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Ting Zhang Jinglin Fu Qun Fang Department of Chemistry, Institute of Microanalytical Systems, Zhejiang University, Hangzhou, P. R. China

Received April 7, 2014 Revised June 5, 2014 Accepted June 5, 2014

Research Article

Improved high-speed capillary electrophoresis system using a short capillary and picoliter-scale translational spontaneous injection Here, we describe an improved high-speed CE (HSCE) system using a short capillary and translational spontaneous sample injection. Several important factors for consideration in system design as well as various factors influencing the performance of the HSCE system were investigated in detail. The performance of this HSCE system was demonstrated in electrophoretic separation of FITC-labeled amino acids. Under optimized conditions, baseline separation of eight amino acids and FITC were achieved in 21 s with the plate heights ranging from 0.20 to 0.31 ␮m, corresponding to a separation rate up to 20 700 theoretical plates per second. The separation speed and efficiency of the optimized highspeed CE system are comparable to or even better than those reported in microchip-based CE systems. Keywords: Amino acids / High-speed CE / Short capillary / Spontaneous sample injection DOI 10.1002/elps.201400186



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction Since Jorgenson’s pioneer work in the 1990s [1–4], high-speed CE (HSCE) has become a powerful and versatile analysis technique, which was used in a variety of applications, e.g. multidimensional separations [3–7], in vivo chemical monitoring [8–13], and reaction dynamics studies [14, 15]. HSCE systems can achieve high-speed separations within a few seconds by using short separation lengths from a few millimeters to centimeters, while maintaining high separation efficiencies. There are three key parameters to achieve highspeed and high-efficiency CE separations, including short separation distances, high electric fields, and narrow sample plug [1–4, 16]. It is relatively simple to adjust separation distance and electric field. Thus, the initial sample plug length plays a more critical role in determining the peak width at the detection point. It is a challenge to construct a HSCE system with narrow sample plug introduction [2, 16, 17]. In

Correspondence: Professor Qun Fang, Department of Chemistry, Institute of Microanalytical Systems, Zhejiang University, Hangzhou, 310058, P. R. China E-mail: [email protected] Fax: +86-571-8827-3572

Abbreviation: HSCE, high-speed CE  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

conventional CE systems, sample injection is required to be 0.1–1% of the volume of the capillary column to minimize the band broadening due to overloading [18]. In most of the reported HSCE systems, capillaries less than 5 cm long were used as separation columns. Thus, 50–500 ␮m (corresponding to 100–1000 pL for a 50-␮m-id capillary) is the typical sample plug length range required to achieve high-speed and high-efficiency separations. A variety of injection techniques including optical gating [1,2], flow gating [3,4], microchip-based injection [19–21], short-end injection [22, 23], and flow injection /sequential injection [24, 25] have been developed to achieve subnanoliter sample introduction for HSCE. For optical gating injection [1, 2], an intense laser is used as a gating beam to photobleach the fluorescent analytes that are continuously introduced into the capillary. Sample introduction is accomplished by blocking the gating beam for a short time, thus creating a narrow sample plug in the capillary for the subsequent separation. Although rapid and efficient CE separation can be obtained using this approach, it could only be applied

Current address: Dr. Ting Zhang, Department of Chemistry, Rutgers University at Camden, Camden, NJ 08102, USA. Current address: Dr. Jinglin Fu, Department of Chemistry, Rutgers University at Camden, Camden, NJ 08102, USA. Colour Online: See the article online to view Figs. 1, 3 and 6 in colour.

