Shear-force methods are an alternative to pressure- and voltage-gradient methods.
Shear-Driven Flow Approaches to LC and Macromolecular Separations David Clicq
Kris Pappaert Sarah Vankrunkelsven Nico Vervoort Gino V. Baron Gert Desmet f one could get rid of the pressure- and voltage-drop limitations (1) prohibiting the application of larger moVRIJE UNIVERSITEIT bile-phase velocities in HPLC and capillary electrochroBRUSSEL (BELGIUM) matography (CEC), would this open up a new range of separation speeds and resolutions? How can the highpressure pumps and high-voltage supplies used to pulse the flow through the columns be avoided? And does the possibility of performing analytical separations in nanochannels that are only a few times larger than the molecules themselves open the road to new separation mechanisms and methods? Undoubtedly, addressing and solving these questions would have a fundamental impact on the future of LC and macromolecular separations. For the past few years, our group has been working on the development of a whole new family of analytical micro- and nanochannel separation methods, all based on the use of shear forces as an alternative to the customary pressure- and voltage-gradient flow-driving methods. In this article, we give an overview of our progress and the remaining hurdles standing between the proof-of-principle prototypes and practical instruments. Detailed descriptions of our setups and experimental procedures are provided, as well as an overview of the key technologies needed to improve the functionality of the current prototypes. We also explain how a conventional upright or inverted fluorescence microscope can be transformed relatively easily into a shear-driven nanochannel chromatograph, in the hopes that enthusiastic readers will attempt to build their own. Whereas various electrically driven (ED) separation techniques could fairly simply be transformed into commercially available labs-on-chips to perform submillisecond and submicrosecond separations, the downscaling of pressure-driven
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(PD) separations has proven to be much more difficult (2–8). When CEC was invented, the analytical community thought it would address these problems; however, researchers now realize that CEC did not outperform HPLC as easily as was anticipated in the early days of its development (9). CEC still faces many unresolved problems, such as frit reproducibility and varying peak residence times caused by slight changes in the electrolyte concentration or by macromolecular sample impurities (10–12).
(a)
Linear velocity profile
Moving channel wall
d
Stationary Absorptive channel layer or receptor wall molecules
df
(b) Step 1
Step 2 Sample
Step 3 Sample
Step 4 Buffer
Step 5
FIGURE 1. How SDF works. (a) Schematic longitudinal cross-sectional representation of the SDF operating principle. (b) Injection procedure. Step 1: The thin layer of buffer solution occupying the front of the channel inlet is aspirated. Step 2: A small drop of the sample mixture is deposited in front of the channel inlet. Step 3: The moving wall is rapidly displaced (black arrows) over a given distance, typically 110–200 µm. Step 4: The noninjected sample is aspirated and replaced by pure buffer solution. Step 5: The translation stage is restarted to perform the actual separation.
