WO2010004236A1

WO2010004236A1 - Material separation device

Info

Publication number: WO2010004236A1
Application number: PCT/GB2008/003855
Authority: WO
Inventors: Gareth Jenkins; Alicia Jenkins
Original assignee: Deltadot Limited
Priority date: 2008-07-11
Filing date: 2008-11-17
Publication date: 2010-01-14
Also published as: EP2334432A1; GB0812781D0

Abstract

Devices and methods are described for separating components in a sample, for example a protein sample, in a branched channel structure, using flow barriers to separate flows between channels and/or providing an interface with an analysis device such as a mass spectrometer. In one example, a flow barrier is provided between a main (4) and a branch (12) channel to reduce diffusional contamination between the channels and cross talk. In another example, contamination of components entering the analysis device is reduced by switching selected components from a main channel (4) into a branch channel (12) and then through an interface into the analysis device.

Description

MATERIAL SEPARATION DEVICE

The present invention relates to a device and method for separating materials comprising components, such as biomolecules. It finds particular although not exclusive application in interfacing an electrophoretic separation with an analysis device such as a mass spectrometer.

Electrophoresis is a technique known for the separation of charged components such as biomolecules including but not limited to proteins, protein fragments, proteinaceous matter and nucleic acids. If an electric current is applied to a sample carried in a microchannel, the sample can be caused to migrate along the microchannel. In doing so, different constituents will migrate at different speeds so that the sample will separate out into constituent molecular bands positioned at varying distances along the microchannel. The use of both gel- filled channels and capillary electrophoresis is known for the electrophoretic separation of different constituents in a sample.

The invention is not limited to molecular separations using electrophoresis but is equally applicable to the separation of molecules or other components which are flowing in some other way along a channel, for example under the influence of a pressure differential as in liquid chromatography.

A branched microchannel is, as its name suggests, a channel which branches into more than one channel. As the constituent molecular bands of a sample migrate along the microchannel, any one band will reach the branch region at a particular time. Either by knowledge of the separation dynamics and appropriate timing or by using a detector providing real time signals, a desired band of the separation can be diverted from a main channel in which the separation has taken place into a branch channel, for example by applying an appropriately directed and timed potential difference at the branch point.

A problem arises with this known arrangement when there is a need to change the potential difference at a branch point relatively rapidly, for instance to switch a specific molecular band into the branch channel from a rapid succession of bands. There is a risk that the leading edge of a constituent molecular band following a selected band may start to follow the previous band into the branch channel, leading to cross contamination of the molecular bands separated into the branch channel.

In a first embodiment, there is provided a separating device for separating components flowing in a channel as defined in claim 1.

The device comprises a main channel, a branch channel and means for generating flows in these channels. The main channel may be a separation channel itself, as such as an electrophoretic separation channel, or may be connected to a further separation channel, such as a capillary for capillary electrophoresis. The branch channel is in fluidic communication with the main channel through an aperture in a wall of the main channel and the device comprises barrier flow means for generating a barrier flow of fluid across the aperture between components flowing in the main channel and the aperture.

The barrier flow, in effect, provides a barrier between fluids in the main channel and the branch channel, limiting cross contamination between the main and the branch channel by diffusion and reducing the risk that a closely following component will inadvertently enter the branch channel behind a selected component. The selected component is attracted into the branch channel through the flow barrier and when the branch tlow is switched off the flow barrier re-establishes itself between the flow in the main channel and the branch channel. It thus acts as a gate in front of the aperture from which the branch channel extends.

The main and barrier flow means may be arranged to generate flow by different mechanisms such that different fluids can be used in the main channel and by the barrier flow means. This provides an increased flexibility in choosing materials, such as buffer fluids or a gel matrix for the main channel and any additional chemistry, for the two flows. This may be particularly advantageous where the main flow is electrokinetically driven such as in gel electrophoresis and the fluid for the barrier flow is not electrically or physically compatible with the main flow. The barrier flow may then be driven by, for example, a suitable pressure differential or other mechanisms, such as electro-osmotic flow (bulk flow induced by a charged mobile layer at the channel wall).

For example, where separation is carried out using electrophoresis, the main channel may be filled with an electrophoresis buffer which contains sodium dodecyl sulphate (SDS), which is often added to the buffer solution in capillary electrophoresis (CE) separation of proteins in order to denature and bind to the proteins and improve the charge to mass ratio, thus allowing better separation performance. However, where the branch channel provides an interface with a mass spectrometer (MS) this can lead to problems because SDS degrades the performance of the MS detection step. Of course, the same concept can equally be useful to limit contamination of the MS (or an alternative analysis device) with other contaminants which may be present in the main channel.