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for fluorescent analytes with fluorescence detectors. Flow gating injection uses the switching of sample and buffer flows to obtain a narrow sample plug for HSCE separation [3, 4]. Compared with optical gating injection, flow gating injection has the advantages of utilizing a simpler setup, ease of building, and compatibility with different types of detectors, such as fluorescence, absorption, and electrochemical detectors. However, the relatively slow switching speed for the gating flow limits the size of achievable sample plug in the hundreds of micrometer range. In addition, both the above-mentioned approaches suffer from the sample biasing effect due to electrokinetic injection. HSCE has also been achieved in microfluidic chips [19–21]. Sample injection for microfluidic chips is typically accomplished by electroosmotically loading the sample into a volume-fixed region forming a sample plug, and then applying the separation voltage to pull the sample plug into the separation channel. However, the fabrication of glass microchips usually requires expensive equipments and complicated operation [19–21, 26, 27]. Shortend injection [22, 23] can be performed with commercial CE instruments. Sample was introduced from the outlet end of the capillary, which reduces analysis time and increases detection sensitivity. Fang and co-workers coupled flow injection/sequential injection devices to CE systems through a split-flow sampling interface [24, 25]. A series of different samples could be continuously introduced into the separation channel by electrokinetic or diffusion injection. In 2005, the authors’ group developed an automated and high-throughput nanoliter-scale sample introduction approach on the basis of a capillary sampling probe and a slottedvial array [28, 29]. Samples/carriers were filled in an array of vials, on the bottom of which slots were fabricated for free passage of the sampling probe. Sample introduction was performed by linearly moving the slotted-vial array to immerse the capillary inlet and electrode in the sample vial, and electrokinetic sample introduction was accomplished by applying voltage between sample and waste vials for a definite time. CE systems using this approach were applied in analysis of drugs [30], amino acids [31], and DNA fragments [32]. However, the amount of sample injected was typically in the nanoliter range, which was excessive for capillaries with only a few centimeters length. Thus, the separation efficiencies of these systems were limited by the excessive sample amount and could not be comparable to those of the above-motioned HSCE systems [19–21]. To address this limitation, in 2009, we reported a novel microfluidic spontaneous sample introduction approach capable of introducing sub-100-pL sample plug using a translational injection system and surface tension driving effect [33]. Using this sample introduction approach, a HSCE system based on a short capillary was developed, with separation speed and efficiency comparable to or even better than those reported in microfluidic chip-based CE systems. This sample introduction approach was further applied in fast separations of DNA fragments [34], proteins [35] and multiple amino acid samples [36]. In this paper, several important factors for consideration in system design as well as various factors in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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fluencing the performance of the short capillary-based HSCE system were studied in detail. The performance of this HSCE system was significantly improved as demonstrated in electrophoretic separation of FITC-labeled amino acids with plate heights ranging from 0.20 to 0.31 ␮m.

2 Materials and methods 2.1 Reagents Unless otherwise stated, analytical grade reagents and deionized water were used in all described experiments. Five micromolar borate buffer (pH 9.2) was used as working electrolyte for CE separation. Solutions containing 10 mM of each amino acid (Kangda Amino Acid Works, Shanghai, China) were prepared in 50 mM borate buffer (pH 9.2). The labeling reagent (10 mM FITC, Sigma Chemicals, St. Louis, USA) was prepared by dissolving 3.89 mg of FITC in 1 mL of acetone. FITClabeled amino acid stock solutions containing each amino acid at 1 mM were prepared by mixing 0.1 mL of stock solutions of each amino acid with 0.1 mL of FITC reagent and 0.8 mL of 50 mM borate buffer (pH 9.2), and the resultant mixtures were allowed to stand overnight in the dark. Before use, sample solutions containing a mixture of 1 ␮M of each amino acid were prepared by diluting the individual stock solutions and mixing them at an equal volume ratio.