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Principle of shear-driven flows Other approaches have been proposed to avoid the problems and limitations of PD and ED flows. Centrifugal (13) and magnetohydrodynamic (14) forces provide solutions to many of these problems, but the solutions all have inherent weaknesses. The approach we are currently investigating is based on a radical modification of the separation channel concept. Instead of considering channels that are hermetically sealed along their longitudinal axes, we started looking at channels that consist of two separate longitudinal walls: One longitudinal wall is shorter and is attached to a stationary frame, and the other is longer and is translated or rotated past the shorter one. The most straightforward format for such an axially split channel is a dual flat-plate system, in one of which a half-open channel is recessed (the top half of the channel is completely open). When the longer plate is moved past the shorter plate, the fluid present in front of this channel is automatically dragged in, through, and out of the channel by the viscous effect present in every fluid, be it a liquid or a gas (Figure 1a). Basic hydrodynamics tells us that the axial velocity in a laminar flow between two flat plates displays a perfectly linear profile, going from u = 0 near the stationary wall to u = uwall near the moving wall (15). As the viscous drag effect sustains this so-called sheardriven flow (SDF) at literally every point of the channel axis, the flow is generated without any drop or buildup in pressure and the fluid will not leak through the axial split. This result implies that the use of axially split channels, which is the basis of the SDF concept, does not pose any sealing problem. In principle, the channels fill up automatically by capillary action if a droplet of the buffer solution is placed in front of the channel inlet. In practice, the capillary fill step is followed by several forward and backward movements of the moving wall to remove any air bubbles. The capillary force effect also causes the buffer solution to pile up near the front and rear sides of the stationary wall plate (Figure 1a). However, this pileup does not disturb the flow or the separation, and it can easily be avoided if the vertical fronts of the stationary channel substrate are properly coated or if micromachined inlet and outlet reservoirs are provided. The fluid-dragging effect in SDF is similar to that in electroosmotic flow (EOF), in which the double layer of cations generates the bulk flow as it is dragged toward the negative electrode. The only difference is that, in an EOF, the entire channel wall acts as the momentum source (it supports the cations of the double layer dragging along the bulk fluid), whereas in SDF, only half of the total channel-wall surface is used to drive the flow. This difference is also reflected in the mean fluid velocity. In EOF, the mean fluid velocity is equal to the wall velocity (i.e., the velocity of the cations of the double layer); in SDF, the mean fluid velocity is only equal to half of the wall velocity, or um = uwall /2.
(a) Wafer
Spacers Holder
Because the 2 can also be considered a geometrical factor (representing the ratio of the moving channel-wall surface to the total channel-wall surface), this equation only works for a channel with a flat, rectangular cross-section that is wide enough that the surface of the sidewalls can be neglected. It also shows that the SDF method intrinsically is an open-tubular technique: The moving wall cannot be used to generate a significant flow through a packed bed because the stationary surface area in a packed bed is typically orders of magnitude larger than that of the moving wall ever could be. At this point, it is also important to note that the SDF method only works if the sidewalls of the channels are perfectly parallel with the displacement direction of the moving wall. If this is not the case, then a tangential flow component is created near the walls and causes excessive band broadening and leakage problems to occur. SDFs are hence much less suited to bending and turning than PD or ED flows are. In addition, the flow is strongly disturbed if the cross-sectional area of the channel systematically increases or decreases along its longitudinal axis. Channels with nonparallel or continuously converging or diverging sidewalls, for example, would induce a dramatic increase in band broadening. The same holds for channels displaying a sloped or stepped depth profile. However, small-scale, stochastic variations pose fewer problems. Because the fluid velocity is independent of the channel diameter, SDFs are also much less prone to lateral depth variations, which were a major cause of the poor success of PD open-tubular LC in flat, rectangular channels (16). The ability to cope with small lateral depth variations implies that very wide channels can be used in the SDF method. Large optical path lengths can thus be created and reasonable flow rates can be achieved, despite the micrometer or submicrometer depth needed to realize the kinetic advantage of SDF. The first major concern in the development of a micro- or nanochannel-based analytical technique is to uniformly inject exactly metered nano- or picoliter sample volumes over the entire channel’s cross-section. The injection process also must occur swiftly enough to prevent noninjected sample from leaking into the channel. In CE- and CEC-on-a-chip, the injection problem has occupied many researchers, who have had to make numerous adaptations to finally reach a suitable solution (17–19). In SDF, the presence of the moving wall greatly simplifies the injection problem. Instead of being injected via a transversely oriented channel (19), the sample can be shifted directly into the separation channel in the same direction as the main flow; with this approach, the entire channel cross-section is addressed at once (Figure 1b). At the start of the five-step injection process, the thin layer of buffer solution occupying the front of the channel inlet is aspirated. A small drop of the sample mixture is then deposited in front of the channel inlet, and the moving wall is rapidly displaced over a given distance. Because the fluid is moving at half the velocity of the moving wall, the width of the injection plug is exactly equal to half the displacement distance. Most commercially available motorized linear-displacement stages can control this distance to within
