Patent Publication Number: US-2003223913-A1

Title: Microfluidic separation devices and methods

Description:
STATEMENT OF RELATED APPLICATION(S)  
       [0001] This application claims benefit of U.S. patent application Ser. No. 10/161,415 filed Jun. 2, 2002 and currently pending.  
       FIELD OF THE INVENTION  
       [0002] The present invention relates to the design and fabrication of microfluidic devices and systems employing the same.  
       BACKGROUND OF THE INVENTION  
       [0003] Chemical and biological separations are routinely performed in various industrial and academic settings to determine the presence and/or quantity of individual species in complex sample mixtures. There exist various techniques for performing such separations.  
       [0004] One separation technique, chromatography, encompasses a number of methods that are used for separating closely related components of mixtures. In fact, chromatography has many applications including separation, identification, purification, and quantification of compounds within various mixtures. Chromatography is a physical method of separation involving a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid, or a supercritical fluid). While carrying the sample, the mobile phase is then forced (e.g., by gravity, by applying pressure, or by applying an electric field) through a separation ‘column’ containing an immobile, immiscible stationary phase. In column chromatography, the stationary phase refers to a coating on a solid support that is typically contained within a tube or other boundary. The mobile phase and stationary phase are chosen such that components of the sample have differing solubilities in each phase. A component that is quite soluble in the stationary phase will take longer to travel through it than a component that is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components become separated from one another as they travel through the stationary phase.  
       [0005] One category of conventional chromatography systems includes pressure-driven systems. These systems are operated by supplying a pressurized mobile phase (typically one or more liquid solvents pressurized with a pump) to a separation column. Standard liquid chromatography columns have dimensions of several (e.g., 10, 15, 25) centimeters in length and between 3-5 millimeters in diameter, with capillary columns typically having internal diameters between 3-200 microns. Columns are typically packed with very small diameter (e.g., 5 or 10 micron) particles. Various types of stationary phase material are commercially available. Some of the more common examples include Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase, Ion Exchange, and Affinity.  
       [0006] It is important to minimize any voids in a packed column, since voids or other irregularities in a separation system can destroy an otherwise good separation. As a result, most conventional separation columns include specially designed end fittings (typically having compressible ferrule regions) designed to hold packed stationary phase material in place and prevent irregular flow-through regions.  
       [0007] As illustrated in FIG. 1, a separation column for use in a conventional pressure-driven chromatography system is typically fabricated by packing particulate material  14  into a tubular column body  12 . A conventional column body  12  has a high precision internal bore  13  and is manufactured typically with stainless steel, although materials such as glass, fused silica, and/or PEEK are also occasionally used. Various methods for packing a column body may be employed. In one example, a simple packing method involves dry-packing an empty tube by shaking particles downward with the aid of vibration from a sonicator bath or an engraving tool. A cut-back pipette tip may be used as a particulate reservoir at the top (second end), and the tube to be packed is plugged with parafilm or a tube cap at the bottom (first end). Following dry packing, the plug is removed and the tube  10  is then secured at the first end with a ferrule  16 A, a fine porous stainless steel fritted filter disc (or “frit”)  18 , a male end fitting  20 A, and a female nut  22 A that engages the end fitting  20 A. Corresponding connectors (namely, a ferrule  16 B, a male end fitting  20 B, and a female nut  22 B) except for the frit  18  are engaged to the second end to secure the dry-packed tube  12 . The contents  14  of the tube  12  may be further compressed by flowing pressurized solvent through the packing material  14  from the second end toward the first (frit-containing) end. When compacting of the particle bed has ceased and the fluid pressure has stabilized, there typically remains some portion of the tube  13  that does not contain densely packed particulate material. To eliminate the presence of a void in the column  10 , the tube  13  is typically cut down to the bed surface (or a shorter desired length) to ensure that the resulting length of the entire tube  12  contains packed particulate  14 , and the unpacked tube section is discarded. Thereafter, the column  10  is reassembled (i.e., with the ferrule  16 B, male end fitting  20 B, and female nut  22 B affixed to the second end) before use.  
       [0008] A conventional pressure-driven liquid chromatography system utilizing a column  10  is illustrated in FIG. 2. The system  30  includes a solvent reservoir  32 , a high pressure pump  34 , a pulse damper  36 , a sample injection valve  38 , and a sample source  40  all located upstream of the column  10 , and further includes a detector  42  and a waste reservoir  44  located downstream of the column  10 . The high pressure pump  34  pumps mobile phase solvent from the reservoir  32 . A pulse damper  36  serves to reduce pressure pulses generated by the pump  34 . The sample injection valve  38  is typically a rotary valve having an internal sample loop for injecting a predetermined volume of sample from the sample source  40  into the solvent stream. Downstream of the sample injection valve  38 , the column  10  contains stationary phase material that aids in separating species of the sample. Downstream of the column  10  is a detector  42  for detecting the separated species, and a waste reservoir  44  for ultimately collecting the mobile phase and sample products. A back pressure regulator (not shown) may be disposed between the column  10  and the detector  42 .  
       [0009] The system  30  generally permits one sample to be separated at a time in the column  10 . Due to its cost, a column  10  is often re-used for several separations (e.g., typically about 100 times). Following one separation, the column  10  may be flushed with a pressurized solvent stream in an attempt to remove any sample components still contained in the stationary phase material  14 . However, this time-consuming flushing or cleaning step rarely yields a completely clean column  10 . This means that, after the first separation performed on a particular column, every subsequent separation may potentially include false results due to contaminants left behind on the column from a previous run. Eventually, columns become fouled to the point that they are no longer useful, at which point they are generally discarded.  
       [0010] From the foregoing description, it is clear that conventional pressure-driven separation columns include numerous components and require numerous manufacturing steps. It would be desirable to reduce the number of parts required to fabricate separation columns, and to simplify their manufacture. It would also be desirable to reduce the cost of a separation column to permit the column to be disposed after a single use, thus eliminating potentially false results and time-consuming cleaning steps. It would be further desirable to provide high-throughput separation systems capable of separating multiple samples using a minimum number of expensive system components (e.g., pumps, pulse dampers, detectors, etc.). Additionally, in an improved separation device, it would be desirable to provide separation media capable of promoting high-quality and repeatable separation results.  
       [0011] Another separation technique utilizes an electric field applied across a column. These systems utilize a separation technique called electrophoresis, which is based on the mobility of ions in an electric field. Upon application of an electric field across a column containing an electrophoretic medium, components of the sample migrate at different rates toward the oppositely charged ends of the column based on their relative electrophoretic mobilities in the medium. Electrochromatography is a combination of chromatography and electrophoresis, in which the mobile phase is transported through the separation system by electroosmotic flow.  
       [0012] Separation systems relying on electric fields are complicated and require integral electrical contacts. Additionally, these systems only function with charged fluids or fluids containing electrolytes. Finally, these systems require voltages that are sufficiently high to cause electrolysis of water, thus forming bubbles that complicate the collection of samples without destroying them. In light of these limitations, there exists a need for devices and systems capable of providing separation utility without utilizing electrical currents.  
       SUMMARY OF THE INVENTION  
       [0013] In a first separate aspect of the invention, a pressure-driven microfluidic separation device includes a microfluidic separation channel having an upstream portion and a downstream portion, both portions containing stationary phase material. A sample loading segment having a sample inlet port is in fluid communication with the separation channel at a sample loading junction. A first mobile phase solvent inlet is in fluid communication with the upstream portion upstream of the sample loading junction. A second mobile phase solvent inlet is in fluid communication with the sample loading segment upstream of the sample inlet port.  