2.2 HSCE system The setup of the HSCE system is similar to that previously reported (Fig. 1). Fused-silica capillaries (50 ␮m id, 335 ␮m od without polyimide coating, Refine Chromatography, Yongnian, China) with a length of 3 or 6.5 cm were used as separation columns. The capillaries were carefully cut using a ceramic cleaving stone (Polymicro Technologies, Phoenix, USA) to get flat ends. The inlet end of the capillary was ground into a cone using sandpapers of various coarseness. The polyimide coating of the capillary was completely removed with hot, concentrated sulfuric acid [33]. The outer surface of the inlet end was silanized by holding an acetonefilled capillary in dimethyldichlorosilane (Sinopharm Chemical Reagent, Shanghai, China) for 10 min and then ethanol for 10 min to produce a hydrophobic surface to reduce sample carryover. The sandwich-type reservoirs for sample and buffer solutions were made by stacking multiple layers of glass. The sample reservoir was composed of a top and bottom plates (1.7 mm thick) sandwiched around a 1-mm-thick plate used as a spacer. This arrangement formed a space of L20 × W10 × D1 mm to hold sample solution. The buffer and waste reservoirs had similar structure as sample reservoir, except that larger top and bottom plates and a thicker spacer were used, forming a space of L20 × W14 × D2 mm to hold buffer solution. The sample and buffer reservoirs were fixed on a computer-programmed translation stage (TSA150-AB, Zolix www.electrophoresis-journal.com

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buffer reservoir and to the waste reservoir through a highvoltage relay (GRL-1202, Hangzhou Zhonghuo Electronic, Hangzhou, China, see Supporting Information for a detailed description), with the buffer reservoir grounded. A lab-made high-voltage control system was used to achieve rapid switching of the high-voltage relay, according to the position of the buffer reservoir. A small piece of black plastic as a mask was fixed on the computer-programmed translation stage in an appropriate place. When the mask was not inserted in the slot between the emitter and receiver of the optical coupler, the emitter lighted, which triggered the optical receiver. The high-voltage electromagnetic relay was opened, and thus the high voltage was cut off. As soon as the capillary inlet was immersed in the buffer reservoir, the mask was just inserted into the slot of the optical coupler, the electromagnetic relay was closed, and high voltage was rapidly supplied to the electrodes.

2.3 CE operation procedure

Figure 1. Schematic diagram of the HSCE system. (A) Sample introduction stage. (B) CE separation stage.

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The procedure for the CE separation was similar to that previously reported [33]. Briefly, before use, the reservoirs and the capillary were sequentially rinsed with 1 M NaOH solution followed by water and then running buffer. Twenty microliter sample solution was filled in the sample reservoir, and 500 ␮L working electrolyte in the buffer and waste reservoirs. Two platinum electrodes were inserted into the buffer and waste reservoirs. The capillary was horizontally fixed onto a glass plate to facilitate better heat dissipation and easier handling. The capillary inlet and outlet were inserted into the working electrolyte solution in the buffer and waste reservoirs positioned on the same level, respectively. The spontaneous sample injection was performed by linearly moving the translation stage to switch the capillary inlet first to the sample reservoir without high voltage applied, and then back to the buffer reservoir. The high voltage for CE separation was applied when the capillary inlet entered the working electrolyte solution of the buffer reservoir. On-capillary detection was performed using an in-house built confocal laser induced fluorescence detection system (see Supporting Information for details).

3 Results and discussion Figure 2. Schematic diagram of the high-voltage control system.

Instruments, Beijing, China), which was controlled by a motion controller (SC300–1B, Zolix Instruments). A home-built high-voltage power supply was used for all CE separations. A 10 k⍀ potentiometer was connected to the high-voltage module (DW-N602–1F, 0 to −6000 V, Tianjin Dongwen High Voltage Power Supply, Tianjin, China) to control the output voltage. As depicted in Fig. 2, the output of the high-voltage power supply was applied directly to the  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.1 Design of the sandwich-type sample and buffer reservoirs As shown in Fig. 3A, the liquid drop in the sandwich-type reservoir was held between the two glass plates and forms a “sandglass” shape. The capillary force generated by the hydrophilic glass surfaces produces a side view profile with a negative R1 , while the retraction force from the liquid–gas interface produces a top view profile with a positive R2 . In the reservoir, the two glass plates are separated with a distance www.electrophoresis-journal.com

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For the buffer drop:  Plb = ␥

Figure 3. Design of the sample and buffer reservoirs (A) and electropherograms obtained when the capillary inlet was immersed in the sample solution for 1 s (B) and 10 s (C). Sample introduction and CE separation conditions: inner diameter of the capillary, 50 ␮m; effective separation length, 15 mm; removing speed of the sample solution, 3.1 mm/s; immersion depth of the capillary tip in the sample solution, 350 ␮m; electric field strength, 900 V/cm; sample, 1 ␮M FITC-labeled amino acids; working electrolyte, 5 mM borate buffer (pH 9.2).