While the switching of components through the flow barrier is expected to result in the reduction of SDS entering the MS, further measures can be taken to reduce contamination of the MS by SDS. For example, an additive selectively binding to SDS may be added to the barrier flow to sequester SDS from the main flow to further reduce the amount of SDS which can enter the branch channel. Similarly, the branch channel may be coated with an SDS binding agent. It will be understood that reference to SDS herein may also include any other contaminants for analytical devices to which the branch channel is connected.

In one particular embodiment, the branch channel may be provided with a side channel to which a voltage can be applied to attract SDS into the side channel, removing it from the flow through the branch channel towards the MS. Because of its lower molecular weight, SDS will be attracted more readily into the side channel than the components, such as proteins, to be studied by MS such that a further reduction of SDS concentration in the branch channel can be achieved.

The side channel may be filled with a buffer which has a lower conductivity than a buffer in the branch channel which is known to lead to protein stacking whereby the proteins accumulate at the interface between the side channel and the branch channel. By carefully controlling a voltage or negative pressure applied to the side channel and the branch flow, proteins can be caused to concentrate at the interface between the side and branch channels with SDS being concentrated further inside the side channel. By then reversing the potential or pressure differential inside the side channel, the proteins can be returned to the branch channel while most or all of the SDS in the side channel is maintained within it.

The device may further comprise interface flow means associated with the branch channel to create a flow in the branch channel in addition to the flow created by the branch flow means. This allows continuous operation of the mass spectrometer interface, independent of the switching state of the branch channel. The interface flow may contain additives such as organic solvents which can aid in the evaporation and ionisation of the components where the interface comprises an electrospray electrode or it may comprise a suitable matrix material where the interface works by matrix assisted laser desorption ionisation (MALDI).

In yet a further embodiment, there is provided a separating device as claimed in claim 8.

Even without arrangements for a barrier flow, the fact of switching selected components from a separation channel to an interface with an analysis device (such as MS) in itself reduces the amount of buffer and hence contaminants within the buffer which is introduced into the analysis device, as compared to a continuous flow of buffer when the separating channel is interfaced directly with the analysis device.

In a further embodiment, there is provided a device for containing fluid for use as or as part of a separation device as described above, as defined in claim 16.

The device comprises a main channel and a barrier flow channel merging from one side with the main channel into a merged portion of the main channel and a branch channel extending from an aperture in a wall of the merged portion on the one side.

The branch channel may be narrower than the merged portion of the main channel and may be in fluidic communication with an interface region on an outer surface of the device. For example, the interface region may include an electrode with an aperture in further communication with the branch channel to provide an electrospray or MALDI electrode for interfacing within a MS. The interfacing region may be hydrophobic.

The device may further include one or more interface flow channels adjacent the interface region for adding additional flow, in addition to the flow from the main channel to the branch channel, to the branch channel adjacent the interface region. The device may further include a side channel extending to one side of the branch channel.

Any of the devices described above may be a microfluidic device, that is a device in which at least one of the channels (whether channels in a chip or capillaries) has at least one dimension in the sub-millimetre range.

In yet a further embodiment, there is provided a method of diverting a component from a main flow into a branch flow as defined in claim 21.

The method includes setting up a main flow of components and, adjacent to it, a barrier flow and diverting a component in the main flow into a branch flow across the barrier flow.

A device for selecting bands of components flowing in an electrophoresis channel and for interfacing the channel with a mass spectrometer will now be described as an embodiment, by way of example only, with reference to the accompanying drawings in which: Figure 1 shows schematically a microfluidic electrophoresis chip having a branch channel branched off a main channel;

Figure 2 shows schematically an electrophoresis chip as in figure 1 including an arrangement for creating a flow barrier in the region of the branch channel; Figure 3 shows an enlarged view of the chip in figure 2;

Figure 4 shows schematically a modified chip as in figure 2 in which the branch channel communicates with an interface region on the outside of the chip; Figure 5 shows an enlarged view of the chip in figure 4;

Figure 6 shows schematically a branch channel including a side channel;

Figures 7 and 8 show schematically electrospray electrodes at the interface region; and

Figure 9 shows schematically an interface region adapted for MALDI.