       [0014] In a second separate aspect of the invention, a pressure-driven microfluidic separation device includes multiple microfluidic separation channels. Each separation channel has an associated sample loading junction, which demarcates a transition between an upstream portion and a downstream portion of the associated microfluidic separation channel. Multiple sample loading segments are provided, with each sample loading segment being in fluid communication with a separation channel at the associated sample loading junction. Each sample loading segment has a sample inlet port adapted to receive a liquid sample. A first mobile phase solvent inlet port is in fluid communication with each upstream portion via a first channel network. A second mobile phase solvent inlet port is in fluid communication with each sample loading segment via a second channel network.  
       [0015] In another separate aspect of the invention, a method for operating a pressure-driven microfluidic separation device includes several steps. A first step includes providing a microfluidic separation channel containing a stationary phase material, the separation channel having a sample loading region positioned between an upstream end and a downstream end of the channel. A second step includes providing a sample loading segment in fluid communication with the separation channel at a sample loading region. A third step includes introducing a sample into the sample loading segment. A fourth step includes supplying a first flow of mobile phase solvent to the separation channel upstream of the sample loading region.  
       [0016] In another separate aspect of the invention, any of the foregoing aspects may be combined for additional advantage. These and other aspects and advantages of the invention will be apparent to the skilled artisan upon review of the following detailed description, drawings, and claims. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0017]FIG. 1 is a cross-sectional view of a conventional packed chromatography column.  
     [0018]FIG. 2 is a schematic showing various components of a conventional liquid chromatography system employing the packed chromatography column of FIG. 1.  
     [0019]FIG. 3A is top view of a portion of a first microfluidic separation device having a separation channel and a sample loading segment. FIG. 3A is a top view of a second microfluidic separation device having a separation channel and a sample loading bypass segment.  
     [0020]FIG. 4 is a schematic showing various components of a microfluidic chromatography system according to the present invention.  
     [0021]FIG. 5A is an exploded perspective view of a pressure-driven microfluidic separation device having eight separation channels, each separation channel having an associated sample input segment and a non-bypass sample loading segment. FIG. 5B is a top view of the assembled device of FIG. 5A.  
     [0022]FIG. 6A is an exploded perspective view of a pressure-driven microfluidic separation device having eight separation channels, each separation channel having an associated bypass sample input segment. FIG. 6B is a top view of the assembled device of FIG. 6A. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION  
     [0023] Definitions  
     [0024] The terms “channel” or “chamber” as used herein is to be interpreted in a broad sense. Thus, such terms are is not intended to be restricted to elongated configurations where the transverse or longitudinal dimension greatly exceeds the diameter or cross-sectional dimension. Rather, such terms are meant to comprise cavities or tunnels of any desired shape or configuration through which liquids may be directed. Such a fluid cavity may, for example, comprise a flow-through cell where fluid is to be continually passed or, alternatively, a chamber for holding a specified, discrete amount of fluid for a specified amount of time. “Channels” and “chambers” may be filled or may contain internal structures comprising, for example, valves, filters, stationary phase media, and similar or equivalent components and materials.  
     [0025] The term “microfluidic” as used herein refers to structures or devices through which one or more fluids are capable of being passed or directed, and which have at least one dimension less than about 500 microns.  
     [0026] The term “separation channel” is used substantially interchangeably with the term “column” herein and refers to a region of a fluidic device containing stationary phase material adapted to separate species of a fluid sample  
     [0027] The term “substantially sealed” as used herein refers to a microstructure having a sufficiently low unintended leakage rate and/or volume under given flow, fluid identity, and pressure conditions. A substantially sealed device may include one or more inlet ports and/or outlet ports.  
     [0028] The term “stencil” as used herein refers to a material layer or sheet that is preferably substantially planar through which one or more variously shaped and oriented portions have been cut or otherwise removed through the entire thickness of the layer, and that permits substantial fluid movement within the layer (e.g., in the form of channels or chambers, as opposed to simple through-holes for transmitting fluid through one layer to another layer). The outlines of the cut or otherwise removed portions form the lateral boundaries of microstructures that are formed when a stencil is sandwiched between other layers such as substrates or other stencils.  
     [0029] The term “column” as used herein refers to a region of a fluidic device containing stationary phase material, typically including packed particulate matter.  
     [0030] The term “slurry” as used herein refers to a mixture of particulate matter and a solvent, preferably a suspension of particles in a solvent.  
     [0031] Microfluidic Devices Generally  
     [0032] Devices according to the present invention are preferably microfluidic devices defining internal channels or other microstructures having at least one dimension smaller than about 500 microns. In an especially preferred embodiment, microfluidic devices according to the present invention are constructed using stencil layers or sheets to define channels and/or chambers. As noted previously, a stencil layer is preferably substantially planar and has a channel or chamber cut through the entire thickness of the layer to permit substantial fluid movement within the stencil layer. Various means may be used to define such channels or chambers in stencil layers. For example, a computer-controlled plotter modified to accept a cutting blade may be used to cut various patterns through a material layer. Such a blade may be used either to cut sections to be detached and removed from the stencil layer, or to fashion slits that separate regions in the stencil layer without removing any material. Alternatively, a computer-controlled laser cutter may be used to cut portions through a material layer. While laser cutting may be used to yield precisely-dimensioned microstructures, the use of a laser to cut a stencil layer inherently involves the removal of some material. Further examples of methods that may be employed to form stencil layers include conventional stamping or die-cutting technologies, including rotary cutters and other high throughput auto-aligning equipment (sometimes referred to as converters). The above-mentioned methods for cutting through a stencil layer or sheet permit robust devices to be fabricated quickly and inexpensively compared to conventional surface micromachining or material deposition techniques that are conventionally employed to produce microfluidic devices.  
     [0033] After a portion of a stencil layer is cut or removed, the outlines of the cut or otherwise removed portions form the lateral boundaries of microstructures that are completed upon sandwiching a stencil between substrates and/or other stencils. The thickness or height of the microstructures such as channels or chambers can be varied by altering the thickness of the stencil layer, or by using multiple substantially identical stencil layers stacked on top of one another. When assembled in a microfluidic device, the top and bottom surfaces of stencil layers are intended to mate with one or more adjacent layers (such as stencil layers or substrate layers) to form a substantially enclosed device, typically having at least one inlet port and at least one outlet port.  
     [0034] A wide variety of materials may be used to fabricate microfluidic devices having sandwiched stencil layers, including polymeric, metallic, and/or composite materials, to name a few. In certain embodiments, particularly preferable materials include those that are substantially optically transmissive to permit viewing and/or electromagnetic analyses of fluid contents within a microfluidic device. Various preferred embodiments may utilize porous materials, including filter materials, for device layers. Substrates and stencils may be substantially rigid or flexible. Selection of particular materials for a desired application depends on numerous factors including: the types, concentrations, and residence times of substances (e.g., solvents, reactants, and products) present in regions of a device; temperature; pressure; pH; presence or absence of gases; and optical properties.  
