sample, comprising: a sheath flow interface; a first separation instrument comprising a separation capillary having an outlet extending in the interface; and a second separation instrument comprising at least one separation capillary each having an inlet extending in the interface and in overlooked alignment with the outlet of the capillary of the first instrument, wherein a separated sample exiting the outlet of the capillary of the first instrument is transferred to the inlet of one of the at least one capillary of the second instrument, the transfer being effected using a non-turbulent flow of a fluid running in the interface, the fluid running in a direction which is first instrument towards second instrument.

(26) System according to (24) or (25) above, wherein the capillaries of the first and second instruments are in vertically arrangement in the interface, the capillaries of the first instrument being positioned above the capillaries of the second instrument.

(27) System according to (24) or (25) above, wherein the capillaries of the first and second instruments are in vertically arrangement in the interface, the capillaries of the first instrument being positioned below the capillaries of the second instrument.

(28) System according to (24) or (25) above, wherein the capillaries of the first instrument are in parallel arrangement and the capillaries of the second instrument are in parallel arrangement; optionally, the capillaries of the second instrument are arranged in a plurality of sets of capillaries, each set of capillaries being supplied with a different fluid.

(29) System according to (24) or (25) above, further comprising at least one motor for moving the capillaries for sequentially positioning an outlet of one capillary of the second instrument in overlooked alignment with an inlet of one capillary of the first instrument.

(30) System according to (24) or (25) above, wherein the sample comprises analytes, a first dimension separation of the analytes being performed in the first instrument using a first dimension separation fluid, and a second dimension separation being performed in the second instrument using a second dimension separation fluid which is the fluid running in the interface.

(31) System according to (30) above, wherein the first dimension separation fluid is selected from buffer, acetonitrile and alcohol; and the second dimension separation fluid is a buffer. (32) System according to (24) or (25) above, wherein the first and second instruments each independently comprises one or more of: capillary electrophoresis (CE), liquid chromatography (LC), capillary electrochromatography (CEC), means for effecting a chemical modification such as enzymatic digestor (ED), flow injection analysis (FIA) etc.

(33) System according to any one of (23)-(25) above, wherein the non-turbulent flow is selected from laminar flow and uniform flow.

(34) System according to (24) or (25) above, wherein the interface comprises an inlet for introducing the fluid and an outlet for removing the fluid.

(35) System according to (24) or (25) above, wherein the direction of the running fluid comprises one or more contours.

(36) System according to (24) or (25) above, wherein the fluid running in the interface comprises at least one agent selected from analyte labeling agents.

(37) System according to (24) or (25) above, wherein at least one internal standard (IS) is introduced into said at least one separation capillary of said first separation instrument and/or said at least one separation capillary of said second separation instrument before and/or after the transfer of the fluid sample.

(38) System according to (24) or (25) above, further comprising at least one of: sensors, detectors, cameras and computer programs.

(39) Method for multidimensional separation of a sample, comprising using a system as defined in any one of (23)-(38) above.

(40) Method for multidimensional separation of a sample, comprising performing sequential separations on at least one first separation instrument and at least one second separation instrument, each instrument comprising at least one capillary, wherein transferred of the sample between the at least one first instrument and the at least one second instrument is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

(41) Method for multidimensional separation of a sample, comprising: (a) providing a sheath flow interface, a first separation instrument comprising a separation capillary having an outlet extending in the interface, and a second separation instrument comprising at least one separation capillary each having an inlet extending in the interface and in alignment with the outlet of the capillary of the first instrument; and

(b) running a non-turbulent flow of a fluid in the interface, in a direction which is first instrument towards second instrument; and

(c) feeding the sample to the first instrument, wherein a separated sample exiting the outlet of the capillary of the first instrument is transferred to the inlet of one of the at least one capillary of the second instrument, the transfer being effected by the non-turbulent flow running in the interface.

(42) Method according to (41) above, wherein at least one internal standard (IS) is introduced into said at least one separation capillary of said first separation instrument and/or said at least one separation capillary of said second separation instrument before and/or after the transfer of the fluid sample.

(43) System according to any one of (23)-(38) above or method according to any one of (39)-(42) above, wherein the sample is a biological sample.

(44) System according to any one of (23)-(38) above or method according to any one of (39)-(42) above, wherein the sample is in sub-microliter amount.

(45) System according to any one of (1)-(15), (23)-(38), (43) and (44) above or method according to any one of (18)-(22) and (39)-(42) above, wherein the fluid sample comprises at least one internal standard.

(46) System according to any one of (16), (17) and (37) above or method according to (21) or (42) above, wherein the introduction of the at least one internal standard after the transfer of the fluid sample is performed following a delay. (47) System according to any one of (16), (17) and (37) above or method according to (21) or (42) above, wherein the transfer of the fluid sample after the introduction of the at least one internal standard is performed following a delay.

(48) System according to any one of (16), (17) and (37) above or method according to (21) or (42) above, wherein the at least one internal standard comprises components that are different from components of the fluid sample and/or the at least one internal standard provides signals that are distinct from signals of components of the fluid sample and/or the at least one internal standard comprises components having electrophoretic mobilities that are different from electrophoretic mobilities of components of the fluid sample.

(49) System according to any one of (16), (17) and (37) above or method according to (21) or (42) above, wherein the at least one internal standard is fluorescein sulfonic acid (FSA).

(50) System according to any one of (16), (17) and (37) above or method according to (21) or (42) above, wherein a duration of the delay is such that the internal standard signals arrive at the detector separated from signals of components of the fluid sample

(51) System according to (50) above, wherein the duration of the delay is between about 5 seconds to about 10 seconds, preferably about 7.5 seconds.

[0019] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the appended drawings:

[0021] FIG. 1 : Conventional capillary electrophoresis.

[0022] FIG. 2: Sample separation in a capillary electrophoresis.

[0023] FIG. 3: Separation of fluorescently labelled serum proteins by capillary electrophoresis. [0024] FIG. 4: Embodiment of the sheath flow interface according to the invention including a capillary.

[0025] FIG. 5: Embodiment of the sheath flow interface according to the invention.

[0026] FIG. 6A-C: Embodiments of the system according to the invention showing the area of the effluent stream.