H, and the contact angle of liquid on glass plate is ␪. Then R1 could be calculated from: R1 = −

H/ 2 . cos ␪

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1 1 + R1 R2

 ,

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where Pl is the pressure difference in the liquid drop, ␥ is the liquid surface free energy, and R1 and R2 are the radii of curvature of the liquid–gas interface in directions vertical (side view) and parallel (top view) to the drop, respectively. Substituting Eq. (1) into Eq. (2), the Laplace pressure difference within the drop could be given by:   1 cos ␪ − . (3) Pl =␥. H/ R2 2 For the sample drop:   1 cos ␪s − Pls = ␥ , Hs/ R2s 2

(4)

where Pls is the Laplace pressure difference in the sample drop, R2s is the radius of curvature in the direction parallel to the sample drop (top view), ␪ s is the contact angle of sample drop on glass, and Hs is the distance between two glass plates of sample reservoir. In our system, Hs = 1 mm, R2s = 2.5 mm. Using the surface free energy of water and the contact angle of water on glass as approximation, ␥ = 0.072 N/m, ␪ s = 25°, the obtained Pls is −101.71 Pa.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1 cos ␪b − Hb/ R2b 2

 ,

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where Plb is the Laplace pressure difference in the buffer drop, R2b is the radius of curvature in the direction parallel to the buffer drop (top view), ␪ b is the contact angle of buffer drop on glass, and Hb is the distance between two glass plates of buffer reservoir. In our system, Hb = 2 mm, R2b = 7 mm. Using the surface free energy of water and the contact angle of water on glass as approximation, ␥ = 0.072 N/m, ␪ b = 25°, the obtained Plb is −54.97 Pa. Considering the difference in liquid levels between the inlet and outlet of the capillary when they are immersed in the sample and waste reservoirs, respectively, the total pressure difference across the capillary during sample introduction is:

⌬ p=Pls − Plb − ␳g h,

(6)

where ␳ is the density of the liquid, g is the gravitational acceleration, and h = (Hb −Hs ) × 0.5. Using the density of water as approximation, ␳ = 997.044 kg/m3 , and g = 9.80665 m/s2 , the obtained ⌬ p = −51.63 Pa. Under this pressure, the buffer solution is driven through the capillary from the outlet to the inlet. The volume rate of flow (dV/dt) in the capillary is given by the Hagen–Poiseuille equation: d V ␲⌬ pr 4 = , dt 8␩L

(7)

where ␩ is the viscosity of the liquid, L is the length of the capillary, r is the inner radius of the capillary, and ⌬ p is the total pressure difference across the capillary. For a 3-cm long capillary (50 ␮m id), with the viscosity of water as ␩ = 0.00089 Pa·s, the calculated volume rate from the outlet of the capillary to the inlet is 0.297 nL/s. As a result, when the capillary inlet is immersed in the sample solution, sample injection caused by hydrostatic pressure, diffusion, or convection can be effectively avoided. Although a tiny amount of buffer solution flows into the sample solution, the dilution effect of buffer solution on sample solution is neglectable. The volume of the buffer solution (297 pL in 1 s) is only ca. 1/70 000 of the sample solution (20 ␮L). The definition of all the equation terms is also summarized in Supporting Information. Figure 3B and C show the electropherograms obtained by translational sample injection and subsequently CE separation, when the capillary inlet was immersed in the sample solution for 1 and 10 s, respectively. The peak shape, migration time, and separation efficiency of the five amino acids and FITC in the two electropherograms do not show obvious differences. Therefore, it is demonstrated that interferences of other sample injection mode caused by hydrostatic pressure, diffusion, or convection can be effectively eliminated by using such a special sample/buffer reservoir design. www.electrophoresis-journal.com