     [0035] Various means may be used to seal or bond layers of a device together, preferably to construct a substantially sealed structure. For example, adhesives may be used. In one embodiment, one or more layers of a device may be fabricated from single- or double-sided adhesive tape, although other methods of adhering stencil layers may be used. A portion of the tape (of the desired shape and dimensions) can be cut and removed to form channels, chambers, and/or apertures. A tape stencil can then be placed on a supporting substrate with an appropriate cover layer, between layers of tape, or between layers of other materials. In one embodiment, stencil layers can be stacked on each other. In this embodiment, the thickness or height of the channels within a particular stencil layer can be varied by varying the thickness of the stencil layer (e.g., the tape carrier and the adhesive material thereon) or by using multiple substantially identical stencil layers stacked on top of one another. Various types of tape may be used with such an embodiment. Suitable tape carrier materials include but are not limited to polyesters, polycarbonates, polytetrafluoroethlyenes, polypropylenes, and polyimides. Such tapes may have various methods of curing, including curing by pressure, temperature, or chemical or optical interaction. The thicknesses of these carrier materials and adhesives may be varied.  
     [0036] In another embodiment, device layers may be directly bonded without using adhesives to provide high bond strength (which is especially desirable for high-pressure applications) and eliminate potential compatibility problems between such adhesives and solvents and/or samples. Specific examples of methods for directly bonding layers of polyolefin (e.g., non-biaxially-oriented polypropylene) materials to form stencil-based microfluidic structures are disclosed in two co-pending U.S. provisional patent applications, No. 60/338,286 (filed Dec. 6, 2001) and No. 60/393,953 (filed Jul. 2, 2002), both of which are hereby incorporated by reference as if set forth fully herein. In one embodiment, multiple layers of 7.5-mil (188 micron) thickness “Clear Tear Seal” polypropylene (American Profol, Cedar Rapids, Iowa) including at least one stencil layer may be stacked together, placed between flat glass platens and compressed to apply a pressure of 0.26 psi (1.79 kPa) to the layered stack, and then heated in an industrial oven for a period of approximately 5 hours at a temperature of 154° C. to yield a permanently bonded microstructure well-suited for use with high-pressure column packing methods. One thin layer of thermally conductive material (e.g., carbon steel) may be optionally inserted along the inside face of each glass platen to contact the outermost device layers using the same process to promote more even heating.  
     [0037] Notably, stencil-based fabrication methods enable very rapid fabrication of devices, both for prototyping and for high-volume production. Rapid prototyping is invaluable for trying and optimizing new device designs, since designs may be quickly implemented, tested, and (if necessary) modified and further tested to achieve a desired result. The ability to prototype devices quickly with stencil fabrication methods also permits many different variants of a particular design to be tested and evaluated concurrently.  
     [0038] Further embodiments may be fabricated from various materials using well-known techniques such as embossing, stamping, molding, and soft lithography.  
     [0039] In addition to the use of adhesives and the adhesiveless bonding method discussed above, other techniques may be used to attach one or more of the various layers of microfluidic devices useful with the present invention, as would be recognized by one of ordinary skill in attaching materials. For example, attachment techniques including thermal, chemical, or light-activated bonding steps; mechanical attachment (such as using clamps or screws to apply pressure to the layers); and/or other equivalent coupling methods may be used.  
     [0040] Pressure-Driven Microfluidic Separation Utilizing On-Column Injection  
     [0041] Performing liquid chromatography in microfluidic volumes provides significant cost savings by reducing column packing materials, analytical and biological reagents, solvents, and waste. Microfluidic separation devices may also be made to be disposable, thus eliminating possible contamination of samples due to re-use of separation columns and eliminating the need to flush columns between separations. Embodiments fabricated with sandwiched stencil layers provide additional advantages, such as rapid and inexpensive prototyping and production, and the ability to use a wide range of materials portions of a device. Additionally, microfluidic devices are well-suited for performing multiple operations in parallel, thus permitting substantial increases in throughput (namely, the number of separations that can be performed within a particular period) to be obtained.  
     [0042] As described in co-pending U.S. patent application Ser. No. 10/161,415 (filed Jun. 3, 2002, commonly assigned to the same assignee as the present application, and incorporated by reference as if set forth fully herein), preferred microfluidic separation devices utilize provide on-column, rather than pre-column, injection of samples onto one or more microfluidic separation columns. In other words, rather than being injected upstream of a separation channel, a sample is injected onto a microfluidic separation channel between an upstream end and a downstream end. On-column injection prevents a sample from ever encountering potential irregularities and manufacturing imperfections that might be found at the upstream end of a separation channel containing a stationary phase material. It is beneficial to avoid sample flow through a poorly packed region to promote high-quality separation, since the quality of separation in chromatography depends heavily on the size of the injection plug, with a small and well-defined plug generally providing better results. Stationary phase regions that are not uniformly packed tend to cause undesirable “smearing” of sample plugs. Thus, as compared to devices employing pre-column sample injection, microfluidic separation columns utilizing on-column injection permit the time-consuming and wasteful step of trimming (or otherwise eliminating) a poorly packed upstream end of a separation channel to be avoided.  
     [0043] One method for fabricating microfluidic separation devices developed by the assignee of the present application includes the steps of flowing slurry at high pressure through a slurry inlet port into a microfluidic separation channel, and then retaining particulate material from the slurry using a porous region disposed at the downstream end of the separation channel. A sample injection region disposed between the upstream end and the downstream end of the separation channel includes an associated porous region to prevent the introduction of particulate material. When such a device is in use, both mobile phase solvent and sample may be supplied through the sample injection region to the separation channel. After the separation channel is packed with particulate material, the slurry inlet port is sealed, such as by using a mechanical plug, using epoxy, or heat-sealing. The effect of sealing the slurry inlet port is to provide a very high upstream impedance to fluid flow. Thus, despite the high impedance provided by the stationary phase material downstream of the sample injection region, when a sample is supplied to the separation channel, it will tend to flow in the desired direction toward the downstream end since the downstream direction provides less impedance to fluid flow than the upstream direction.  
     [0044] A potential drawback of employing on-column injection in a device as just described, however, is that during operation the addition of pressurized fluid to the stationary phase material tends to cause any particles unrestrained by a porous material or frit to move, thus permitting the stationary phase material within the separation channel to become “unpacked.” For example, referring to FIG. 3A, a microfluidic device  50  includes a separation channel  52  and a sample loading segment  55  having a sample inlet port  56 . The sample loading segment  55  is in fluid communication with the separation channel  52  at a junction  58  that preferably includes a porous membrane (not shown). The junction  58  is disposed between an upstream portion  52 A and a downstream portion  52 B of the separation channel  52 , with both the upstream portion  52 A and the downstream portion  52 B containing a packed stationary phase material  54 . If it is assumed that the upstream portion  52 A includes a sealed upstream end (not shown) and that both mobile phase and sample are supplied to the separation channel  52  by way of the sample loading segment  55 , then such conditions may give rise to problems with maintaining the stationary phase material  54  uniformly packed near the junction  58 .  
