Development Of Automated High Speed Electrophoretic Separation Systems For Multiple Samples Based On Short Capillaries

As early as1990s, Jorgenson and Lukacs initiated the use of High speed Capillary Electrophoresis (HSCE). HSCE systems based short separation lengths could obtain both high speed and high efficiency over conventional CE systems. Most HSCE systems are built on the basis of microfluidic chips or short capillaries. Microchip-based HSCE systems usually require expensive microfabrication equipments and complicated operation, which may limit their broad application in routine analysis. In contrast to microchips, capillary-based HSCE systems have several practical benefits of easy to build, inexpensive, and convenient to use. Currently, the ability of CE systems to analyze multiple samples and multiple small amount of samples has attracted considerable interest, which is important in a range of applications, including drug discovery, inhibitor screening, catalyst screening.In Chapter1, the applications of short capillary-based high speed capillary electrophoresis systems, as well as four basic sample injection techniques of hydrodynamic, electrokinetic, diffusion and spontaneous injection are reviewed.. The recent progress of sample injection approaches for capillary-based HSCE for multiple samples and small amount of samples is also reviewed.In Chapter2, an automated HSCE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multiwell plate and an x-y-z translation stage. The spontaneous injection method was used to achieve picoliter-scale sample injection from different sample wells. Under the optimized conditions, a40-μm-long sample plug (corresponding to78-pL plug volume) was obtained in a50-μm i.d. capillary, which ensured both the high separation speed and high separation efficiency. Five Fluorescein5-Isothiocyanate (FITC)-labeled amino acids including arginine, phenylalanine, glycine, glutamic acid and asparagine were separated within15s with an effective separation length of1.5cm. Separation efficiencies ranged from7.96×105/m to1.12×106/m (corresponding to1.26μm-0.89μm plate heights) were obtained. The repeatability of the peak heights calibrated with an inner standard for different sample wells were2.4%and2.7%(n=20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of20different samples of FITC-labeled amino acids. The whole separation was achieved in less than6min.In Chapter3, a HSCE system coupled to a two-dimensional droplet array was developed to achieve automated analysis for multiple small amount of samples. The nanoliter-scale droplets containing different samples immersed by oil were formed on a nanowell array chip fixed on an x-y-z translation stage. A novel method for interfacing the HSCE system with droplet array was developed by using the capillary for CE separation as a sampling probe to pass through the oil layer and directly sampling from the droplet without the need of special droplet extraction interface. The influences of the oil type, oil thickness and sample droplet volume were investigated. Picoliter-scale sample injection was achieved using the spontaneous injection method, which ensured both the high separation speed and high separation efficiency with a short separation length. The analysis for different samples was performed by moving the x-y-z stage to sequentially switch different sample droplets to the capillary sampling probe. The performance of the system was demonstrated in the separation of FITC-labeled amino acids with laser-induced fluorescence detection. Three FITC-labeled amino acids including arginine, phenylalanine and glycine were separated within25s with an effective separation length of2cm. The separation efficiencies ranged from5.86×105/m to9.22×105/m, corresponding to1.71μm-1.08μm plate heights. The present system was also applied in consecutive separations of 25different samples of amino acids with a whole separation time less than15min. Moreover, an on-line monitoring to in-droplet derivatizing reaction of amino acids was also achieved.