With reference to figure 1, a microfluidic chip 2 defines a main channel 4 extending between a buffer 6 and waste reservoir 8. The chip further comprises a sample injection structure 10 and a branch channel 12 extending from the main channel 4. In operation, a sample containing components such as proteins, digested protein fragments or any other proteinaceous matter, nucleic acid or any other biomolecule is injected into the main channel using the sample injection structure 10 and. under the influence of an electric field generated by electrodes in the sample reservoir 6 and waste reservoir 8 migrate through the main channel 4 towards the branch channel 12.

Typically, the main channel 4 may be about 50 micrometers wide and 50 to 200 micrometers deep. Although the intersections between channels are depicted in the figures as sharp angles, this may not necessarily be so in a practical implementation. In certain circumstances, it may be desirable for the angles between channels to be to a certain extent rounded. The microfluidic chip may be manufactured, for example, using lithographic patterning of a poly-dimethyl-siloxone substrate. The main channel 4 is filled with a suitable electrophoretic buffer, which may contain additives to aid electrophoretic separation, such as SDS for denaturing proteins, such that components travelling through the channel under the influence of the electric field travel at different velocities for different charges/mass ratios. Consequently the components injected at the sample structure 10 separate as they travel through a separation region 14 of the main channel 4 between the sample injection structure 10 and the branch channel 12 to form spaced bands of components having the same charge/mass ratio.

As the bands of components arrive at the junction with the branch channel 12, selected bands can be made to migrate into the branch channel 12 by switching a voltage applied to the waste reservoir 8 off and switching on a voltage applied to a branch reservoir 16 at the end of the branch channel 12. Once the band has migrated into the branch channel 12, the voltage can be switched back to the waste reservoir 8. The timing of this switching operation can be controlled either by a predetermined timer if the migration dynamics of a selected band of components to be collected is known, or otherwise a detector may be provided at or near the end of separation region 14 to detect components as they travel past in the main channel 4 and control the voltage switching accordingly.

A voltage controller for controlling the respective applied voltages can be connected to the reservoirs in various ways including but limited to: wired electrodes inserted directly into the reservoirs (or channels), electrodes may be patterned lithographically onto one or both surfaces of a planar microfluidic chip, or by using fluidic connections such that voltage can be applied through a side channel containing conductive medium, the channel being connected to an external reservoir into which an electrode is inserted. The voltage controller may control the voltages to be constant or may control the voltage to achieve constant current flow instead. The latter may be advantageous since it allows flow rates to be controlled independent of channel geometries and conductivities.

A problem with this arrangement, as discussed above, is that components from the main channel will tend to diffuse into the branch channel even if the voltage is applied at the waste reservoir 8 rather than the branch reservoir 16. Further, if the component bands are close together, switching of the voltages will have to be sufficiently rapid to direct a selected band into the branch channel 12 without also directing the following band, at least partially, into the branch channel.

With reference to figures 2 and 3, a chip as discussed above in relation to figure 1 but with a modified branch region 18 is now described. The branch region 18 is indicated schematically in figure 2 by a dashed ellipse and is depicted in figure 3 in an enlarged view.

The modified chip defines a further channel 20 which merges with the main channel 4 to define a merged portion 22 of the main channel having a cross section which is larger than the cross section of the main channel 4 upstream of the junction between the main channel 4 and the further channel 20. The further channel 20 merges into the main channel 4 at an oblique angle in the direction of flow in the main channel 4 such that, in use, there are two regions of flow in the merged portion 22 of the main channel.

The first region 24 contains flow of components through the main channel 4. For electrophoresis, this main flow comprises a flow of charged particles, for example inside a bulk medium or polymer gel in gel electrophoresis. A barrier region 26 between the first region 24 and the further and branch channels 20, 12 contains flow from the further channel 20 to one side of the first region 24. For electrokinetically induced barrier flow, this may again be a flow of charged components in a bulk medium such as a gel, for example the same gel as in the main channel 4. Alternatively, the flow in the barrier region 26 can be a bulk flow of not necessarily charged medium, for example induced by pressure differential or electro-osmotically. Since the junction between channels 4 and 20 is situated upstream of an aperture 28 in a wall 30 of the merged portion 22 to the side of the channel 20, through which the branch channel 12 is connected to the main channel 4, components flowing in the main channel 4 from the region 24 to the branch channel 12 must cross the barrier region 26. The distance between the junction between the further channel 20 and the main channel 4 and the aperture 28 is short relative to the length of the main channel 4 and may be in the region of 10 to 500 or over micrometers, for example 10 to 100 micrometers or 10 to 200 micrometers.