     [0045] As noted previously, after the separation channel  52  is packed with a slurry, it is extremely difficult to achieve uniform and complete packing near the slurry inlet or upstream end (not shown) of the separation channel  52 . Due to the method of chip fabrication, the upstream end of the separation channel  52 A lacks a porous material intended to retain packed stationary phase material. Upon sealing the slurry inlet to impede fluid flow in the upstream direction in the upstream portion  52 A of the separation channel  52 , it is believed that a small pocket of air typically becomes trapped within the upstream portion  52 A. When pressurized fluid is supplied to the separation channel  52  by way of the sample loading channel  55  and junction  58 , this pressure may be sufficient to push the stationary phase material  54  disposed in the upstream portion  52 A in an upstream direction, away from the junction  58  and toward the upstream end. Because the air pocket disposed at the upstream end is compressible, this permits some upstream migration of the stationary phase material  54  contained in the upstream portion  52 A, thus reducing the local packing density of the stationary phase material  54 . The problem is exacerbated upon release of the fluid pressure within the sample loading channel  55  and separation channel  52 , since the air pocket compressed near the upstream end tends to expand to an equilibrium state, thus pushing the stationary phase material  54  contained in the upstream portion  52 A in a downstream direction, toward the junction  58 . What results is slight but observable back-and-forth movement of this stationary phase material  54  for each pressure cycle experienced by the separation channel  52  that serves to reduce the packing density of (or “unpack”) the stationary phase material  54  contained in the upstream portion  52 A. As discussed previously, this condition tends to interfere with obtaining high-quality separations, since stationary phase regions that are not uniformly packed tend to cause undesirable “smearing” or broadening of sample plugs. Such a condition may make it difficult to ensure that the entire volume of a discrete sample plug will flow from a sample loading segment  55  and into the downstream portion  52 B of the separation channel without migrating into a region of lower packing density along the junction  58 .  
     [0046] Improved Microfluidic Separation Devices And Methods  
     [0047] To avoid undesirable packing density variations in a microfluidic separation device employing on-column sample injection by way of a sample loading segment, embodiments of the present invention permit pressurized mobile phase (solvent) to be communicated simultaneously to both the upstream portion of a separation channel and to a sample loading segment. For example, referring to FIG. 3A, the upstream end of a microfluidic separation channel  52  may be left unsealed, and when operating the device  50 , mobile phase may be supplied simultaneously to both the upstream portion  52 A (e.g., through upstream end of the separation channel  52 ) and the sample loading channel  55  upstream of the sample inlet port  56 . Preferably, a first mobile phase inlet (not shown) in fluid communication with the upstream portion  52 A, and a second mobile phase inlet (not shown) disposed upstream of the sample inlet port  56 , are provided. The terms “first” and “second” in this context refer to the inlets, not to any particular solvent. The first mobile phase inlet and the second mobile phase inlet preferably receive the same solvent or solvent mixture (i.e., mobile phase). In one embodiment, the first mobile phase inlet and the second mobile phase inlet each comprise an inlet port. Distinct mobile phase inlet ports may be used, or the first and second mobile phase inlet ports may comprise a single common inlet port. A first mobile phase inlet and a second mobile phase inlet may or may not be in fluid communication with one another within the microfluidic device. Preferably, the sample inlet port  56  is selectively sealed, such as by using a removable mechanical seal (not shown), to allow a sample to be loaded into the sample inlet port  56  when the mechanical seal is removed, and to permit pressure-driven separation to commence when the mechanical seal is applied (i.e., by retaining mobile phase pressure within the sample loading segment  55  and separation channel  52 ).  
     [0048] In another embodiment, a sample loading segment may comprise a bypass segment in fluid communication with a separation channel at more than one location. For example, referring to FIG. 3B, a microfluidic device  60  includes a microfluidic separation channel  62  having an upstream portion  62 A and a downstream portion  62 B, both portions  62 A,  62 B containing packed stationary phase material  64 . The terms “upstream” and “downstream” as applied to portions  62 A,  62 B of the separation channel  62  indicate positions relative to the sample loading junction  68 , with the boundary between the upstream portion  62 A and the downstream portion  62 B being defined at this junction  68 . A sample loading segment  65  is in fluid communication with the separation channel  62  at both a solvent splitting junction  63  and the sample loading junction  68 , such that the sample loading segment  65  comprises a bypass segment. The solvent splitting junction  63  serves as a mobile phase inlet to the sample loading channel  65 , with a portion of the mobile phase proceeding through the separation channel  62  and a portion of the mobile phase being diverted into the sample loading segment  65 . A porous region (not shown) preferably disposed at each junction  63 ,  68  to prevent the passage of stationary phase material  64  into the sample loading segment  65 . The sample loading segment  65  is in further communication with two sample ports  66 ,  67 . The use of multiple sample ports  66 ,  67  helps facilitate the injection of a small but repeatable volume of sample, since upon injection through the upstream port  66 , the sample will flow into the sample loading segment  65  toward the downstream port  67  to define a sample plug in the portion of the loading segment  65  between the two ports  66 ,  67 , with the excess flowing out of the downstream port  67 . A mechanical seal (not shown) is preferably associated with the two sample ports  66 ,  67 . After loading the sample plug, the mechanical seal is closed to disallow further flow through the ports  66 ,  67 , and then mobile phase flow may be re-established to push the sample plug through the sample loading segment  65 , through the sample loading junction  68 , and into the separation channel  62  to be eluted in the downstream portion  62 B.  
     [0049] In both of the foregoing microfluidic separation devices  50 ,  60 , it is believed that providing pressurized mobile phase simultaneously to both the (upstream portion of the) separation channel and associated sample loading segment prevents stationary phase material from unpacking within the separation channel, and ensures that sample plugs flow exclusively from the sample loading channel toward the downstream portion of the separation channel.  
     [0050] In another embodiment, a microfluidic separation device permitting simultaneous communication of pressurized mobile phase to both the upstream portion of a separation channel and to a sample loading segment may be included in a separation system. For example, FIG. 4 provides a schematic illustrating various components of a separation system  70  adapted to separate species of a sample using a technique such as liquid chromatography with a pressure-driven microfluidic separation device  80  permitting on-column sample injection. A solvent reservoir  72  contains mobile phase solvent. While a single reservoir  72  is shown, multiple reservoirs  72  may be provided to perform gradient separation. A solvent pump  74  pressurizes mobile phase solvent supplied from the reservoir  72 . If additional solvent reservoirs  72  are provided, then preferably one or more additional pump  74  are also provided. IN an alternative embodiment, the pump(s)  74  may be replaced by a pressure source such as pressurized gas supplied directly to the solvent reservoir(s)  72  to motivate a flow of mobile phase solvent through the microfluidic separation device  80 . One or more pulse dampers  76  are preferably provided to reduce pressure pulses generated by the solvent pump(s)  72 . The microfluidic separation device  80  includes a separation channel  82  having an upstream portion  82 A and a downstream portion  82 B, with both portions  82 A,  82 B containing stationary phase material  84 . Preferably, the stationary phase material includes packed particulate matter, such as may be supplied to the device in slurry form through a mobile phase inlet  81 . The upstream portion  82 A includes a trailing edge  84 A of stationary phase material  84  adjacent to the mobile phase inlet  81 . A sample loading segment  85  includes a sample inlet port  86  (in fluid communication with an external sample source  78 ) and another mobile phase inlet  83  disposed upstream of the sample inlet port  86 . The sample loading segment  85  is in fluid communication with the separation channel  82  at a sample loading junction  88  that preferably includes a porous material (not shown) adapted to permit the passage of fluid but retain stationary phase material  84  within the separation channel  82 . Another porous material (not shown) is preferably provided at the downstream end of the downstream portion  82 B of the separation channel and adapted to permit the passage of fluid but retain stationary phase material within the separation channel  82 . A common solvent may be supplied to both the first mobile phase inlet  81  and the second mobile phase inlet  83  such as by using a splitter  89  located either on-board or external to the microfluidic device  80 . Downstream of the separation channel  82 , a detector  92  may be provided to detect one or more properties of the separated species, and a waste reservoir  94  may be provided to collect mobile phase and solvent exiting the microfluidic device  80 . Preferably, the sample inlet port  86  is selectively sealed, such as by using a removable mechanical seal (not shown), to allow a sample to be loaded into the sample inlet port  86  when the mechanical seal is removed, and to permit pressure-driven separation to commence when the mechanical seal is applied.  