[0027] FIG. 7A-C: Sheath flow interface according to the invention including two capillaries and showing the transfer of sample between the capillaries.

[0028] FIG. 8: Embodiment of the system according to the invention.

[0029] FIG. 9: Embodiment of the system according to the invention.

[0030] FIG. 10: Embodiment of the system according to the invention.

[0031] FIG. 11 : Embodiment of the system according to the invention showing two sets of second dimension separation capillaries each set supplied with a different buffer.

[0032] FIG. 12: Overview of an embodiment of the system according to the invention.

[0033] FIG. 13: Overview of an embodiment of the system according to the invention.

[0034] FIG. 14: Embodiment of the system according to the invention showing transfer of a brain dialysate.

[0035] FIG. 15: Data from separation of a fluorescently labelled tryptic digest of a model protein.

[0036] FIG. 16: Plot of separation efficiency (plate number) of 100 nM fluorescein injected onto the eight second dimension capillaries as a function of injection time (where the first dimension capillary is aligned with the second dimension capillary). Both dimensions are using borate 25 mM, sodium dodecyl sulfate 5 mM at pH 9.2 as mobile phase/background electrolyte.

[0037] FIG. 17: Plate number per meter. This figure demonstrates the efficiency of the injection and separation process on the second dimension capillaries. Capillary electrophoresis operates with a maximum efficiency of about 500000 to 1000000 theoretical plates/m.

[0038] FIG. 18: This figure establishes that the signal measured in the second dimension is proportional to the mass injected. The mass was calculated based on the concentration (50 nM fluorescein) and the injection time (500-3500 ms). This data also allows calculation of the detection limit of 3-17 attomoles (3-17 x 10^"18 Moles) of fluorescein but. This may be improved with a more sensitive detector system (increased laser power, higher NA optics, more sensitive photon detector).

[0039] FIG. 19: Data collected from the eight second dimension capillaries demonstrating the separation of Atto-tag FQ labelled 0.7 mg/mL β-casein tryptic digest. Traces have been offset for clarity. Note that all separations are the same except for the influence of differing electroosmotic flows in the capillaries. Differences in sensitivities (peak heights) are believed to be the result of small misalignments of the capillaries in the excitation laser beam rather than inherent differences in the mass injected onto the second dimension capillary.

[0040] FIG. 20: Conceptual diagram illustrating a separation in the first dimension (3 mg/ml of Atto-tag FQ labelled bovine serum albumin in borate buffer) being sub-sampled into the second dimension capillaries by injecting approximately 3.3 minutes of first dimension effluent onto each second dimension capillary.

[0041] FIG. 21 : Top trace, separation of bovine serum albumin in borate buffer. Bottom trace, reconstructed separation of the bovine serum albumin separation as collected at the end of the eight second dimension separations demonstrating that a separation begun on the first dimension may be sub-sampled and directed to multiple second dimension separations without destroying the separation. Both dimensions utilized 25 mM borate buffer as mobile phase/background electrolyte.

[0042] FIG. 22: Demonstration that a broad peak eluting from the first dimension (peak width is approximately 2.5 minutes) may be sub-sampled by the second dimension and the second dimension separations reproduce the first dimension peak shape. Eight picomoles fluorescein injected onto the first dimension and "separated" using 10 mM sodium acetate, pH 5.76, second dimension separated using 25 mM borate pH 9.2. [0043] FIG. 23: Separation of Atto-tag FQ labelled bovine serum albumin (BSA) tryptic digest in 10 mM sodium acetate, pH 5.75.

[0044] FIG. 24: Separation of bovine serum albumin (BSA) tryptic digest in 25 mM borate, pH 9.2.

[0045] FIG. 25: Multidimensional separation of bovine serum albumin (BSA) tryptic digest separated in the first dimension by sodium acetate buffer and borate buffer in the second dimension. Each of the eight second dimension capillaries was reused for approximately 32 separations. The data demonstrate that overlapping peaks in the first separation may often be resolved through the application of the second dimension separation conditions.

[0046] FIG. 26: Multidimensional separation of a tryptic digest of β-casein that has been fluorescently labelled. Individual second dimension separations are represented by the columns.

[0047] FIG. 27: Same data as shown in FIG. 26. The individual separations (columns) in the second dimension have been aligned by internal standardization (IS) using the two prominent internal standard peaks appearing at the top of the data.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0048] Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments described below, as variations of these embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

[0049] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. [0050] As used herein, the term "non-turbulent fluid flow" is intended to refer to a flow that is characterized by non-chaotic fluid movement.

[0051] As used herein, the term "laminar flow" is intended to refer to a flow that occurs when fluid is moving in a non-turbulent regime. A laminar flow is characterized by a flow that is largely non-mixing, follows smooth streamlines, and possesses a parabolic velocity profile.

[0052] As used herein, the term "uniform flow" is intended to refer to a flow that occurs when fluid is moving in a non-turbulent regime. A uniform flow is characterized by a flow that is largely non-mixing, follows smooth streamlines, and possesses a flat velocity profile.

[0053] As used herein, the term "sheath flow channel" or "sheath flow interface" is intended to refer to a recipient comprising an outlet or outlets of a first instrument and an inlet or inlets of a second instrument, and a fluid running in the recipient.

[0054] As used herein, the term "capillary" is intended to refer to a tube, usually of small internal and external dimensions, that carries a fluid flow. It is not used to designate a separation exclusively.

[0055] As used herein, the term "separation capillary" is intended to refer to a tube, usually of small internal and external dimensions, that carries a fluid flow, and wherein separation of a sample occurs.

[0056] Herein, the terms "first dimension capillary", "donor capillary" and "first separation capillary" are used interchangeably.

[0057] Herein, the terms "second dimension capillary", "receiver capillary" and "second separation capillary" are used interchangeably.

[0058] As used herein, the term "internal standard (IS)" is intended to refer to a substance that may be used in conjunction with a sample prior to separation. The IS may be introduced into the separation system separately from the sample or mixed with the sample. Also, the IS may be mixed with the separation solvent/background electrolyte. Such IS produces one or more peaks that can be readily distinguished from peaks of the sample components. This allows for alignment and normalization of the analytical data obtained, in particular the analytical data of the sample components.