Although figure 3 depicts a boundary between flow regions 24 and 26 as a dashed line, it will be understood that in practice there will be a smooth transmission between the flows in these two regions. The individual flow rates and geometries of the channels can be adjusted to provide a substantially laminar flow along the wall 30 which acts as a barrier in front of the aperture 28. It will, however, be understood that the flow may also act efficiently as a barrier if it is somewhat turbulent.

As described above, components may be switched from flowing through the main channel to the waste reservoir 8 to flowing into the branch channel to the branch reservoir 16 by applying a voltage to one or the other of the two reservoirs. The switching duration may be between microseconds to 10s of seconds to ensure the correct amount of material is switched into the branch channel, as required. After switching for this period of time, the voltage is switched back to the reservoir 8 of the main channel, following which the flow from the channel 20 along the wall 30 re-establishes itself to form a barrier in front of the aperture 28, preventing or at least limiting cross contamination between the main and branch channels. To aid switching, the flow from channel 20 can be reduced and/or a slight back flow from the waste reservoir 8 can be induced at the time of switching by appropriately adapting the flow controls.

In an alternative approach to controlling the voltages, rather than switching the voltage or current between reservoirs 8 and 16 to direct the flow, components may be diverted into the branch channel while the voltage remains applied to the main reservoir 8. In this approach, the voltage applied to the main reservoir 8 need not be changed but rather the voltage (or current) applied to the branch reservoir 16 is set such that the potential in the region of the aperture 28 is sufficiently high to attract components from the flow in region 24 across the boundary flow in the region 26 into the branch channel 12. This may allow smoother, more continuous operation of the switching of components.

In the examples described above, the flows in the main channel 4 and the branch channel 12 are induced electrokinetically. Similarly, the flow from channel 20 along wall 30 in barrier flow region 26 can also be driven electrokinetically by applying a potential difference between the further reservoir 32 and the waste reservoir 8, for example controlling the potentials to obtain roughly equal current flows in the main channel 4 and the further channel 20. Of course, the potential difference can be tuned to achieve a desired flow rate of the barrier flow from channel 20. In this mode of operation, the flow from channel 20, together with the flow from channel 4 will be diverted into the branch channel as the voltages switches to it (or the branch channel voltage is switched on in addition to the main channel voltage as the case may be), thereby facilitating switching of a band of components across the region 26.

In some embodiments, the main channel 4 is 150 μm wide in the branch region and 100 μm upstream, the further channel 20 is 50 μm wide and the branch channel 12 is 50 μm wide with all channels having the same depth, typically 50-100 μm. All channels are filled with a polymer gel and flows induced electrokinetically as follows (with the waste reservoir 8 at ground, negatively charged flow, and applied voltages controlled to give roughly constant current flows): 20μA in the main channel 4 (typically - 500V applied at buffer reservoir 6) and lOμA in the further channel 20 (typically - 200V applied at further reservoir 32) with the branch reservoir 16 held at the same potential as the buffer reservoir 6 for separation. To switch components into the branch channel 12, the potential of the reservoirs 8 and 16 are swapped. Corresponding flow rates (of charged species in the bulk polymer gel) are approximately 0.5μl/s for the further channel 20 and lμl/s for the main channel 4 with a total with a total flow of about 1.5μl/s to the reservoir 8 or 16, as applicable. To improve switching into the channel, the flow from the further channel 20 can be temporarily reduced, for example to about 0.25 μl/s and a small back flow, for example 0.25μl/s, can be induced from the waste reservoir 8.

In some circumstances, however, it may be desirable to make the components cross through an established flow in the region 26 into the branch channel 12. In an embodiment adapted for such applications, flow from the channel 20 is induced by pressure deferential, for example applying positive pressure (using a pump or other source of positive pressure) to the further reservoir 32, or inducing electro-osmotic flow inside the further channel 20. Advantageously, this creates a flow in the barrier region 26 which is to some extent independent of the barrier flow in the region 24 of the main channel. This allows the barrier flow in the region 26 to act as a diffusion barrier even while switching is in progress, with components to be switched having to traverse this flow.

While it is preferable to use the same buffer solution for all three channels 4, 20 and 16 if the same, electrokinetic, flow mechanism is used in each of these, using different mechanisms of generating flow for the various channels (or at least the further channel 20 and main channel 4, for example pressure driven flow in channel 20 and electrokinetic flow in channel 24) allows the respective buffer solutions to be selected more flexibly. In an exemplary application where the main channel is filled with a polymer gel for gel electrophoresis, the flow in channel 20 may be selected independently, for example a bulk flow of a suitable buffer may be used. Further, where the flow in a barrier region 26 is driven by a pressure differential, it is not necessary to stop the flow for switching as long as the potential applied to the branch channel 12 is high enough to attract the components across the flow in the barrier region 26. In some embodiments, the barrier flow is hence generated continuously both during separation and switching. As will be described in more detail below, this may be advantageous when interfacing the branch channel 12 with a mass spectrometer.