     [0051] To permit operate of the separation system  70 , solvent (mobile phase) is preferably supplied first to the microfluidic device  82  to thoroughly wet the stationary phase material  84  contained in the separation channel  82 . During the wetting step, the sample inlet port  86  should be mechanically sealed to prevent solvent from escaping the device  80  through the sample inlet port  86 . After the separation channel  82  has been thoroughly wetted, the solvent pump  74  is preferably deactivated and the separation channel  82  is preferably depressurized to permit sample injection. With the mechanical seal removed from the sample inlet port  86 , a fluidic sample from the sample source  78  (e.g., a pipettor) may be injected through the sample inlet port  86  and into the sample loading segment  85 . After the mechanical seal is reapplied to the sample inlet port  86 , the solvent pump  74  is preferably activated to reinitiate mobile phase flow into the first and the second mobile phase inlets  81 ,  83 . Notably, mobile phase provided to the second mobile phase inlet  83  serves to push the fluid sample from the sample loading segment  85  through the sample loading junction  88  and into the separation channel  82 , where the sample is eluted by the stationary phase material  84  contained in the downstream portion  82 B.  
     [0052] Following elution, one or more properties of the separated species may be detected by the detector  92 . The detector  92  may be adapted to detect fluid properties of the separated species either within or outside the microfluidic device  80 , and materials with suitable optical and mechanical properties are preferably selected for portions of the microfluidic device  80  (e.g., windows) to facilitate such detection. While various detection technologies may be used, preferred technologies include optical spectroscopies including absorbance, fluorescence, Raman scattering, polarimetry, circular dichroism and refractive index detection. Window materials can also be used to permit other analytical techniques such as scintillation, chemilluminescence, electroluminescence, and electron capture. A range of electromagnetic energies can be used including ultraviolet, visible, near infrared, and infrared. Additionally, techniques such as electrochemical detection, capacitive measurement, conductivity measurement, mass spectrometry, nuclear magnetic resonance, evaporative light scattering, ion mobility spectrometry, and matrix-assisted laser desorption ionization may be performed in conjunction with the separation system  70 .  
     [0053] Downstream of the detector  92  is a waste reservoir  94 . In an alternative embodiment, a sample collector (not shown) may be substituted for the waste reservoir  94 . Although not shown, the system  70  preferably further includes a controller (e.g., a microprocessor-based controller) for controlling the various components of the system  70 .  
     [0054] In a preferred embodiment, a pressure-driven microfluidic separation device includes multiple separation channels and multiple discrete sample inputs to permit multiple different samples to be separated simultaneously using a minimum number of expensive system components such as pumps, pulse dampers, etc. For example, FIGS.  5 A- 5 B illustrate a pressure-driven microfluidic separation device  100  including eight separation channels  161 - 168 . The device  100  may be constructed with nine device layers  101 - 109 , including multiple stencil layers  103 - 108 . Each of the nine layers  101 - 109  defines two alignment holes  180 ,  181 , which may be used in conjunction with fixed external pins (not shown) to aid in aligning the layers  101 - 109  during construction and/or to aid in aligning the device  100  with an external interface such as a mechanical seal (not shown) or slurry packing apparatus (not shown).  
     [0055] The first layer  101  defines several fluidic ports: three solvent inlet ports  112 ,  114 ,  116  that are used to admit mobile phase solvent to the device  100 ; eight pairs of sample ports  110 A- 110 H and  111 A- 111 H that permit samples to be supplied to sample loading segments  135 A- 135 H (defined in the third layer  103 ); and eight outlet ports  118 A- 118 H to permit mobile phase and separated sample species to exit the device  100  downstream of the separation channels  161 - 168 . Due to the sheer number of elements depicted in FIGS.  5 A- 5 B, numbers for selected elements within alphanumeric series groups (e.g., sample ports  110 B- 110 G,  111 B- 111 G, sample loading segments  135 B- 135 G, outlet ports  118 B- 118 G) are omitted for clarity. Notably, of the three solvent inlet ports  112 ,  114 ,  116 , the first solvent inlet port  112  is additionally used to admit slurry to the device  100  during a column packing procedure. The first layer  101  further defines eight apertures  117 A- 117 H that, along with identical apertures  177 A- 177 H defined in the fifth through ninth device layers  105 - 109 , facilitate optical detection by locally reducing the thickness of material bounding (from above and below) the detection regions  139 A- 139 H of channels  138 A- 138 H defined in the third layer  103 .  
     [0056] The second through sixth layers  102 - 106  each define a first solvent via  122  for communicating a mobile phase solvent from a first mobile phase inlet port  112  to a transverse channel  171  defined in the seventh layer  107 . A second solvent via  124  is defined in each of the second through fourth device layers  102 - 104  for communicating a mobile phase solvent from the second mobile phase inlet port  114  to a channel segment  151  defined in the fifth layer  105 . A third solvent via  126  is defined in the second layer  102  to communicate a mobile phase solvent from the third mobile phase inlet port  116  to an initial solvent mixing channel  131  defined in the third layer  103 . Eight pairs of sample vias  120 A- 120 H and  121 A- 121 H defined in the second layer  102  are interposed between the sample ports  110 A- 110 H and  111 A- 111 H and the sample loading segments  135 A- 135 H defined in the third layer. Additionally, eight outlet vias  128 A- 128 H are interposed between the outlet ports  118 A- 118 H and the channels  138 A- 138 H defined in the third layer  103 .  
     [0057] In addition to the structures described previously, the third layer  103  defines a series of six transverse segments  132 A- 132 F and a curved channel  133 . The transverse segments  132 A- 132 F and curved channel  133 , when coupled with the longitudinal segments  142 A- 142 G defined in the fourth layer  104 , form continuous flow path between the initial solvent mixing channel  131  and the large forked channel  143  defined in the fourth layer  104 . Further defined in the third layer  103  are two transverse segments  134 A,  134 B that fluidically couple the large forked channel  143  to four small forked channels  144 A- 144 D defined in the fourth layer  104 . In addition to the structures described previously, the fourth layer  104  defines eight sample loading vias  145 A- 145 H and eight effluent vias  147 A- 147 H. A first porous membrane  140  is disposed between the third and fourth layers  103 ,  104 , between the sample loading channels  135 A- 135 H (defined in the third layer  103 ) and the small forked channels  144 A- 144 D in the fourth layer  104 . The purpose of this first porous membrane  140  is to impede the flow of samples (which are injected into the device  100  through sample input ports  110 A- 110 H) into the small forked channels  144 A- 144 H, thus preventing undesirable cross-talk or contamination between samples.  