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3.2 Optimization of the separation electric field Both theory and practice show that the separation electric field largely affects the speed, efficiency, and resolution of CE separations. Application of a high electric field will reduce the analysis time, but may result in increases or losses of separation efficiency and resolution. Normally, the higher the electric field applied, the higher the plate number and resolution that can be attained, unless excessive Joule heating generated within the capillary at the higher applied electric fields. We optimized the operating electric field first by measuring the current I passing through the capillary as a function of the electric field strength E in the short capillary. The I-E curve was linear between 100 and 900 V/cm. Significant deviations from linear behavior were observed at the time the electric field exceeded 1000 V/cm. Thus, electric field strengths up to 900 V/cm could be used without serious Joule heating effect of the buffer solution. Figure 4 shows electropherograms of a mixture of amino acids obtained under different separation electric fields. As

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Figure 4. Electropherograms obtained at different separation electric fields in the range of 100–1300 V/cm. Other conditions as in Fig. 3B.

expected, significant decrease of migration time was observed when increasing the electric field in the range of 100–1300 V/cm. In Fig. 5, the plate height of the peak of phenylalanine and the resolution of adjacent peaks of phenylalanine and glycine are plotted as functions of the electric field strength. With the increase of the electric field in the range of 100–900 V/cm, both the separation efficiency and resolution significantly increased. A minimum plate height of 0.49 ␮m was observed when the field strength was 900 V/cm. At higher field strengths, the separation efficiency and resolution decreased as a consequence of Joule heating induced plug dispersion. Therefore, in the following HSCE experiments, a separation electric field of 900 V/cm was employed, unless mentioned otherwise.

3.3 Effect of the fixing method for the capillary When high electric fields are applied in electrokinetic separation systems, Joule heat will be inevitably generated. Joule

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heat will influence physical properties of the working electrolyte such as temperature and viscosity, and thus reduce separation efficiency of CE systems. Most conventional CE instruments are equipped with cooling devices to dissipate heat generated during the separation. Typical operating electric field ranges from 100 to 500 V/cm. Capillary cooling devices usually have complicated structure and require good insulation and sealing designs. On the contrary, it is relatively difficult to make cooling devices for HSCE systems with short capillaries of a few centimeters lengths. Because the power generated by Joule heating is proportional to the square of the inner diameter of the capillary, in most of the HSCE systems, capillaries with smaller inner diameter (20 ␮m) are used to reduce the Joule heating effect at high electric fields. However, such finer capillaries increase the difficulty of detection and the risk of capillary blockage. The influence of Joule heat on the separation efficiency has been greatly reduced in microchip-based CE systems, due to the larger surface-to-volume ratio of the microchannels and the larger surface area of the glass chip. As a result, high electric field in the range of 500–2000 V/cm can be used for glass CE chips. In this work, the polyimide coating of the capillary was completely removed by hot, concentrated sulfuric acid [33] and horizontally fixed onto a glass plate as shown in Fig. 6A–C. Fixation of the capillary on a glass plate facilitated better heat dissipation as well as easier handling. This permitted higher electric fields than that applied in an unfixed capillary, which resulted in more efficient and rapid separation (Fig. 6D and E). We also compared the performance of CE separations obtained under the same electric field using capillaries fixed and unfixed on a glass plate, and the electropherograms are shown in Fig. 6E and F. The separation efficiency and resolution of Fig. 6F is obviously inferior to that of Fig. 6E. Some peaks of unknown impurities, which could be separated from the main peaks of amino acids in Fig. 6E, could not be effectively resolved in Fig. 6F. This indicates that the heat generated within the capillary unfixed under high electric field could not effectively dissipate through the capillary walls, which led to the decreases in separation efficiency and resolution.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 6. The method for fixing the capillary and its influence on CE separation. (A) Front view, (B) top view, and (C) side view of the capillary fixed on a glass plate by epoxy. Electropherograms obtained using capillaries fixed on a glass plate (E) or not (D and F). Electric field strength for (D), 700 V/cm; electric field strength for (E and F), 900 V/cm; effective separation length, 50 mm; other conditions as in Fig. 3B.

