Binary Electroosmotic-Pump Nanoflow Gradient Generator for

Nov 17, 2014 - pump can be manufactured at a cost of a few hundred dollars. However, it is ... The electroosmotic pump (EOP) is one of the most promis...
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Binary Electroosmotic-Pump Nanoflow Gradient Generator for Miniaturized High-Performance Liquid Chromatography Lei Zhou,† Joann Juan Lu,‡ Congying Gu,‡ and Shaorong Liu*,‡ †

College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States

‡

S Supporting Information *

ABSTRACT: High-performance liquid chromatography (HPLC) plays an important role in biotechnology, and a majority of chromatographic separations use gradient elution. While gradient generators can be built in different formats, binary pumps or quaternary pumps are most frequently used for gradient generator constructions. We have recently developed a high-pressure electroosmotic pump (EOP); the pump can be manufactured at a cost of a few hundred dollars. However, it is challenging to use this pump to deliver a gradient eluent directly. In this study, we first improve the monolith preparation by applying a pressure to the monomer solution during polymerization. We assemble a binary EOP gradient generator and discuss the relationship between the gradient profile and voltage applied to the EOP. We demonstrate the feasibility of the binary EOP gradient generator for generating a smooth and reproducible nanoflow gradient. After integration of the gradient generator into a miniaturized HPLC system, we use the HPLC system for separating peptide mixtures from trypsin-digested proteins. The performance comparison between the above miniaturized HPLC system and an Agilent 1200 HPLC system exhibits comparable efficiencies, resolutions, and peak capacities.

T

prefilled with a series of eluents with increasing elution power. The valve is operated in such a manner that the weakest eluent is delivered to the column first, and then the next stronger eluent, and so on. Because only a limited number of injection loops can be incorporated into the valve, the choices of gradient profiles are constrained. Deguchi et al.14 addressed this issue by utilizing a ten-port switching valve with two injection loops, combined with a conventional gradient delivery system, for delivering an unlimited number of (gradient) eluent segments. Basically, a conventional gradient delivery system was employed to form a gradient eluent, and the gradient was delivered to the ten-port switching valve, loading one of the two loops. As the valve switched, the eluent loaded into the first loop was delivered to the column, while the second loop was loaded with a slightly stronger eluent. Since these operations could be repeated without constraints, any desired gradients could be obtained. Brennen et al.15 tested a microchip gradient generator to particularly reduce the dead volume between the gradient front and the HPLC column; the volume would contribute directly to the gradient delay time. The chip used a few microliters of two eluents to passively form a gradient, and the

he development and application of miniaturized liquid chromatographic systems have received increasing attention for the benefits of reduced cost, portability, and low consumption of sample and reagent.1−3 Compared to a conventional liquid chromatography (LC) system, a miniaturized LC system is particularly advantageous when it is used in conjunction with a mass spectrometer, because all of its effluent can be directly introduced into the mass spectrometer. To construct such an LC instrument, a miniaturized highpressure pump is essential. Although numerous micropumps have been developed,4 not many can produce high pressures. The electroosmotic pump (EOP) is one of the most promising micropumps that have shown potential for practical highpressure liquid chromatography HPLC separations.5,6 We have recently developed some high-pressure EOPs,7−10 and the pump could be made at a cost of a few hundred dollars, compared to $20K−30K for a commercial high-pressure nanoflow pump. However, owing to the nature of electroosmotic pumping, it is challenging to use these pumps to deliver gradient eluents directly. Gradient elution is almost required when researchers use HPLC for separating molecules in complex biological samples.11,12 Various nanoflow gradient generators have been developed, but none are completely satisfactory. Cappiello et al.13 applied a switching valve holding a set of injection loops © 2014 American Chemical Society

Received: August 27, 2014 Accepted: November 17, 2014 Published: November 17, 2014 12214