     [0058] The fifth layer  105  defines a channel segment  151 , eight junction vias  155 A- 155 H disposed below the vias  145 A- 145 H defined in the fourth layer  104 , and eight effluent vias  157 A- 157 H disposed at the downstream end of each separation channel  161 - 168 . Two porous materials  150 ,  160  are disposed between the fourth layer  104  and the fifth layer  105 . These materials  150 ,  160  serve as frits to retain stationary phase material  169  within the separation channels  161 - 168  defined in the sixth layer  106 . In other words, the frits  150 ,  160  permit the passage of liquid solvent, but impede the passage of stationary phase material  169 . If the stationary phase material  169  includes packed particulate matter, then the frits  150 ,  160  preferably have a pore size that is smaller than each particle to be retained. Although various materials may be used for the frits  150 ,  160  (and the porous membrane  140 ), a preferred material for constructing these elements  140 ,  150 ,  160  is a permeable polypropylene membrane such as, for example, 1-mil (25 microns) thickness Celgard 2500 membrane (55% porosity, 0.209×0.054 micron pore size, Celgard Inc., Charlotte, N.C.). This is particularly preferred when the device layers  101 - 109  are fabricated with a substantially adhesiveless polyolefin material, such as non-biaxially-oriented polypropylene, using direct (e.g. thermal) bonding methods such as discussed herein. Devices  100  constructed according to such methods may be readily capable of withstanding (internal) operating pressures of 10 psi (69 kPa), 50 psi (345 kPa), 100 psi (690 kPa), 500 psi (3450 kPa), or even greater pressures.  
     [0059] The sixth layer  106  defines eight parallel separation channels  161 - 168  and a transverse channel segment  172 . The seventh layer  107  defines an elongate transverse channel  171 , one large forked channel  171 , and four small forked channels  176 A- 176 D. The eighth layer  108  defines two intermediate forked channels  174 A- 174 B that permit fluid communication between the large forked channel  171  and the four small forked channels  176 A- 176 D. The eighth layer  109 , which serves to bound the forked channels  174 A- 174 B from below, defines no channels; rather it defines only alignment holes  180 - 181  and apertures  177 A- 177 H that facilitate optical detection.  
     [0060] Stationary phase material  169  is preferably added to the device  100  after the various layers  101 - 109  and porous elements  140 ,  150 ,  160  are laminated or otherwise bonded together to form an integral structure. While various types of stationary phase material may be used, preferred types include packed particulate material, and preferred packing methods employ slurry. One preferred slurry includes silica powder having surface chemical groups (e.g., Pinnacle II™ C-18 silica, 5-micron, catalog no. 551071, Restek Corp., Bellefonte, Pa.) and acetonitrile (MeCN), such as in a ratio of 1.00 grams particulate to 500 ml of solvent. Pressurized slurry may be supplied to the device  100  by way of a solvent inlet port  112 . From the solvent inlet port  112  and associated vias  122 , the slurry is split to the eight separation channels  161 - 168  through a microfluidic channel network that includes an elongate transverse channel segment  171 , a reversing transverse channel segment  172 , the large forked channel  173 , the intermediate forked channels  174 A- 174 B, and the small forked channels  176 A- 176 D. Upon filling the separation channels  161 - 168 , particulate matter within the slurry is prevented from leaving by way of the frits  150 ,  160 , and solvent separated from the slurry emerges from the device  100  through the downstream frit  160 , vias  147 A- 147 H, channels  138 A- 138 H, vias  128 A- 128 H, and finally the outlet ports  118 A- 118 H. Preferably, pressurized slurry is added to the device  100  until not only the separation channels  161 - 168  are filled, but also the forked channels  176 A- 176 D,  174 A- 174 D,  173  and the transverse segments  171 - 172  are filled so as to leave a trailing edge of stationary phase material disposed adjacent to the solvent inlet port  112 .  
     [0061] In operation of the device  100 , a first mobile phase solvent may be supplied to one solvent inlet port  114  and a second mobile phase solvent may be supplied to another solvent inlet port  116 . These two solvents meet within the mixing channel  131  adjacent to the slit  141  (defined in the fourth layer  104 ), after which the two solvents are laminated one atop the other to promote mixing. Preferably, each solvent is supplied by an independently controlled pressure source (e.g., a pump) to permit gradient separation to be performed within the device  100 . From the mixing channel  131 , the combined solvents flow through a compact composite channel composed of seven longitudinal segments  142 A- 142 G alternated with six transverse segments  132 A- 132 F that provide a relatively long channel structure within a compact area. From the last longitudinal channel  142 G, the solvent mixture flows into a curved channel  133  leading to a composite splitter including a large forked channel  143 , a two intermediate channel segments  134 A- 134 B, and four small forked channels  144 A- 144 D that in turn supply the solvent mixture to the sample loading channels  135 A- 135 H upstream of the sample loading ports  110 A- 110 H. The sample loading channels  135 A- 135 H are in fluid communication with the separation channels  161 - 168  at sample loading junctions  155 A- 155 H disposed between the upstream portions  161 A- 168 A and downstream portions  161 B- 168 B of the separation channels  161 - 168 . The junctions  155 A- 155 H demarcate the transition from the upstream portions  161 A- 168 A and the downstream portions  161 B- 168 B.  
     [0062] Another pressurized solvent stream is supplied to the device  100  through the solvent inlet port  112  leading to the provided the solvent mixture flows into a curved channel  143  leading to a composite splitter including the large forked channel  173 , two medium forked channels  174 A- 174 B, and four small forked channels  176 A- 176 D that divide the solvent mixture among the eight separation channels  161 - 168 . This second solvent stream provided through the solvent inlet port  112  helps prevent stationary, phase material from unpacking within the separation channel, and also helps to ensures that sample plugs injected into the sample loading channels  135 A- 135 H flow exclusively through the downstream portions  161 B- 168 B of the separation channels  161 - 168 . Preferably, the solvent supplied to this solvent inlet port  112  comprises one of the multiple solvents that may be supplied through the sample loading channels  135 A- 135 H. For example, if gradient separation is desired using water and acetonitrile as solvents and C-18 silica stationary phase material  169 , then the solvent to be supplied to the solvent inlet port  112  preferably comprises water or acetronitrile (MeCN). More preferably, the solvent to be supplied to the solvent inlet port  112  is the solvent that will encounter the greater fluidic impedance by traveling through the fluidic network including channels  171 ,  172 ,  143 ,  144 A- 144 B,  145 A- 145 D that contain stationary phase material  159 . Thus, in the same example as just mentioned above, where water and acetonitrile are solvents supplied through the sample loading channels  135 A- 135 H, the solvent supplied to the device  100  through the solvent inlet port  112  would preferably be water since it is more difficult to flow water through packed C- 18  silica particulate matter.  
     [0063] After the stationary phase material  169  is fully wetted with solvent, samples may be added to the device  100  through the sample ports  110 A- 110 H and  111 A- 111 H. Preferably, solvent flow is interrupted and the device is temporarily depressurized (e.g., by disengaging a removable mechanical seal (not shown) from the sample loading ports  110 A- 110 H,  111 A- 111 H) to permit the samples to be loaded. Two sample ports (e.g.,  110 A,  111 A) correspond to each sample loading segment (e.g.,  135 A) of the eight sample loading segments  135 A- 135 H defined in the third layer  103 . Preferably, different samples are provided to each upstream port ( 110 A- 110 H) and each sample flows within a portion of a sample loading segment  135 A- 135 H to emerge through the downstream port  111 A- 111 H so as to define a sample plug of a repeatable volume in each sample loading segment between the upstream port (e.g., port  110 A) and downstream port (e.g.,  111 A). After the samples are loaded, the sample loading ports  110 A- 110 H,  111 A- 111 H are preferably re-sealed (e.g., by disengaging a removable mechanical seal (not shown)) and solvent flow through solvent ports  112 ,  114 ,  116  is re-initiated. The solvents supplied into the sample loading segments  135 A- 135 H from the sample inlet ports  114 ,  116  sweep the sample plugs onto the separation columns  161 - 168  where they flow into the through downstream portions  161 B- 168 B and are eluted.  