In the case of pressure driven flow in the barrier region 26, the channel geometries are chosen to ensure the correct flow path in the barrier region 26 from the channel 20 along the channel wall 30 and across aperture 28 is maintained. For example, the merged portion 22 can be made sufficiently wider than the main channel 4 before the junction with the channel 70 to ensure that the flow from the channel 20 is directed along the channel wall 30 and does not backup the main channel 4. This can further be aided, or achieved alternatively, by making the length from the junction in the merged region 18 to the waste reservoir 8 sufficiently small. In these embodiments, the branch channel 12 is made relatively narrow as compared to the merged portion 22 to reduce flow from the barrier region 26 into the branch channel 12 rather than further along the channel wall 30.

In some embodiments, the microfluidic chip described above can be modified by adapting the branch channel 12 to interface capillary electrophoresis separation (CE), such as gel capillary electrophoresis (although capillary zone electrophoresis is equally envisaged) in the main channel 4 or a CE channel or capillary connected to the main channel, with a mass spectrometer (MS). Known CE-MS systems tend to suffer in performance due to additives of the buffer used for CE continually contaminating the MS system. For example, SDS is often added to the buffer solution in CE separation of proteins, as discussed above. However, contamination of the MS with SDS degrades the performance of the MS detection step. By selectively switching components for interest out of the CE separation step using a microfluidic chip as described above with reference to figure 1 or figures 2 and 3, the exposure of the MS system to SDS (or other contaminants) can be reduced.

With reference to figure 4, the chip is modified for interfacing with an MS in that the branch channel 12 is extended up to an interface region 34 at or near an outer surface of the chip for interfacing with an MS. The branch channel 12 extends right up to the interface region 34, dispensing with the branch reservoir 16. Instead, an electrode 36 is provided around the branch channel 12 in the interface region 34. The electrode 36 could equally be implemented as an internal electrode inside the branch channel 12, as a coating applied to it υi in any other appropriate way. Although figure 4 shows a modified version of the figure 2 chip, the figure 1 chip can be modified in a similar fashion.

In operation, a voltage is applied to the electrode 36 as described above for the reservoir 16. The resulting potential in the branch channel acts to both attract selected components into the branch channel 12 through the barrier region 26 and at the same time to generate an electrospray (as described below) to inject, vaporise and ionise the component selectively diverted into the branch channel

12 into the MS. The voltage is applied for a sufficiently long time to the electrode 36 such that the component band continues to travel towards it. At the same time, the electrode 36 generates an electrospray to inject the band into the MS as it arrives at the electrodes 36.

To form the electrospray and direct it into the MS, the chip 2 is arranged next to the MS with the outlet 34 adjacent to the inlet of the MS. For example, the inlet may comprise a cone and skimmer arrangement (not shown) with a vacuum pump communicating with a space between the cone and skimmer. The cone is electrically connected to the electrode 36 and held at an appropriate potential relative to it to attract the ionised spray of the components.

In some embodiments, the voltage applied to the electrodes 36 is switched to a continuous background level, which is sufficiently low so as not to attract the components across the barrier flow. An electrospray is continuously generated from fluid being diverted from the flow in region 26 across the aperture 28 to allow a stable electrospray to be maintained without stopping and starting the spray for each band to be injected. An appropriate potential of the electrode 36 will be high enough to create the electrospray but lower than the level required to divert components flowing in the main channel 4 into the branch channel 12. Alternatively, the electrospray can be made to operate only when components are diverted into the branch channel by keeping the electrodes 36 at an appropriate potential, for example a floating potential or one approximately equal to the potential in the barrier region.

In the device described above with reference to figures 4 and 5, it can be seen from the above discussion that it can be advantageous that the flow in channel 20 (and subsequently region 26) is driven by a pressure differential rather than electrokinetically (as, in some embodiments, the flow in the main channel 4) as this allows more flexibility in setting a continuous electrospray potential. Further, this arrangement allows greater flexibility in selecting the buffer for creating the barrier flow. This facilitates the use of a MS compatible fluid for the barrier flow to reduce the concentration of SDS or other contaminants from the electrophoresis buffer in the main channel 4 inside the branch channel 12 and, consequently, could reduce the amount of contaminant being injected into the MS. This will be of course particularly advantageous where the voltage controller is adapted for a continuous electrospray, as described above.