[0059] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more.

[0060] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0061] As used herein the term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0062] The inventors have designed and constructed a system for transferring a fluid sample from one flow to another using a non-turbulent flow of a fluid running in a sheath flow interface. The system according to the invention also allows for multidimensional separation of a sample. The source of the sample / fluid sample may be from an organism, a bulk solution or a sub-sample thereof held in a container such as a vial. The present invention is illustrated in further details by the following non-limiting embodiments.

[0063] In conventional capillary electrophoresis (CE), a capillary (typical dimensions are: 30-100 cm long, 180-365 μηι outside diameter (OD) and 25-150 μηι inside diameter (ID)) is filled with an electrolyte buffer. Such buffers may be for example borate- and/or formate- based buffers. Other mobiles phases including for example a wide variety of aqueous and non-aqueous mobile phases may also be used.

[0064] Referring to the figures, FIGs. 1 -3 pertain to the prior art. In FIG. 1 , a conventional capillary electrophoresis 10 is illustrated. A short plug (approximately 1-3% of the column length; not illustrated) is introduced into the separation capillary 14 by replacing the separation buffer vial 12b with the sample vial 12a, and either applying a small positive pressure or a voltage. Thus, the short plug comprises the injected sample. The separation capillary 14 is then returned to the separation buffer vial 12b and a high voltage 11 (high electric field E) is applied across the separation capillary via the two platinum electrodes 16a, 16b immersed in the separation buffer vial 12b and the waste buffer vial 18. The insulated high voltage wires are 17a, 17b. This electric field generates an electroosmotic flow (EOF) in the separation capillary 14. An interesting aspect of the EOF is that the flow inside of the separation capillary 14 is essentially uniform. This minimizes dispersion and aids in achieving a high-efficiency separation. This flow is directed towards the detector (exit) 13a, 13b, i.e., end of the capillary, by choosing a suitable polarity. In some instances, hydrodynamic flow may be used to alter separation speed by assisting or hindering the EOF. This may be generated through siphoning or by applying a small pressure.

[0065] Charged analytes / species in the sample plug possess electrophoretic mobilities proportional to their charge and inversely proportional to their hydrodynamic size, which are often sufficiently different to permit separation of the analytes.

[0066] Referring to FIG. 2, a capillary-based separator 20 is illustrated. The sample comprises analytes A, B. At the beginning of the separation, all the analytes in the sample are mixed together at the capillary inlet 22a of the capillary 24. As the separation process progresses, the analytes A, B begin to separate due to velocity differences that arise from the summation of the flow velocities derived from the EOF, any hydrodynamic flow and the individual electrophoretic mobilities. Eventually, the analytes A, B in the sample injected onto the capillary 24 migrate past the detector 26 and exit the capillary 24 through the capillary outlet 22b. The various times indicated on the figure are as follows: t=0, sample comprising analytes A, B is injected; t=1.0, analytes start to separate; t=2.5, analytes continue to separate; t=4, analytes continue to separate, analyte B is detected; t=5, analytes continue to separate, A and B are passed detector; t=6, analyte B has left the capillary, analyte A is near the end.

[0067] FIG. 3 shows an example of a separation of fluorescently labelled serum proteins by capillary zone electrophoresis as detected when the proteins exit the capillary. Many other separation techniques that may be employed, all attempting to separate complex samples into various fractions. The technique of the invention is described in more detail below. [0068] The volume of sample injected onto the capillary is typically about 4-10 x 10^"9 I (4-10 nl_). Manipulating such small volumes without destroying the separation presents some challenges. Mechanical valves, tubing, vials, etc. may not be suitable for this manipulation.

[0069] The invention uses non-turbulent fluid flow to transfer material from one capillary to another. More specifically, in order to manipulate separated analytes (i.e. pass material from one capillary to the next) the ends of the capillaries are suspended in a flowing stream of liquid.

[0070] Typically, fluid under flow falls into three distinct flow regimes: turbulent, uniform or laminar. Turbulent flow is characterized by chaotic fluid movement and is unsuitable for transferring material from one capillary to another due to significant mixing, dispersion and loss. Uniform and laminar flow is encountered when fluids flow near stationary surfaces such as walls, capillary surfaces etc. Laminar flow is characterized by long streamlines that follow the contours of neighboring surfaces. In laminar flow, the highest velocities are encountered in the part of the stream most distant from the stationary surfaces (walls, capillary surfaces, etc.). This type of flow is generally considered to have a parabolic flow profile. In uniform flow, all streamlines possess the same velocity; this is an uncommon flow situation in pressure generated flow regimes and is most often encountered at the entrance of a channel prior to laminar flow being established. Uniform flow and laminar flow are each non-turbulent flow, and are capable of transporting analytes from one capillary to another with minimal mixing or dispersion.

[0071] Referring to FIG. 4, a sheath flow interface / sheath flow channel 40 with a donor separation capillary 44 suspended therein is illustrated. The arrows in the interface and outside the capillary 44 represent the streamlines (velocity vectors) of a fluid flowing in the interface. The influence of the walls 42a, 42b is to slow down the fluid. The capillary 44 is suspended in the flow, and due to its small surface area, has minimal impact on the overall flow in the interface. Fluid emerging from the capillary 44 through capillary exit 46 quickly accelerates to the same velocity as the surrounding flow. This results in a stream of effluent that is embedded in the surrounding flow with minimal mixing. The sample comprises analytes A, B. [0072] As will be understood by a skilled person, if the fluid flows around a corner, the separation capillary effluent will also be entrained in the flow and deviate around the corner. This is illustrated in FIG. 5. A fiber optic light source 58 is used to monitor the movement of the fluorescent fluid in the separation capillary 54 and the channel with walls 52. As can be seen, the separation capillary 54 is in the middle arm of a Tee. The fluid flows towards the left as illustrated by arrows 55, over the capillary and around the corner towards the bottom. It should be noted that no fluid is being introduced from the top arm. Also, it should be noted that the stream of fluorescent dye 59 flows around the corner with little mixing and dispersion into the neighboring fluid. This illustrates that flow of the effluent may be controlled by the geometry of the surrounding walls / structure, and that the effluent flows directly "downstream" of the capillary tip exit 56.