     [0064] Another embodiment, similar in many respects to the device  100  just described, is shown in FIGS.  6 A- 6 B. This pressure-driven microfluidic separation device  200  including eight separation channels  261 - 268 . The device  200  may be constructed with nine device layers  201 - 209 , including multiple stencil layers  202 - 207 . Each of the nine layers  201 - 209  defines three alignment holes  280 - 282 , which may be used in conjunction with fixed external pins (not shown) to aid in aligning the layers  201 - 209  during construction and/or to aid in aligning the device  200  with an external interface such as a mechanical seal (not shown) or slurry packing apparatus (not shown).  
     [0065] The first layer  201  defines several fluidic ports: one slurry inlet port  212  (that is used during a column packing procedure to supply stationary phase material  269  to the separation channels  261 - 268 ); two solvent inlet ports  214 ,  216  that are used to admit mobile phase solvents (e.g. for gradient separation) to the device  200 ; eight pairs of sample ports  210 A- 210 H and  211 A- 211 H that permit samples to be supplied to sample loading segments  235 A- 235 H (defined in the third layer  203 ); and eight outlet ports  218 A- 218 H that permit mobile phase and separated sample species to exit the device  200  downstream of the separation channels  261 - 268 . As before, numbers for selected elements within alphanumeric series groups are omitted for clarity. The first layer  201  further defines eight apertures  217 A- 217 H that, along with identical apertures  279 A- 279 H defined in the fifth through ninth device layers  205 - 209 , facilitate optical detection by locally reducing the thickness of material bounding (from above and below) the detection regions  239 A- 239 H of outlet channels  238 A- 238 H defined in the third layer  203 .  
     [0066] The second through fifth layers  202 - 205  each define a slurry via  222  for communicating a stationary phase material (e.g., combined with a liquid in slurry form) from the slurry inlet port  212  to a transverse channel  248  defined in the sixth layer  207 . A first solvent via  224  is defined in each of the second and third device layers  202 - 203  for communicating a first mobile phase solvent from the first mobile phase inlet port  214  to a channel segment  241  defined in the fourth layer  204 . A second solvent via  226  is defined in the second through fifth device layers  202 - 205  to communicate another mobile phase solvent from the second mobile phase inlet port  216  to an initial solvent mixing channel  258  defined in the sixth layer  206 . Eight pairs of sample vias  220 A- 220 H and  221 A- 221 H defined in the second layer  202  are interposed between the sample ports  210 A- 210 H and  211 A- 211 H and the sample loading segments  235 A- 235 H defined in the third layer  203 . Additionally, eight outlet vias  228 A- 228 H are interposed between the outlet ports  218 A- 218 H and the channels  238 A- 238 H defined in the third layer  203 . The second layer further defines six longitudinal segments  227 A- 227 F that, coupled with the transverse segments  242 A- 242 F (defined in the fourth layer  204 ) and small vias  231 A- 231 K (defined in the third layer  203 ) provide additional contraction/expansion regions (marked by vias  231 A- 231 K) downstream of the primary mixing channel  258 . That is, after two mobile phase solvents are laminated one atop another in the mixing channel  258 , further and more complete mixing between the two solvents is promoted by passage through the series of small vias  231 A- 231 K (which cause the solvent flow to contract) and transverse segments  242 A- 242 F and longitudinal segments  227 A- 227 F (which permit the solvent flow to expand upon exiting each small via  231 A- 231 K).  
     [0067] The third layer  203  defines (in addition to the structures described previously) a large forked channel  232 , eight bypass sample loading channels  235 A- 235 B, and eight outlet channels  238 A- 238 H each having an enlarged region  239 A- 239 H to facilitate optical detection of species following separation of samples in the separation channels  261 - 268 . The fourth layer defines six transverse segments  242 A- 242 F, a curved channel  243 , two solvent vias  244 A- 244 B (in fluid communication with the large forked channel  232 ), eight sample loading inlet vias  245 A- 245 H, and eight sample loading outlet vias  246 A- 246 H. Each sample loading inlet via  245 A- 245 H is in fluid communication with a separation channel  261 - 268  by way of a first porous frit  250 A and a solvent splitting junction  255 A- 255 H (defined in the fifth layer  205 ), while each sample loading outlet via  246 A- 246 H is in fluid communication with a separation channel  261 - 268  by way of a second porous frit  250 B and a sample loading junction  256 A- 256 H (also defined in the fifth layer  205 ).  
     [0068] The fifth layer  205  defines (in addition to the structures described previously) a slit  251  that permits fluid communication between channel segment  241  (defined in the fourth layer  204 ) and the mixing channel  258 , and facilitates lamination of one solvent atop the other to promote mixing within the mixing channel  258 . The fifth layer  205  further defines a longitudinal channel segment  252  that permits fluid communication between the mixing channel  258  and the first transverse segment  242 A defined in the fourth layer  204 . Fluid communication between the downstream end of the separation channels  261 - 268  and the outlet channels  238 A- 238 H is established by way of vias  247 A- 247 H (defined in the fourth layer  204 ), a frit  260 , and vias  257 A- 257 H (defined in the fifth layer  205 ).  
     [0069] In addition to defining the separation channels  261 - 268 , the sixth layer  206  defines two intermediate forked channels  259 A- 259 B, a mixing channel  258 , and a transverse segment  248 . The seventh layer  207  defines a large forked channel  271  (for splitting a flow of slurry during a column packing procedure) and four small forked channels  272 A- 272 D. The eighth layer  208  (which serves to bound the forked channels  274 A- 274 B from below) and ninth layer  209  are identical, and could be combined into a single layer if desired. Neither of these layers  208 ,  209  define any channels; rather, they define only alignment holes  280 - 281  and apertures  279 A- 279 H that facilitate optical detection.  
     [0070] As was the case with the device  100  according to the previous embodiment, the device  200  is preferably fabricated with a substantially adhesiveless polyolefin material, such as non-biaxially-oriented polypropylene, using direct (e.g. thermal) bonding methods such as discussed herein. Such a device  200  is preferably adapted to withstand (internal) operating pressures of 10 psi (69 kPa), 50 psi (345 kPa), 100 psi (690 kPa), 500 psi (3450 kPa), or even greater pressures. With regard to the porous materials  240 ,  250 A,  250 B,  260 , various porous materials may be used but a preferred material for constructing these elements  240 ,  250 A,  250 B,  260  includes a permeable polypropylene membrane such as, for example, 1-mil (25 microns) thickness Celgard 2500 membrane (55% porosity, 0.209×0.054 micron pore size, Celgard Inc., Charlotte, N.C.)—particularly if the device layers  202 - 209  are fabricated with nonbiaxially-oriented polypropylene.  
     [0071] If the stationary phase material  269  includes packed particulate matter, then the frits  240 ,  250 A,  250 B,  260  preferably have a pore size that is smaller than each particle to be retained. Although various materials may be used for the frits  240 ,  250 A,  250 B,  260 , a preferred material for constructing these elements  240 ,  250 A,  250 B,  260  is a permeable polypropylene membrane such as, for example, 1-mil (25 microns) thickness Celgard 2500 membrane (55% porosity, 0.209×0.054 micron pore size, Celgard Inc., Charlotte, N.C.).  