In some embodiments, the barrier flow is pressure driven and a continuous electrospray potential of 1.5kV is applied to electrode 36, increased to about 3kV to attract the components across the barrier flow.

Further, decoupling the buffer of the barrier flow- in region 26 from the electrophoresis buffer isolates the buffer in the branch channel from the buffer in the main channel and allows the addition of an additive to the barrier flow which removes SDS or other contaminants from the bands diverted into the branch channel 12. For example, in some embodiments a chemical with a high affinity to SDS is used as an additive to displace SDS from being bound to the proteins in the band as they cross the barrier region 26. Further, in some embodiments the channel wall 38 of the branch channel 12 could be provided with a coating which preferentially binds to SDS or other contaminants, thus removing contaminants from solution and preventing them from entering the MS.

In a further measure to reduce contamination of the MS, now described with reference to figure 6, a side channel 40 extending away from the branch channel 12 is added to the device. In this arrangement, after the band has been drawn into the switching channel either electrokinetically (applying a suitable potential to the branch channel 12 either via the electrode 36 or the side channel 40), SDS or other contaminants from the electrophoretic buffer are displaced from the components such as proteins in the band, which is now in the MS compatible environment of the buffer in the barrier region 26 and the branch channel 12. With a carefully controlled voltage applied to the side channel 40, the SDS or other contaminants could then be attracted into the side channel 40, leaving the protein in the branch channel 12. Because SDS or similarly contaminants are significantly lighter than protein molecules, they travel faster than proteins and would therefore enter the side channel 40 before the proteins in response to the applied voltage. After the SDS or other contaminants have thus been drawn into the side channel 40, a voltage applied to the electrodes 36 attracts the components and electrosprays them into the MS. The distance between the side channel 40 and the outlet 34 should be sufficiently small such that the components reach it before the SDS or other contaminants catch up with the components (as they have to travel farther from the side channel but travel faster to their lower weight).

To aid in the separation of SDS or other contaminants from the components, the side channel 40 may be filled with a lower conductivity buffer (for example a lower conductivity polymer gel) than the rest of the branch channel 12. This would cause the heavier components, such as proteins, to accumulate (in a process known as sample stacking) at the interface between the high and lower conductivity regions, while the SDS and other lighter contaminants would, as before, be drawn further into the side channel than the components. The voltage in the side channel can then be switched off and the electrospray electrodes 36 switched on high enough to attract the components towards it. Alternatively or additionally, the potential of (or pressure applied to) the side channel could be reversed to aid directing the proteins towards the electrodes 36.

The process just described can further be used for concentration and accumulation of components by running sequential separations of samples and repeatedly switching out the band of interest from the separation at the main channel 4 into the branch channel 12 where they can be collected at the interface between the branch channel 12 and the side channel 40, as described above. After enough components have been collected and accumulated in that way, the output could then be switched towards the mass spectrometer, as described above.

Alternatively, or additionally, an electrophoretic flow towards a side channel reservoir 41 is controlled such that any remaining SDS or other contaminants are continuously removed from the branch channel 12 and/or maintained in the side channel 40.

One such exemplar mode of operation is now described with reference to the following table which schematically sets out a sequence of voltages applied to the reservoirs of the chip 2.

As discussed above, during separation (A) the buffer reservoir 6 is held at a negative potential (all polarities given apply to negatively charged components, the reverse signs apply to positively charged components) -HV and the waste reservoir 8 is held at ground (GND). The electrode 36 and the side channel reservoir 41 are held at a respective positive potential +HV which is, respectively, large enough to maintain an electrospray and maintain charged contaminants in the side channel 40 (or attract them to the reservoir 41 for disposal) but in both cases low enough such as not to attract the compoenets across the barrier flow (which in some embodiments is pressure driven).

During switching (B) of a component band into the branch channel 12, the voltage of the waste reservoir 8 is changed to -HV, slightly negative to create a back flow, or floating and the voltage of the side channel reservoir 41 is increased to ++HV, sufficiently large to create a potential in the branch channel 12 which attracts the components across the barrier flow and into the side channel. Since lighter contaminants such as SDS travel faster, they will be attracted deeper into the side channel 40 in a given amount of time.