[0073] Depending on the relative velocities between the ensheathing flow (used in the present invention as the source of second dimension mobile phase) and the flow in the capillary / first dimension mobile flow, different diameters of flow are observed when the material exits the capillary (effluent stream). This is illustrated in FIGs. 6A-C. The area of the effluent stream is dictated by conservation of volume and the flow velocity of the ensheathing fluid. When the velocity in the capillary 64 is high (high mass flow rate) then the resulting cross-sectional area of the effluent stream 67 is larger once the fluid decelerates to the same velocity as the ensheathing fluid 65 (FIG. 6A). When both the velocity of the ensheathing flow 65 and the velocity of the effluent stream match, the area of the effluent stream matches the cross-sectional area of the capillary's inside diameter (ID) (FIG. 6B). When the flow rate of the effluent stream 67 is lower the flow "pinches" into a narrow stream (FIG. 6C).

[0074] Each of the flow regimes illustrated in FIGs. 6A-C presents some advantages and disadvantages with respect to transferring material from one capillary to another. For example if the two capillaries (donor capillary and receiver capillary) differ in ID, the effect generated by the variation of velocity between the ensheathing flow 65 and the effluent stream 67 may be used to optimize the transfer. In some instances when it is desired to insure that the maximum concentration sample is transferred into the receiver capillary, it may be advantageous to "overflow" the receiver capillary. In some instances, it may be advantageous to use a "pinched" stream to insure that the sample is enveloped by the surrounding stream to maximize interactions with agents in the ensheathing fluid. Such agents may be for example analyte labelling agents, buffers of a different pH, ion-pairing agents, organic solvents, etc. Also, in the pinched transfer, its cross-sectional area may be small enough to insure a nearly 100% transfer. FIG. 6A also shows the walls of the sheath flow interface 62.

[0075] In embodiments of the invention, transfer between two separation capillaries is achieved by placing a "donor" capillary upstream of a "receiver" capillary. This is illustrated in FIGs. 7A-C. In the example shown, the donor capillary 74a is located directly above the receiver capillary 74b. In other embodiments as shown above, flow may occur around corners and follow contours (FIG. 5).

[0076] In the example shown in FIGs. 7A-C, two analytes / species A, B are being separated in the donor capillary 74a. In this example, the goal is to transfer the first analyte, analyte A into the receiver capillary 74b. To accomplish this, the velocities of the ensheathing flow and the effluent have been matched to allow substantially nearly 100% of the effluent to be swept from the exit of the donor capillary 74a to the entrance of the receiver capillary 74b. A small percentage of the analyte may "spill-over" the tip and is ultimately lost (FIGs 7A-B). To prevent the second analyte, analyte B from being swept into the receiver capillary 74b, the donor capillary 74a is moved into a part of the stream that is not directly upstream of the receiver capillary 74b (arrow in FIG. 7C). Once the donor capillary 74a is no longer upstream, the receiver capillary 74b takes-in the ensheathing solution (second dimension mobile phase) that is flowing through the sheath flow interface / sheath flow channel 72 (FIG. 7C). FIG. 7A also shows the walls of the sheath flow interface 72.

[0077] As will be understood by a skilled person, relative positioning of the donor capillary and the receiver capillary may be accomplished by either moving the donor capillary in the ensheathing solution (FIG. 7C, arrow), or by moving the receiver capillary in the ensheathing solution (not illustrated). In embodiments of the invention, using an arrangement wherein the receiver capillary is moved in the ensheathing solution allows for the possibility of fixing a camera on the donor capillary. This facilitates alignment and observation of the transfer.

[0078] In embodiment of the invention illustrated in FIG. 8, the sheath flow interface 82 embodies a second dimension buffer outlet/waste 83. The first dimension (donor) capillary 84a is located above the second dimension (receiver) capillary 84b. As will be understood by a skilled person, many such second dimension (receiver) capillaries 84b may be used as described below. An XYZ three-axis stepper motor 81 is used as controlled positioner.

[0079] In embodiments of the invention, a plurality of receiver capillaries (second dimension capillaries) is used (FIGs. 9-14). The array of second dimension (receiver) capillaries (94b, 104b, 114b, 124b, 134b, 144b) may be manually assembled. There are small deviations between the capillaries. Adjusting the alignment is facilitated by observing when the effluent from the donor capillary "hits" the receiver capillary. The XY coordinates (currently the Z axis is manually set) are recorded and used by the control software on a computer (97, 117, 127, 137, 147) to return to the aligned position(s) for a transfer from the donor (94a, 104a, 114a, 124a, 134a, 144a) to the receiver (94b, 104b, 114b, 124b, 134b, 144b). An XYZ three-axis stepper motor 91 is used as controlled positioner.

[0080] FIG. 9 illustrates a face view of the sheath flow interface 92 showing one first dimension capillary 94a (donor capillary) and 12 second dimension capillaries 94b (receiver capillaries). The motion control software 97 allows any of the second dimension (receiver) capillaries 94b to be aligned with the first dimension (donor) capillary 94a. Under normal usage, the capillaries are aligned sequentially so that a minimum of sample is lost into the ensheathing fluid. When only a few second dimension (receiver) capillaries are used, a cyclic pattern may reasonably be used, for example 1 ,2, 3, 4...9, 10, 1 1 , 12 back to 1 ,2,3,4... . However, when many second dimension (receiver) capillaries are used (e.g. 32), then an interleaved pattern may be used, for example 1 ,3, 5, 7...31 , 32, 30...8, 6, 4, 2, 1 ,3... so that there is a uniform time in-between channels and the risk of introducing artifacts during the rapid transit of the sheath flow interface is minimized. FIG. 9 also illustrates a second dimension buffer supply 95 and a second dimension buffer out 93.

[0081] In embodiments of the invention as described above, the first dimension separation employs capillary zone electrophoresis with a low pH separation buffer as mobile phase and the second dimension separation is also a capillary zone electrophoresis separation using a high pH buffer as mobile phase. As will be understood by a skilled person, a wide variety of separation mechanisms and mobile phases may be employed in the first dimension and second dimension. In embodiments of the invention as described above the first dimension mobile phase is a formate buffer and the sheath fluid / second dimension mobile phase is a borate buffer.