     [0072] Stationary phase material  269  is preferably added to the device  200  after the various layers  201 - 209  and porous elements  240 ,  250 A,  250 B,  260  are laminated or otherwise bonded together to form an integral structure. While various types of stationary phase material may be used, preferred types include packed particulate material, and preferred packing methods employ slurry. One preferred slurry includes silica powder having surface chemical groups (e.g., Pinnacle II™ C-18 silica, 5-micron, catalog no. 551071, Restek Corp., Bellefonte, Pa.) and acetonitrile (MeCN), such as in a ratio of 1.00 grams particulate to 500 ml of solvent. Pressurized slurry may be supplied to the device  200  by way of the slurry inlet port  212 . From the slurry inlet port  212  and associated vias  222 , the slurry is split to the eight separation channels  261 - 268  through a microfluidic channel network that includes an elongate transverse channel segment  248 , the large forked channel  271 , the intermediate forked channels  259 A- 259 B, and the small forked channels  272 A- 272 D. Upon filling the separation channels  261 - 268 , particulate matter within the slurry is retained by the frits  240 ,  250 A,  250 B,  260 , and solvent separated from the slurry emerges from the device  200  through the downstream frit  260 , vias  247 A- 247 H, channels  238 A- 238 H, vias  228 A- 228 H, and finally the outlet ports  218 A- 218 H. Preferably, pressurized slurry is added to the device  200  until not only the separation channels  261 - 268  are filled, but also the forked channels  272 A- 272 D,  259 A- 259 B,  271  and the transverse segment  248  are substantially filled so as to leave a trailing edge of stationary phase material disposed adjacent to the solvent inlet port  212 . After the packing procedure is complete, the slurry inlet port  212  is preferably sealed, either temporarily (e.g., with a screw or other removable means) or permanently (e.g., with epoxy or localized heating). This is in marked contrast to the device  100  according to previous embodiment, since that device  100  was intended to receive a flow of pressurized solvent into the solvent inlet port  112   
     [0073] While the device  100  (described previously in connection with FIGS.  5 A- 5 B) included multiple sets of both intermediate and small forked channels (each set in fluid communication with only one large forked channel  143 ,  173 ), the device  200  according to the present embodiment provides two large forked channels  232 ,  271  that are both in fluid communication with the same set of intermediate forked channels  259 A- 259 B and small forked channels  272 A- 272 D. To prevent the passage of slurry into one large forked channel  232 , a frit  240  is fluidically disposed between the large forked channel  232  and the medium forked channels  259 A- 259 B.  
     [0074] Another notable difference between the device  200  and the device  100  described in connection with FIGS.  5 A- 5 B is that the device  200  according to the current embodiment includes bypass sample loading channels  235 A- 235 H rather than non-bypass sample loading segments  135 A- 135 H. Each bypass sample loading channel  235 A- 235 H is in fluid communication with a separation channel  261 - 268  through both a solvent splitting junction  255 A- 255 H and a sample loading junction  256 A- 256 H. When solvents supplied to the upstream portion  261 A- 261 H of each separation channel  261 - 268  encounter the solvent splitting junction  255 A- 255 H, a portion of the solvents will flow into the bypass sample loading segments  235 A- 235 H while the remainder of the solvents will continue traveling down the separation channels  261 - 268  toward the sample loading junction  256 A- 256 H. That is, for each solvent stream supplied to the upstream end of a separation channel  261 - 268 , a first portion of the stream continues to flow within the channel  261 - 268 , while the remaining portion of the stream is split into a bypass sample loading segment  235 A- 235 H that reconnects with the first portion at a sample loading junction  256 A- 256 H. This design causes solvent flow to push the stationary phase material  269  within each separation channel  261 - 268  toward the outlet vias  257 - 257 H, thus ensuring that the stationary phase material  269  remains packed not only in the downstream portions  261 B- 268 B but also the upstream portions  261 A- 268 A of each separation channel  261 - 268 . Each sample loading junction  256 A- 256 H demarcates the transition from an upstream portions  261 A- 268 A to a downstream portion  261 B- 268 B of the eight separation channels  261 - 268 . Preferably, the portion of each separation channel  261 - 268  disposed between a solvent splitting junction  255 A- 255 H and a sample loading junction  256 A- 256 H provides a greater resistance to fluid flow than corresponding bypass path (i.e., through a via  255 A- 255 H, a frit  250 A, another via  245 A- 245 H, a bypass sample loading segment  235 A- 235 H, another via  246 A- 246 H, another frit  250 B, and finally another via  256 A- 256 H).  
     [0075] In operation of the device  200 , a first mobile phase solvent may be supplied to one solvent inlet port  214  and a second mobile phase solvent may be supplied to another solvent inlet port  216 . These two solvents meet within the mixing channel  258  adjacent to the slit  251  (defined in the fifth layer  205 ), after which the two solvents are laminated one atop the other to promote mixing. Preferably, each solvent is supplied by an independently controlled pressure source (e.g., a pump) to permit gradient separation to be performed within the device  200 . From the mixing channel  258 , the combined solvents flow through a longitudinal segment  252  into further mixing regions formed by seven longitudinal segments  242 A- 242 G, the small vias  231 A- 231 K, and six transverse segments  227 A- 227 F. From the last longitudinal channel  242 G, the solvent mixture flows into a curved channel  243  leading to a composite splitter including a large forked channel  232 , a two intermediate channel segments  2594 A- 259 B, and four small forked channels  272 A- 272 D that in turn supply the solvent mixture to the upstream end of each of the eight separation channels  261 - 268 .  
     [0076] After the stationary phase material  269  is fully wetted with solvent, samples may be added to the device  200  using the sample ports  210 A- 210 H,  211 A- 211 H. Preferably, solvent flow is interrupted and the device is temporarily depressurized to permit the samples to be loaded. Two sample ports (e.g.,  210 A,  211 A) correspond to each sample loading segment (e.g.,  235 A) of the eight sample loading segments  235 A- 235 H defined in the third layer  203 . Preferably, different samples are provided to each upstream port ( 210 A- 210 H) and each sample flows within a portion of a sample loading segment  235 A- 235 H to emerge through the downstream port  211 A- 211 H so as to define a sample plug of a repeatable volume in each sample loading segment between the upstream port (e.g., port  210 A) and downstream port (e.g.,  211 A). After the samples are loaded, solvent flow is re-initiated, and solvent flow through each bypass sample loading segment  235 A- 235 H serves to carry each sample through a sample loading junction  256 A- 256 H into the separation channels  261 - 268  where they are eluted to separate individual species.  
     [0077] While FIGS.  5 A- 5 B and FIGS.  6 A- 6 B each depict microfluidic devices  100 ,  200  having eight microfluidic separation channels  161 - 168 ,  261 - 268  and appurtenant microstructures, it will be readily apparent to one skilled in the art that any number of separation channels may be provided in a microfluidic separation device. Higher density microfluidic separation devices, each having many more than eight separation channels, are specifically contemplated.  
     [0078] It is also to be appreciated that the foregoing description of the invention has been presented for purposes of illustration and explanation and is not intended to limit the invention to the precise manner of practice herein. It is to be appreciated therefore, that changes may be made by those skilled in the art without departing from the spirit of the invention and that the scope of the invention should be interpreted with respect to the following claims.