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ready for the monolith preparation. To produce a monolith inside the above capillary, a solution containing 5 mg of AMPS (1% (w/w unless otherwise indicated), 115 mg of BMA (23%), 80 mg of EDMA (16%), 210 mg of 1-propanol (42%), 60 mg of 1,4-butanediol (12%), and 30 mg of water (6%) was prepared, and 2 mg of AIBN was added into the solution as an initiator. The solution was mixed ultrasonically into a homogeneous solution and purged with helium for 10 min. This solution was then introduced into a 20 cm long vinylized capillary. The two ends of the capillary were connected to a microtee (VICI, Valco Instruments, Houston, TX), and the third port of the microtee was connected to an HPLC pump (see Figure S1 in the Supporting Information for details). A pressure of ∼1000 psi was applied to the polymerizing solution while the reaction was allowed to proceed at 60 °C in a water bath for 20 h, forming a negative monolith immobilized inside the capillary. The monolith was then washed with acetonitrile under ∼1500 psi for 12 h to remove the unreacted monomers and porogens and equilibrated with a pump solution (PS; 3.0 mM sodium acetate, pH 5.0) for 30 min. The ends of the capillary were cut off to obtain the desired length for experimentation. Scanning electron micrographs were acquired to examine the column morphology and pore structure using a Zeiss NEON high-resolution scanning electron microscope after the monolith was coated with approximately 4 nm of Ir using an Emitech K575D sputter coater. The maximum flow rate (Qmax, the flow rate at a zero backpressure) and maximum pumping pressure (Pmax, the backpressure at a zero flow rate) of an EOP at a given high voltage were measured as described previously.8,10 Briefly, an empty capillary with a 200 μm i.d. was connected by a union to the outlet of an EOP. As the EOP was turned on, the meniscus of the PS inside the empty capillary was monitored and measured using a microscope. The maximum flow rate of the EOP was calculated by Qmax = 314L/t (nL/min), where L (cm) is the length the meniscus moved within a time period of t (min). A flow-through pressure sensor (model 602192-2, Senso-Metrics Inc., Simi Valley, CA) was used for measuring the maximum pumping pressure. The sensor channel was first flushed with methanol at a flow rate of 1 mL/min for 10 min to remove possible trapped air bubbles. After the sensor was rinsed with the PS, the outlet of the pump assembly was connected to the sensor inlet while the sensor outlet was blocked using a stainless steel plug from IDEX (Lake Forest, IL). A high voltage (HV) was then applied to the pump assembly, and the pressure number on the sensor indicator increased gradually. The Pmax value was recorded as the pressure became stabilized. Preparation of a Polyacrylamide-Based Bubbleless Electrode. A polyacrylamide-based bubbleless electrode was prepared as previously described, but with minor modifications.17 A degassed solution containing 10% T (total weight concentration of acrylamide and Bis in the solution), 2% C (Bis concentration relative to that of acrylamide), 0.2% (v/v) TEMED, and 0.1% APS was pressurized into a 20 cm long vinylized capillary. The polymerization reaction was allowed to proceed inside the capillary at 0 °C overnight under a pressure of ∼300 psi and then at 4 °C for another 24 h. After both ends of the capillary were trimmed off by 1 cm, the capillary was cut into 2 cm long segments and stored in a pump solution; each segment served as a polyacrylamide-based bubbleless electrode. Integration of the Bi-Egg into a Miniaturized HPLC System. Figure 1 presents a schematic diagram of a

dead volume was reduced to a few nanoliters. While these approaches were utilized successfully for several applications, there were several drawbacks, including a complicated system design, tedious operations, stepwise gradient profiles, etc. We have developed two approaches for generating an eluent gradient and performed gradient HPLC separations successfully.10,16 In one approach,10 we preloaded two eluent segments consecutively into a capillary (with the weaker eluent in front), and this capillary was then connected to the outlet of the EOP. As the EOP drove the eluent segments forward, the two solutions were mixed, forming a gradient eluent at the interfacial region. We then used this gradient for peptide separations. In a second approach,16 we utilized an approach to that of Deguchi et al.,14 but we replaced Deguchi’s conventional gradient delivery system with an autosampler. While these approaches reduced the physical size of the LC system, their operations were still tedious, and obtaining a specific gradient profile or tuning a gradient profile precisely was difficult. To address this problem, here we construct a binary EOP gradient generator (called bi-Egg) using a negatively charged polymer monolith as the pumping element. We develop formulas that guide us to apply voltages on the EOPs to achieve the desired gradient profiles. After integration of the bi-Egg into a miniaturized nanoflow HPLC system, we utilize this HPLC system for separating peptide mixtures from trypsin-digested proteins.