[0082] FIG. 10 illustrates an embodiment of the sheath flow interface 102 according to the invention. In this embodiment, the fluid in the capillary / first dimension mobile phase is of lower density than the sheath fluid / second dimension mobile phase. This is frequently encountered with reversed phase liquid chromatography solvents such as ACN, MeOH, etc. The effluent naturally rises in the higher density aqueous sheath flow solvent, and placing the second dimension (receiver) capillaries 104b in the same direction facilitates effective transfer. It should be noted that the second dimension (receiver) capillaries 104b are located above the first dimension (donor) capillary 104a. Accordingly in this embodiment, the first dimension mobile phase may be a liquid having a density lower than the ensheathing fluid / second dimension mobile phase. Common first dimension mobile phases according to this embodiment may include for example acetonitrile, alcohols such as methanol, ethanol, etc. Second dimension mobile phases according to this embodiment may include aqueous buffers or high density solvents such as dichloromethane. FIG. 10 also illustrates a second dimension buffer supply 105 and a second dimension buffer out 103 as well as an XYZ three-axis stepper motor 101 used as controlled positioner.

[0083] In embodiments of the invention, the second dimension capillaries may be operated in a different manner. This is illustrated in FIG. 11. For example two or more sets of second dimension capillaries 114b may be provided with each set being supplied by a different buffer C, D. A first dimension capillary 114a is also illustrated. The effluent flow from the first dimension to the second dimension is constrained by the non-turbulent flow of the second dimension fluid. This same constraint also applies to the second dimension fluid itself once it enters the non-turbulent flow region. Therefore, by partitioning the source of the second dimension fluid, distinct flows of second dimension fluid may be created which will ensheath specific second dimension capillaries. This allows a variety of second dimension operational modes to operate in parallel as illustrated in FIG. 11 where 5 capillaries are ensheathed by buffer C while the remaining 5 capillaries are ensheathed by buffer D. Due to the non-turbulent flow there is little intermixing of the buffers. FIG. 11 also illustrates the sheath flow interface 112 embodying two second dimension buffer out 113a, 113b, one for each of the two second dimension buffers C, D. [0084] An overview of an embodiment of the system according to the invention is illustrated in FIG. 12. This embodiment illustrates an example using conventional capillary electrophoresis (CE) for the first dimension separation. The first dimension separation is accomplished using a commercial HP-3D (now Agilent) capillary electrophoresis separation system 123, which embodies a sample vial 1 a and separation buffer 1 b. The system has been modified to allow the capillary 124a to exit the instrument. The system has also been modified to bring-out the high voltage current return 120 which is used to ground the sheath flow interface 122 and capillary exit. The array of second dimension separation capillaries 124b is illustrated. FIG. 12 also illustrates a second dimension detector (laser) 126, a Nikon lens and Interference Filter 128, a CCD camera 129 as well as a computer control and data acquisition 127.

[0085] Not shown in FIG. 12 is a computer controlled relay in the high voltage line that allows the separation to be stop-started in order to minimize sample lost in-between injections onto the 8 second dimension (receiver) capillaries used in the sheath flow interface responsible for the data presented herein. This feature may not be needed when additional capillaries are installed, since additional capillaries may allow a higher duty cycle (number of injections/minute) that matches the first dimension separation better.

[0086] An overview of another embodiment of the system according to the invention is illustrated in FIG. 13. This embodiment is similar to the embodiment of FIG. 12 with the difference that in the embodiment of FIG. 13, the first dimension separation is accomplished using liquid chromatography. The liquid chromatography system embodies a column 2, a pump and auto-sampler 3, samples 4 and an optional detector 5. As will be understood by a skilled person, if the chromatography system uses a low density mobile phase compared to the density of the sheath fluid, an inverted sheath flow interface may be used (FIG. 10). Typical separation columns operate at relatively high flow rates (0.1-2.0 mL/min) which would produce relatively high velocities in the interface capillary if it were directly connected to the chromatograph. Accordingly, in embodiments of the invention as illustrated in FIG. 13, a flow splitter 7 is used to reduce / match the sheath flow rate and the flow originating from the chromatograph. Excess flow may be collected in a fraction collector or sent to waste 6. FIG. 13 also illustrates the sheath flow interface 132, a second dimension detector (laser) 136, a Nikon lens and Interference Filter 138, a CCD camera 139 as well as a computer control and data acquisition 137. [0087] An overview of another embodiment of the system according to the invention is illustrated in FIG. 14. This embodiment is similar to the embodiment of FIG. 12 with the difference that in the embodiment of FIG. 14, the first dimension is not functioning as a separation mechanism but rather a means of transferring material from a source into the interface. In the example illustrated in FIG. 14, the source is a micro-dialysis probe 144a that is sampling the biochemical fluid in the brain of a rat 7b and transferring the fluid towards the sheath flow interface 142 and to the array of second dimension capillaries 144b. The transfer is effected without loss and with minimal dispersion. FIG. 14 also illustrates a second dimension detector cell 146, an array of electrochemical sensors 148 and as well as a computer control and data acquisition 137.

[0088] As will be understood by a skilled person, the source of the fluid may be from a wide variety of sources such as a cell culture, a fermentation, a bulk fluid, a pipe monitoring an industrial process etc. The flow, in the first dimension capillary, may be generated electroosmotically (as shown) or may be from hydrodynamic processes (pressure driven or siphoning). In this configuration, the time course of the brain's biochemical activity may be followed. The array of second dimension separation capillaries allows periodic analyses of the brain dialysate. Advantages in this embodiment are for example the ability to analyze specific time points in the brain's activity since the transit time from the brain to the interface is controlled, and to utilize / manipulate sub-microliter samples which is critical since the volume available from a brain, for example a rat's brain, is very small (μΙ_/ηιίη).