3.4 Effect of the sample solution volume filled in the sample reservoir We varied the volume of the sample solution filled in the sample reservoir to study its influence on translational sample injection and CE separation. The initial volume filled in the sample reservoir was 40 ␮L then half of the sample www.electrophoresis-journal.com

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mediately to start the electrophoretic separation. Otherwise, sample loss would also occur due to molecular diffusion or convection. Figure 7A–C shows the electropherograms obtained with different time delay between immersing the capillary inlet in the buffer solution and applying the separation electric field. Obvious decrease in peak areas was observed when there was a time delay of 1 or 2 s (Fig. 7B–C), which indicated that sample loss occurred during these short periods. Therefore, we developed a high-voltage controller to control the ON/OFF state of the high voltage (Fig. 2), according to the position of the buffer reservoir. The rapid and accurate switching of the electric field could be achieved by adjusting the position of the mask on the translational stage, so that the diffusion- or convection-induced sample loss could be avoided maximally.

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solution was successively removed to reduce the sample solution volume to 20, 10, 5, and 2.5 ␮L. The electropherograms obtained with different volume of sample solution filled in the sample reservoir did not show any obvious differences in migration time, peak height, peak width, and other performances. It was observed that, when the volume was reduced to 2.5 ␮L, the sample solution would be drawn by the moving capillary inlet immersed in it. In addition, considering the influence of evaporation of the sample solution, 20 ␮L of the sample solution was filled in the sample reservoir in the HSCE experiments. 3.5 Effect of the electrospray interference on the sample injection In the preliminary studies, the electric field strength applied between the buffer and waste reservoirs was kept constant during the sample injection and electrophoretic separation process without interruption. After the sample injection, when the buffer solution was moved back to its initial position and close to the capillary inlet, we observed an electrospray from the capillary inlet tip to the buffer solution. Such an electrospray of the sample solution led to the loss of sample plug in the capillary tip. In the present system, when the electric field strength exceeded 300 V/cm, the influence of electrospray on sample loss was obvious; no sample peak was obtained in the CE separation. Therefore, the electric field should be cut off before the capillary inlet was reimmersed in the buffer solution. On the other hand, once the capillary inlet was immersed in the buffer solution, the electric field should be applied im C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

A capillary with smaller inner diameter (25 ␮m) (Fig. 8B1) was used to reduce the Joule heat effect, thus higher electric field could be applied in electrophoretic separation. Figure 8B2 shows a typical electrophoregram at an electric field of 1700 V/cm. High separation efficiencies ranging from 2 880 000 to 3 730 000 theoretical plates per meter were obtained within 3.5 s. Compared with results in Fig. 8A2, higher separation efficiency was obtained with less separation time by using a narrower capillary column. This indicates that more effective and faster CE separation could be achieved by further reducing the inner diameter of the capillary. However, such finer capillaries increase the difficulty of detection, and the risk of capillary blockage.

3.7 Performance of the HSCE system To evaluate the repeatability of the system, consecutive CZE separations of a mixture of FITC-labeled amino acids were

Figure 8. Images of tapered capillary tips with different inner diameters and electropherograms obtained using them as separation columns. The capillary inner diameters in (A) and (B) are 50 and 25 ␮m, respectively; electric field strengths for (a2) and (b2) are 900 and 1700 V/cm, respectively; other conditions as in Fig. 3B.