Once switching is complete (C), the waste reservoir 8 is set back to GND for separation to continue and the side channel reservoir 41 is also set to GND to stop attracting components across the barrier flow and cause the components and contaminants to travel to the outlet to be electrosprayed, with electrode 36 continuously held at a positive elctrospray voltage such as +HV. However, since the proteins have less far to travel from the side channel 40 into the branch channel 12 the timing and voltage can be arranged such that they reach the branch channel 12 and engage it towards the out let 34 before the contaminants.

At this point in time (D) the side channel reservoir 41 is set back to +HV to prevent or reduce backflow into the branch channel 12 of contaminants while the components continue towards electrode 36 and are electrosprayed while separation continues in the main channel 4.

It will be understood that the pressure driven barrier flow and voltages are adjusted so as to prevent components flowing from the main channel 4 to the branch channel 12 other than at the switching time (B). Further, in some embodiments the electrospray is not operated to be continuous, and electrode 36 is set to FLT at times A and B. Finally, in some embodiments, waste reservoir 8 is maintained at GND at all times for continuous separation in the main channel 4.

As described above, a continuous flow may be maintained at the electrospray electrode 36 by maintaining the electrode 36 continuously at a relatively low potential. To facilitate a continuous electrospray where flow in a branch channel 12 from the barrier region 26 is insufficient to sustain the electrospray, one or more channels to provide to additional fluid flow may be added to the device adjacent the electrospray electrode 36. In one specific embodiment, two channels 42 are arranged symmetrically adjacent to the electrospray electrode 36 to provide additional flow to sustain the electrospray. Advantageously, in some embodiments to achieve efficient electrospray conditions including a smooth laminar flow from the channels 42 surrounding the flow in the branch channel 12 as it reaches the electrospray electrode 36, the channels 42 merge at an oblique angle with the branch channel 12. As depicted schematically in figure 7 by a dotted line, the resulting flows merge smoothly with flow from channels 42 surrounding flows from the branch channel 12, to produce a Taylor cone 44. As an alternative to a flat electrospray electrode 36 on the side of the chip, a micromachined tip 46 could be incorporated into the structure to improve the performance of the electrospray by increasing the field strength at the tip of the electrode structure facing away from the interface regions 34. With both the embodiments of figure 7 and figure 8, a hydrophobic coating may be applied to electrode structures to ensure stable formation of the Taylor cone 44 to assist in the formation of an efficient electrospray.

In both the figure 7 and figure 8 embodiments, the flow inside the channels 42 may be driven electrokinetically, by pressure differential or electrosmotically.

This flexibility allows suitable MS compatible fluids for the channels 42 to be used for example a solution to assist efficient vaporisation and ionisation at the electrospray electrodes, such as a volatile organic solvent. Examples are methanol, acetonitrile and halogenated solvents. The channels 42 are thus particularly advantageous if the rest of the device requires different buffer solutions for best operation since the channels 42 allow to deliver a solution designed to aid in electrospray formation directly to the electrospray electrodes without requiring modification of the buffer solutions in the remainder of the device.

In an alternative embodiment for interfacing the device with a mass spectrometer, a matrix assisted laser desorption ionisation (MALDI) interface could also be coupled to the branch channel 12. In this embodiment, an electrospray electrode is of course not required, although a similar electrode structure 36 (or an internal or coated electrode), with channels 42, as described above may be used. Similarly, as above, the interface region 34 may have a hydrophobic coating to facilitate droplet formation. Rather than creating an electrospray, the electrode 36 is controlled to attract the components into a droplet 48 in the interface region. The droplet may be formed by the action of the components being attracted to the electrodes 36 and/or pressure driven flow from the channels 42 and/or the barrier region. A suitable matrix to allow good laser absorption is introduced into the droplet through the flow from channels 42 to allow good absorption of laser energy in a pulse of laser light 50 supplied from an external laser to evaporate the MALDI droplet.

It will be understood that the above description is of specific embodiments by way of example only and that many modifications, juxtapositions and alterations will be within the skilled person's reach and are intended to be covered by the scope of the appendent claims.

For example, separation techniques other than electrophoresis or capillary electrophoresis, such as liquid chromatography, which would use a pressure driven flow in the main channel, could be used. Likewise, analysis devices other than mass spectrometers may be interfaced with the device with similar benefits, that is a reduction of contaminants associated with the separation process entering the analysis device. One example is inductively coupled plasma atomic emission spectroscopy. Although the interface has been described above in relation to embodiments implementing a barrier flow, it may equally be applied to embodiments without such arrangements, where appropriate. The device is further not limited to a microfluidic chip as described above but other ways of manufacturing the device such as using capillaries for the channels or a combination of capillaries and microfluidic chips are equally possible. For example, a capillary can be used for separation by capillary electrophoresis and can be interfaced with the main channel 4 of the microfluidic device describe above such that components leaving the capillary enter the main channel 4. Further, any appropriate combination of mechanisms for driving flow in the various channels may be used, including but not limited to electrokinetic, electroosmotic, pressure driven, gravitational, and centrifugal flow generation.