[0089] Also as will be understood by a skilled person, our invention utilizes the phenomena of laminar flow to allow liquid, for example the effluent from a first dimension capillary (typically, inside diameter (ID) ~ 25-100 μηι, outside diameter (OD) ~ 180-365 μηι) to be swept / injected into the inlet of a second dimension capillary. An aspect of the invention relates to the fact that the position of the first dimension (donor) capillary is precisely controlled, allowing: 1- controlled injection times, and 2- injections onto an array of second dimension (receiver) capillaries.

[0090] As outlined above, laminar flow occurs when fluid is moving in a non-turbulent regime and is characterized by flow that is, largely, non-mixing and follows smooth streamlines. We have constructed a laminar flow interface cell that uses a separation buffer / mobile phase as the fluid so that all the second dimension (receiver) capillaries in the array are continuously supplied with the second dimension separation buffer / mobile phase. The flow cell / flow interface is mounted on a stepper-motor driven translation stage (FIG. 8) to control positioning. In embodiments of the invention, the first dimension (donor) capillary is positioned in the flow cell upstream from the array of second dimension (receiver) capillaries so that as the effluent emerges from the first dimension (donor) capillary it is swept directly downstream from the tip with minimal mixing / disturbance.

[0091] Under software control (FIGs. 12-14), the position of the flow cell is set so that the effluent from the first dimension (donor) capillary is swept into a second dimension (receiver) capillary. This in an "injection", and it lasts for only a short period (0.3-1 s). Then the cell is repositioned to align another second dimension (receiver) capillary so it may receive an injection. In embodiments of the invention, this injection-move-injection process is carried-out round-robin style, meaning that each second dimension capillary is reused for multiple separations.

[0092] Depending on the number of second dimension (receiver) capillaries the amount of time in-between injections on a given second dimension (receiver) capillary varies, but the time is long enough for the separation to proceed with high efficiency.

[0093] In an embodiment of the invention, we have separated a fluorescently labelled tryptic digest of a model protein, β-casein was digested, labelled using Atto-tag FQ and separated in the first dimension capillary using 13 mM formate buffer pH 3 on a 78 cm, ID = 100 μηι, OD ~ 180 μπι capillary at 30kV and ~ 3% injected volume. As the separation proceeded the effluent from the first dimension capillary was injected onto the 8 second dimension capillaries (15.4 cm, ID = 50 μηι, OD = 365 μηι) using 1s injection times. In- between each injection an idle time of ~ 13s was used because we only have 8 capillaries and want to avoid overlapping separations. Additional second dimension capillaries will address this shortcoming. The second dimension separation used 25 mM borate pH 9.16 with 5 mM SDS. This separation also employed fluorescein internal standards. This is seen on FIG. 15 as a set of three peaks common to all separations.

[0094] The data on FIG. 15 show that each second dimension capillary (separation channel in the figure) received 4 or 5 injections. Each "cluster" of peaks is separated by 112s (8 capillaries x (1 s injection + 13s idle) = 1 12s). [0095] Neighboring capillaries are offset by 14 seconds. The variation in the peaks is a result of sampling the changing composition of the first dimension separation. With appropriate software treatment, this type of data may be "unwound" and aligned to reconstruct a two-dimensional map of the proteins (FIG. 25). On one axis would be the separation due to low pH and on the other axis is due to a high pH.

[0096] As will be understood by a skilled person, by exploiting laminar flow to constrain the effluent, the invention allows dissimilar separation modes to be combined to maximize the separation power (peak capacity). Calculations show that the ideal combination of liquid chromatography (150-peak capacity) and capillary electrophoresis (50) should produce a peak capacity of 7500, which is higher than in typical multidimensional separation methods.

[0097] Also as will be understood by a skilled person, use of precise positioning allows a single first dimension separation to be interfaced to multiple second dimension separations operating in parallel. Operating in parallel improves time efficiency so that the total time is the sum of the first dimension separation (minutes to an hour) plus the second dimension separation time (minutes) while maintaining separation efficiency. In embodiments of the invention, we interfaced to 8 capillaries. As will be understood by a skilled person, 16, 32 or more capillaries may be readily interfaced. Additional second dimension capillaries will improve overall peak capacity and may provide redundancy if a few capillaries fail.

[0098] The invention allows for a micro-separation, for example a capillary electrophoresis separation, to be interfaced to another micro-separation without destroying the separation efficiency. The volumes transferred are orders of magnitude smaller than what can be manipulated by mechanical valves (about 1/1000^th). This may be advantageous for biological samples, for example when it is desired to analyse single cells or precious samples such as brain dialysate.

[0099] As will be understood by a skilled person, by exploiting laminar flow to constrain the effluent, the invention allows dissimilar processes to be combined to provide an efficient interface between the processes that is difficult to obtain using other conventional methods.

[00100] Also as will be understood by a skilled person, use of precise positioning allows the effluent from one, or more, the first dimension capillary(ies) to be interfaced to multiple second dimension capillaries operating in parallel. Each second dimension capillary may be used for a separation (as demonstrated above) or for a selective analysis such as, but not limited to, an analyte specific electrochemical measurement for dopamine (as shown in FIG. 14). The ability to manipulate small volumes in combination with operating the second dimension in parallel increases the frequency that the effluent from the first dimension may be sampled and reduces the volume required per sampling. This results in a significant increase in the resolution of time varying processes (such as brain chemistry studies) and is less likely to perturb the subject of the study due to the minimal sample volume required. As will be understood by a skilled person, 16, 32, or more, capillaries may be readily interfaced. Additional second dimension capillaries will improve overall resolution and may provide redundancy if a few capillaries or detectors fail.