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performed using different separation lengths. As shown in Fig. 9A, using a short separation length of 5 mm, peak height precisions ranging from 3.0% RSD for fluorescein thiocarbamyl (FTC)-Asp to 2.1% RSD for FTC-Arg were achieved in 30 consecutive separations with a high analysis throughput of 159 h−1 . In this case, we sacrificed much of the separation efficiency and resolution to increase the overall analysis speed. Two acidic amino acids, glutamic acid and aspartic acid, could not be effectively separated. In order to balance the separation efficiency and speed, the effective separation length was increased to 50 mm. As shown in Fig. 9B, baseline separation of eight amino acids and FITC were achieved in 21 s with the plate heights ranging from 0.20 to 0.31 ␮m. Arginine was separated at a rate up to 20 700 theoretical plates per second. Although the high-voltage supply was not very stable at the output near its maximum, the peak height precisions  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 9. Electropherograms of consecutive separations with effective separation length of 5 mm (A) and 50 mm (B). Other conditions as in Fig. 3B.

ranging from 5.9% RSD for FITC to 4.6% RSD for FTC-Arg were still achieved in ten consecutive separations.

4 Concluding remarks In this work, the HSCE system using a short capillary has been developed and optimized, without resorting to microfabrication techniques. Several important factors including the structure of reservoirs, the fixation of capillary column, and the interference of electrospray effect, were investigated and optimized to improve sample introduction and CE separation. Using FITC-labeled amino acids as sample, the separation speed and efficiency of the HSCE system are comparable to or even better than those reported in microchip-based CE systems.

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Electrophoresis 2014, 35, 2361–2369

Due to its simplicity in system building and operation, its ability in high-speed and high-efficiency separation, and its versatility in use, this HSCE system is an attractive option in a range of applications and a valuable complement to the repertoire of currently available HSCE systems based on various sample introduction techniques [1–4, 19–25]. Moreover, the present system has great potential to be further simplified to a portable HSCE instrument with integrated automated sample injector, miniaturized detector, and high-voltage power supply. It could be applied to broad CE-based diagnosis and bioanalysis areas, especially in field analysis or point-of-care testing. This work was supported by the National Natural Science Foundation of China (Grants 20825517, 21027008, and 21227007) and the Ministry of Science and Technology of China (Grant 2007CB714503). The authors are grateful to Drs. Wenbin Du, Tao Zhang, and Jianzhang Pan for inspiring discussions. The authors have declared no conflict of interest.

CE and CEC

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[13] Yang, P., Kennedy, R. T., J. Chromatogr. A 2008, 1194, 225–230. [14] Moore, A. W. Jr., Jorgenson, J. W., Anal. Chem. 1995, 67, 3464–3475. [15] Yang, P., Whelan, R. J., Mao, Y., Lee, A. W. M., Carter-Su, C., Kennedy, R. T., Anal. Chem. 2007, 79, 1690–1695. [16] Kennedy, R. T., German, I., Thompson, J. E., Witowski, S. R., Chem. Rev. 1999, 99, 3081–3131. [17] Kennedy, R. T., Anal. Chim. Acta 1999, 400, 163–180. [18] Gordon, M. J., Huang, X., Pentoney, S. L., Zare, R. N., Science 1988, 242, 224–228. [19] Harrison, D. J., Fluri, K., Seiler, K., Fan, Z. H., Effenhauser, C. S., Manz, A., Science 1993, 261, 895–897. ¨ [20] Jacobson, S. C., Hergenroder, R., Koutny, L. B., Ramsey, J. M., Anal. Chem. 1994, 66, 1114–1118. ¨ [21] Jacobson, S. C., Koutny, L. B., Hergenroder, R., Moore, A. W. Jr., Ramsey, J. M., Anal. Chem. 1994, 66, 3472–3476. [22] Altrial, K. D., Kelly, M. A., Clark, B. J., Chromatographia 1996, 43, 153–158. [23] Glatz, Z., Electrophoresis 2013, 34, 631–642.