Claims

1. A separating device for separating components flowing in a channel, the device comprising: a main channel and main flow means for generating flow of the components in the main channel; a branch channel in fluidic communication with the main channel through an aperture in a wall of the main channel and branch flow means for selectively generating flow of the components from the main channel into the branch channel; and barrier flow means for generating a barrier flow across the aperture between components flowing in the main channel and the aperture.

2. A separating device as claimed in claim 1 in which the main and branch flow means include voltage control means for applying respective voltages to the main and branch channels and are operable to apply a first voltage to the main channel to generate flow in the main channel in a first mode of operation and to additionally apply a second voltage to the branch channel in addition to the first voltage to generate flow of the components from the main channel into the side channel in a second mode of operation.

3. A separating device as claimed in claim 1 or claim 2 in which the main flow means and barrier flow means are arranged to generate their respective flows by different mechanisms.

4. A separating device as claimed in claim 3 in which the main flow means are arranged to generate flow electrokinetically and the barrier flow means are arranged to generate flow by a pressure differential.

5. A separating device as claimed in claim 3 in which the main flow means are arranged to generate flow electrokinetically and the barrier flow means are arranged to generate flow by electro-osmosis.

6. A separating device as claimed in any one of the preceding claims in which the branch channel is in fluidic communication with an outlet for generating an electrospray.

7. A separating device as claimed in claim 6 further including flow control means operable to apply a first potential to the branch channel to create an electrospray in a first mode of operation and to apply a second, higher potential to the branch channel to generate the flow of components from the main channel into the side channel in a second mode of operation.

8. A separating device for separating components flowing in a separation channel, the device comprising a branch channel in fluidic communication with the separating channel; an outlet interface for interfacing the branch channel with an analysis device; and flow control means for selectively diverting components flowing in the separation channel into the branch channel and to the outlet interface.

9. A device as claimed in any one of claims 1 to 8 in which the device further includes a side channel in fluidic communication with the branch channel and flow control means for controlling flow from the branch channel into the side channel.

10. A device as claimed in claim 9, the device including flow control means arranged to produce flow into the side channel for a predetermined period of time to preferentially divert lighter constituents of the fluid flow into the side channel while preferentially leaving heavier constituents of the fluid flow in the branch channel.

1 1. A device as claimed in claim 9 or claim 10 when dependent on claim 7 in which the flow control means are arranged to apply the second potential by applying a potential to the side channel.

12. A device as claimed in any one of claims 9 to 1 1 in which the side channel is filled with a material having a conductivity lower than a material in the branch channel.

13. A device as claimed in any one of claims 8 to 12 further comprising at least one additional flow channel in fluidic communication with the branch channel through an aperture adjacent the outlet interface.

14. A device as claimed in claim 13 in which the additional flow channel is arranged to deliver a matrix for matrix assisted laser desorption or an additive for assisting electrospray formation to the outlet.

15. A device as claimed in any one of claims 8 to 14 in which the analysis device is a mass spectrometer.

16. A device for containing fluid for use with a separating device as claimed in any one of claims 1 to 15, the device defining a main channel, a barrier flow channel merging into a merged portion of the main channel at a junction and a branch channel in fluidic communication with the main channel through an aperture in a wall of the merged portion.

17. A device as claimed in claim 16 in which the branch channel is narrower than the merged portion.

18. A device as claimed in claim 16 or claim 17 in which the main channel across the junction from the merged portion is narrower than the merged portion.

19. A device as claimed in any one of claims 16 to 18 in which the aperture is spaced from the junction.

20. A device as claimed in any one of claims 16 to 19 in which the branch channel is coated with a coating specifically binding a selected chemical for removing the selected chemical from a flow through the branch channel.

21. A method of diverting a flow of a component from a main channel of a separating device for separating components into a branch channel in communication with the main channel through an aperture in the main channel, the method including generating a barrier flow across the aperture and diverting the component into lhe branch channel through the barrier flow.

22. A method as claimed in claim 21 in which the barrier flow comprises a fluid compatible with injection into a mass spectrometer.

23. A method as claimed in claim 21 or claim 22 in which the fluid flow comprises an additive for removing contaminants contained in the main channel as the component traverses the barrier flow.