[00101] As will be understood by a skilled person, features of embodiments of the invention may be combined in various ways leading to other embodiments of the invention. In this regard, the first dimension may be selected from a diverse group of modalities that can provide samples in a continuous stream or samples that have undergone a separation or other fractionation process. Examples of where a continuous supply of sample would be appropriate ^sampling) are in-vivo monitoring of brain chemistry FIG. 14 monitoring cell growth and activity such as embryo development, industrial processes such as chemical synthesis etc. In the case shown, the "first dimension" is operated as a means of transferring material while preserving the chronological / time course information. Each of the second dimension separations would provide a separation/analysis of a specific time point. For application in multidimensional separations, the first dimension separation may be from a wide diversity of separation mechanisms such as capillary electrophoresis (CE), liquid chromatography (LC), enzymatic digestors (ED), flow injection analysis (FIA), etc. The second dimension may be selected, independently from the first dimension, from a wide diversity of modalities including common ones such as capillary electrophoresis (CE), capillary electrochromatography (CEC), liquid chromatography (LC), enzymatic digestors, etc. Accordingly, embodiments of the invention may comprise combinations such as the following, non-exhaustive list: μ33ΓηρΙ^-(0Ε or LC or CEC or ED, etc.), FIA-(CE or LC or CEC or ED, etc.), CE-ED, ED-CE, CE-CEC, CE-LC, LC-CE, LC-LC.

[00102] Other embodiments and examples of the invention are outlined in FIGs. 16-25. FIG. 20 presents a conceptual diagram illustrating a separation in the first dimension (3 mg/mL of Atto-tag FQ labelled bovine serum albumin in borate buffer) being sub-sampled into the second dimension capillaries by injecting approximately 3.3 minutes of first dimension effluent onto each second dimension capillary. The illustration in FIG. 20 also shows a high voltage supply 200, a multidimensional interface 204 and a multi-channel detector 206.

[00103] As outlined above, the present invention allows for the introduction of the effluent from one capillary onto a selected second capillary by controlled positioning of the first upstream of the selected second capillary. To introduce the IS into the second dimension separation capillaries, an additional capillary was placed in the sheath flow interface close to the capillary used for the first dimension separation and continuously infused with IS. Through controlled positioning, effluent from the first dimension separation was introduced into a given second dimension capillary (i.e. sample injection). This was then followed by repositioning to permit injection of the IS onto the same capillary (secondary injection) after a delay. The delay between the sample and secondary injections is chosen to allow the IS to be present, within the capillary during the separation of the sample, yet not migrate within the separation window. This produces an IS signal that is distinct from the analytes and prevents loss of peak capacity.

[00104] FIG. 26 shows the two-dimensional separation of a fluorescently labelled tryptic digest of β-casein along with an IS. The internal standard chosen in this case was fluorescein sulfonic acid (FSA) and was injected 7.5 seconds after the sample injection. FSA, and its degradation product, produced the two prominent peaks at the end of the separation window (from 76-90s). As can be seen in the figure, there is a periodic structure in the positions of the IS peaks that repeats every eighth separation (column) since there are 8 second dimension capillaries. There is also a more gradual shift in the position of the IS peaks as the overall separation proceeds (left to right) with a trend towards later elution (upward), possibly due to temperature drift during the 1/2 hour separation.

[00105] By aligning the data so that the IS peaks appear at uniform times the remaining β-casein peaks show an improvement in alignment as shown in FIG. 27. As will be understood by a skilled person, further improvements may be obtained by injecting additional internal standards such that they migrate ahead of the analyte separation window and provide references for alignment both before and after the separation window. The aligned data may also now be processed by alignment algorithms such as correlation optimized warping which is intolerant of large mismatches between datasets, but is capable of aligning data with small mismatches.

[00106] Also as will be understood by a skilled person, additional internal standardization may be performed by mixing other internal standards into the sample and/or first dimension background electrolyte so that peaks distinct from those of the sample components and those used for the above alignment procedures are produced. These additions allow for the correction of sample-to-sample variations or injection-to-injection variations, respectively.

[00107] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[00108] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

REFERENCES

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Claims

1. System for transferring a fluid sample from one flow to another, wherein transfer of the fluid sample is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

2. System for transferring a fluid sample from one flow to another, wherein the fluid sample is fed to the system through at least one first tube and is transferred to at least one second tube using a non-turbulent flow of a fluid running in a sheath flow interface.

3. System according to claim 1 or 2, wherein the fluid sample is fed to the system from a source which is an organism, a bulk solution or a sub-sample thereof.

4. System according to claim 2, wherein an outlet of the at least one first tube and an inlet of the at least one second tube extend in the sheath flow interface, in overlooked alignment, the fluid running in a direction which is first tube towards second tube.

5. System according to claim 2, wherein the first tubes are in parallel arrangement and the second tubes are in parallel arrangement; optionally, the second tubes are arranged in a plurality of sets of tubes, each set of tubes being supplied with a different fluid.

6. System according to claim 2, wherein the tubes are in vertical arrangement in the interface, the first tubes being positioned above the second tubes.

7. System according to claim 2, wherein the tubes are in vertical arrangement in the interface, the first tubes being positioned below the second tubes.

8. System according to claim 2, wherein the at least one first tube and the at least one second tube are capillaries.

9. System according to claim 2, wherein the at least one first tube and the at least one second tube each independently comprises one or more of: capillary electrophoresis (CE), liquid chromatography (LC), capillary electrochromatography (CEC), means for effecting a chemical modification such as enzymatic digestor (ED), flow injection analysis (FIA) etc.

10. System according to claim 1 or 2, wherein the non-turbulent flow is selected from laminar flow and uniform flow.

11. System according to claim 2, wherein the interface comprises an inlet for introducing the fluid and an outlet for removing the fluid.

12. System according to claim 2, wherein the direction of the running fluid comprises one or more contours.

13. System according to claim 2, wherein the fluid running in the interface comprises at least one agent selected from analyte labeling agents.

14. System according to claim 2, further comprising at least one motor for moving the tubes for sequentially positioning an inlet of one of the second tubes in overlooked alignment with an outlet of one of the first tubes.

15. System according to claim 2, further comprising at least one of: sensors, detectors, cameras and computer programs.

16. System according to claim 1 , wherein at least one internal standard (IS) is introduced into said one flow and/or said other flow.

17. System according to claim 2, wherein at least one internal standard (IS) is introduced into said at least one first tube and/or said at least one second tube before and/or after the transfer of the fluid sample.

18. Method for transferring a fluid sample from one flow to another, comprising using a system as defined in any one of claims 1-17.

19. Method for transferring a fluid sample from one flow to another, comprising: feeding the fluid sample to at least one first tube; and transferring the fluid sample to at least one second tube, wherein the transfer of the fluid sample is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

20. Method for transferring a fluid sample from one flow to another, comprising:

21. Method according to claim 19 or 20, wherein at least one internal standard (IS) is introduced into said at least one first tube and/or said at least one second tube before and/or after the transfer of the fluid sample.