5 References

[24] Fang, Z. L., Fang, Q., Fresenius J. Anal. Chem. 2001, 370, 978–983.

[1] Monning, C. A., Jorgenson, J. W., Anal. Chem. 1991, 63, 802–807.

[25] Fang, Q., Wang, F. R., Wang, S. L., Liu, S. S., Xu, S. K., Fang, Z. L., Anal. Chim. Acta 1999, 390, 27–37.

[2] Moore, A. W. Jr., Jorgenson, J. W., Anal. Chem. 1993, 65, 3550–3560.

[26] Gabriel, E. F. M., Coltro, W. K. T., Garcia, C. D., Electrophoresis 2014, 35, 2325–2332.

[3] Lemmo, A. V., Jorgenson, J. W., Anal. Chem. 1993, 65, 1576–1581.

[27] Fang, Q., Xu, G. M., Fang, Z. L., Anal. Chem. 2002, 74, 1223–1231.

[4] Hooker, T. F., Jorgenson, J. W., Anal. Chem. 1997, 69, 4134–4142.

[28] Du, W. B., Fang, Q., He, Q. H., Fang, Z. L., Anal. Chem. 2005, 77, 1330–1337.

[5] Larmann, J. P. Jr., Lemmo, A. V., Moore, A. W. Jr., Jorgenson, J. W., Electrophoresis 1993, 14, 439–447.

[29] Fang, Q., Shi, X. T., Du, W. B., He, Q. H., Shen, H., Fang, Z. L., Trends Anal. Chem. 2008, 27, 521–532.

[6] Moore, A. W. Jr., Jorgenson, J. W., Anal. Chem. 1995, 67, 3448–3455.

[30] Liu, J., Fang, Q., Du, W. B., Chin. J. Anal. Chem. 2005, 33, 1799–1802.

[7] Moore, A. W. Jr., Jorgenson, J. W., Anal. Chem. 1995, 67, 3456–3463.

[31] Xu, Z. R., Lan, Y., Fan, X. F., Li, Q., Talanta 2009, 78, 448–452.

[8] Lada, M. W., Vickroy, T. W., Kennedy, R. T., Anal. Chem. 1997, 69, 4560–4565.

[32] Fan, X. F., Li, Q., Wang, S. L., Xu, Z. R., Du, W. B., Fang, Q., Fang, Z. L., Electrophoresis 2008, 29, 4733–4738.

[9] Thompson, J. E., Vickroy, T. W., Kennedy, R. T., Anal. Chem. 1999, 71, 2379–2384.

[33] Zhang, T., Fang, Q., Du, W. B., Fu, J. L., Anal. Chem. 2009, 81, 3693–3698.

[10] Bowser, M. T., Kennedy, R. T., Electrophoresis 2001, 22, 3668–3676.

[34] Cheng, Y. Q., Yao, B., Zhang, H. D., Fang, J., Fang, Q., Electrophoresis 2010, 31, 3184–3184.

[11] Shou, M., Smith, A. D., Shackman, J. G., Peris, J., Kennedy, R. T., J. Neurosci. Methods 2004, 138, 189–197.

[35] Lin, Q. H., Cheng, Y. Q., Dong, Y. N., Zhu, Y., Pan, J. Z., Fang, Q., Electrophoresis 2011, 32, 1–6.

[12] Shou, M., Ferrario, C. R., Schultz, K. N., Robinson, T. E., Kennedy, R. T., Anal. Chem. 2006, 78, 6717–6725.

[36] Li, Q., Zhang, T., Zhu, Y., Cheng, Y. Q., Lin Q. H., Fang, Q., Electrophoresis 2013, 34, 557–561.

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Improved high-speed capillary electrophoresis system using a short capillary and picoliter-scale translational spontaneous injection.

Here, we describe an improved high-speed CE (HSCE) system using a short capillary and translational spontaneous sample injection. Several important fa...
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