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August/September 2011


Droplet-Based Sample Injection for Chip-Based Analytical Separations


Fiona Pereira1 , Xize Niu1 , Petra S. Dittrich2 & Andrew J. deMello2 *


1. Department of Chemistry, Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, United Kingdom 2. Department of Chemistry and Applied Biosciences, ETH Zürich, CH-8093 Zürich, Switzerland. *Corresponding Author; Andrew.demello@chem.ethz.ch


The advent of capillary and chip-based electrophoresis has opened up new possibilities for high-throughput analysis of a diversity of biological systems. The high-resolution separations typical of chip-based electrophoresis are in large part defined by the ability to introduce small sample volumes into the separation channel. This is not a trivial process and standard electrokinetic and hydrodynamic methods are far from ideal. Herein, we review recent studies that integrate droplet-based and continuous flow microfluidics to provide an alternative injection mechanism that is simple to implement and enables reproducible transport of defined sample volumes without bias.


Electrophoresis remains one of the most powerful tools in separation science. In recent years capillary and chip-based electrophoresis (CE/MCE) formats have been developed and refined to provide rapid, high-throughput and automated analysis of a broad range of targets, including nucleic acids, proteins and peptides, small molecules, enzymes and cells. [1-3] Indeed, benefits including reduced sample and reagent volumes, facile automation and integration with other analytical techniques (such as chromatography and mass spectrometry), improved efficiency and high analytical throughput are inherent to both formats.[4,5] Interestingly, however, whilst there have been extensive studies aimed at controlling separation conditions and capillary/channel surface chemistries, sample injection techniques and post-separation fractionation vary little from the original formats reported over 20 years ago.


As with conventional CE, controlled injection of a small sample volume into a chip-based separation channel is critical when performing high-efficiency electrophoresis. Extra-column band broadening is primarily defined by the quality of the on-column injection and detection procedures. In simple terms, the total peak variance, σ2


tot, is given by pathlengths, σ2 injdominates the extra-column


contribution. Accordingly, larger injected sample plugs will significantly impact σ2


col


and will result in a decrease in the theoretical plate number. In short, higher theoretical plate numbers are contingent on small injection volumes and associated plug lengths.


where σ2


separation channel, σ2 injection and σ2


colis the variance generated in the injis the variance due to


detis the variance originating


from the (finite volume) detection system. Since most on-line optical detection schemes provide for sufficiently small detector


Unfortunately, electrokinetic injection schemes are normally biased towards the introduction of small, high-mobility molecules, even when


The primary methods for introducing a sample plug into a separation channel are hydrodynamic or electrokinetic in nature. During hydrodynamic injection, a defined sample plug is either driven directly into the capillary or into a cross-channel intersection on a planar chip by means of a pressure difference. Hydrodynamic injections are reproducible and also permit accurate quantification of the contained components (due to a lack of injection bias).[6] However, pressure injections require external pumps and valves for on-chip separations, thus increasing system complexity and instrumental footprint. During electrokinetic injections, sample is driven into the separation channel under an applied electric field. A variety of schemes for injection are possible in chip-based systems, however the majority involve an orthogonal crossed channel geometry where sample is electrokinetically conveyed across the separation channel after which the applied field is switched so that only the sample within the intersection is injected for subsequent separation.[7] Importantly, this general approach allows the reproducible injection of extremely small volumes of sample.


using cross-channel injectors in chip-based systems.[8] Accordingly, quantitative analysis of resulting electropherograms can often misrepresent the sample content. To illustrate the hidden complexities of electrokinetic injection schemes Jin and Luo presented a numerical analysis of electroosmotic flow in such systems.[9] Figure 1 illustrates calculated electric potential distributions during a pinched injection (Figure 1A) and subsequent separation (Figure 1B) using a standard cross- channel injection scheme. It can be seen that during the loading step the potential gradient acts to drive sample towards the sample waste vial while preventing it from leaking into the separation channel. During the dispensing and separation stages the potential distribution shifts to permit the plug at the intersection to enter the separation channel with excess sample being “pushed back” towards the sample and sample waste reservoirs. Accordingly, the ratio of electric field strengths in the separation and sample channels control both the confinement of the sample at the intersection and the volume of each species injected. Additionally, it should also be noted that variable surface conditions (a common scenario) on each arm of the cross-piece will cause significant variations in local electroosmotic flow and complicate optimisation of injection protocols. Experimental support of these ideas was provided by Alarie and co-workers who found that pinched injection using a cross-channel injector exhibited a bias towards neutral molecules.[8] They found that bias occurs during both loading and dispensing steps. Indeed, a variation in injected volume of up to 27% was observed, with the degree of bias


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