22. Method according to claim 19 or 20, wherein the source of the fluid sample is an organism, a bulk solution or a sub-sample thereof.

23. System for multidimensional separation of a sample, comprising at least two separation instruments, wherein transfer of the sample between the instruments is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

24. System for multidimensional separation of a sample, comprising first and second separation instruments each comprising at least one separation capillary, wherein a separated sample exiting an outlet of the at least one separation capillary of the first separation instrument is transferred to the second instrument through an inlet of the at least one separation capillary thereof, and wherein the transfer is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

25. System for multidimensional separation of a sample, comprising: a sheath flow interface; a first separation instrument comprising a separation capillary having an outlet extending in the interface; and a second separation instrument comprising at least one separation capillary each having an inlet extending in the interface and in overlooked alignment with the outlet of the capillary of the first instrument, wherein a separated sample exiting the outlet of the capillary of the first instrument is transferred to the inlet of one of the at least one capillary of the second instrument, the transfer being effected using a non-turbulent flow of a fluid running in the interface, the fluid running in a direction which is first instrument towards second instrument.

26. System according to claim 24 or 25, wherein the capillaries of the first and second instruments are in vertically arrangement in the interface, the capillaries of the first instrument being positioned above the capillaries of the second instrument.

27. System according to claim 24 or 25, wherein the capillaries of the first and second instruments are in vertically arrangement in the interface, the capillaries of the first instrument being positioned below the capillaries of the second instrument.

28. System according to claim 24 or 25, wherein the capillaries of the first instrument are in parallel arrangement and the capillaries of the second instrument are in parallel arrangement; optionally, the capillaries of the second instrument are arranged in a plurality of sets of capillaries, each set of capillaries being supplied with a different fluid.

29. System according to claim 24 or 25, further comprising at least one motor for moving the capillaries for sequentially positioning an outlet of one capillary of the second instrument in overlooked alignment with an inlet of one capillary of the first instrument.

30. System according to claim 24 or 25, wherein the sample comprises analytes, a first dimension separation of the analytes being performed in the first instrument using a first dimension separation fluid, and a second dimension separation being performed in the second instrument using a second dimension separation fluid which is the fluid running in the interface.

31. System according to claim 30, wherein the first dimension separation fluid is selected from buffer, acetonitrile and alcohol; and the second dimension separation fluid is a buffer.

32. System according to claim 24 or 25, wherein the first and second instruments each independently comprises one or more of: capillary electrophoresis (CE), liquid chromatography (LC), capillary electrochromatography (CEC), means for effecting a chemical modification such as enzymatic digestor (ED), flow injection analysis (FIA) etc.

33. System according to any one of claims 23-25, wherein the non-turbulent flow is selected from laminar flow and uniform flow.

34. System according to claim 24 or 25, wherein the interface comprises an inlet for introducing the fluid and an outlet for removing the fluid.

35. System according to claim 24 or 25, wherein the direction of the running fluid comprises one or more contours.

36. System according to claim 24 or 25, wherein the fluid running in the interface comprises at least one agent selected from analyte labeling agents.

37. System according to claim 24 or 25, wherein at least one internal standard (IS) is introduced into said at least one separation capillary of said first separation instrument and/or said at least one separation capillary of said second separation instrument before and/or after the transfer of the fluid sample.

38. System according to claim 24 or 25, further comprising at least one of: sensors, detectors, cameras and computer programs.

39. Method for multidimensional separation of a sample, comprising using a system as defined in any one of claims 23-38.

40. Method for multidimensional separation of a sample, comprising performing sequential separations on at least one first separation instrument and at least one second separation instrument, each instrument comprising at least one capillary, wherein transferred of the sample between the at least one first instrument and the at least one second instrument is effected using a non-turbulent flow of a fluid running in a sheath flow interface.

41. Method for multidimensional separation of a sample, comprising:

(a) providing a sheath flow interface, a first separation instrument comprising a separation capillary having an outlet extending in the interface, and a second separation instrument comprising at least one separation capillary each having an inlet extending in the interface and in alignment with the outlet of the capillary of the first instrument; and (b) running a non-turbulent flow of a fluid in the interface, in a direction which is first instrument towards second instrument; and

42. Method according to claim 41 , wherein at least one internal standard (IS) is introduced into said at least one separation capillary of said first separation instrument and/or said at least one separation capillary of said second separation instrument before and/or after the transfer of the fluid sample.

43. System according to any one of claims 23-38 or method according to any one of claims 39-42, wherein the sample is a biological sample.

44. System according to any one of claims 23-38 or method according to any one of claims 39-42, wherein the sample is in sub-microliter amount.

45. System according to any one of claims 1-15, 23-38, 43 and 44 or method according to any one of claims 18-22 and 39-42, wherein the fluid sample comprises at least one internal standard.

46. System according to any one of claims 16, 17 and 37 or method according to claim 21 or 42, wherein the introduction of the at least one internal standard after the transfer of the fluid sample is performed following a delay.

47. System according to any one of claims 16, 17 and 37 or method according to claim 21 or 42, wherein the transfer of the fluid sample after the introduction of the at least one internal standard is performed following a delay.

48. System according to any one of claims 16, 17 and 37 or method according to claim 21 or 42, wherein the at least one internal standard comprises components that are different from components of the fluid sample and/or the at least one internal standard provides signals that are distinct from signals of components of the fluid sample and/or the at least one internal standard comprises components having electrophoretic mobilities that are different from electrophoretic mobilities of components of the fluid sample.

49. System according to any one of claims 16, 17 and 37 or method according to claim 21 or 42, wherein the at least one internal standard is fluorescein sulfonic acid (FSA).

50. System according to any one of claims 16, 17 and 37 or method according to claim 21 or 42, wherein a duration of the delay is such that the internal standard signals arrive at the detector separated from signals of components of the fluid sample

51. System according to claim 50, wherein the duration of the delay is between about 5 seconds to about 10 seconds, preferably about 7.5 seconds.