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EXPERIMENTAL SECTION Reagents and Materials. [(Methacryloyloxy)propyl]trimethoxysilane (γ-MAPS; 98%) was purchased from Acros (Fairlawn, NJ). Ethylene glycol dimethacrylate (EDMA; 98%) and butyl methacrylate (BMA) were purchased from Alfa Aesar (Ward Hill, MA). 2-Acrylamido-2-methylpropanesulfonic acid (AMPS; 99%) was obtained from Sigma-Aldrich (St. Louis, MO). 1-Propanol and 2,2′-azobisisobutyronitrile (AIBN; 98%) were obtained from Aldrich (Steinheim, Germany). 1,4Butanediol (99%) was supplied by Emerald BioSystems (Bainbridge Island, WA). Acrylamide, N,N′-methylenebisacrylamide (Bis), N,N,N′,N′-tetraethylmethylenediamine (TEMED), and ammonium perfulfate (APS) were obtained from Bio-Rad Laboratories (Hercules, CA). Cytochrome c and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, Mo). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). LC−MS (mass spectrometry) grade acetonitrile (ACN) was purchased from Fisher Scientific (Fair Lawn, NJ). Fused silica capillaries were purchased from Polymicro Technologies Inc. (Phoenix, AZ). All other reagents used in this study were analytical grade or above and used as received. All solutions were prepared with ultrapure water purified by a Nanopure infinity ultrapure water system (Barnstead, Newton, WA). Preparation and Characterization of the EOP Monolith. The inner wall of a capillary was first vinylized with γMAPS using the following procedure. A 1 m long × 100 μm i.d. (365 μm o.d.) capillary was flushed with acetone for 10 min and 1.0 M NaOH for 10 min. Then the capillary was filled with 1.0 M NaOH, sealed with rubber septa, and put in an oven at 100 °C for 2 h. After the capillary was taken out of the oven, it was washed with water, 0.1 M HCl, water, and acetone, each for 20 min. The capillary was then dried with N2 for 1 h at 60 psi, and the inner wall was reacted with a solution containing 30% (v/v) γ-MAPS in acetone at 50 °C for 14 h. After the capillary was flushed with acetone and dried with N2, the capillary was 12215

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protein/mL using 2% ACN/0.1% TFA without any additional cleanup for analysis.

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RESULTS AND DISCUSSION Effect of Pressure on the Monolith Characteristics. Applying a pressure (∼1000 psi) to the monomer solution during polymerization seemed to have improved the performance of the monolith considerably. Figure 2 presents the

Figure 1. Schematic configuration of the binary EOP gradient generator (bi-Egg) and miniaturized high-performance liquid chromatographic system: PS, pump solution (3 mM sodium acetate at pH 5.0); HV, high voltage; GND, ground; MONOLITH, 100 μm i.d. × 13 cm long monolith column; pump solution, 3 mM sodium acetate at pH 5.0; LOOP A/B, 200 μm i.d. × 365 μm o.d. × 100 cm long capillary; MA/MB, mobile phase A/B; W, waste; V, injection valve (60 nL); C, Waters Atlantis 75 μm i.d. × 10 cm and dC18 (3.0 μm) packed capillary column; D, UV absorbance detector (210 nm).

miniaturized HPLC system with the bi-Egg integrated into it. The bi-Egg consisted of two high-pressure EOPs, one 12-port valve (VICI, Valco Instruments), and a microtee (or micro-T). The EOP outlet was connected to the 12-port valve via a 150 μm i.d. × 365 μm o.d. × 20 cm long capillary. Two capillary loops (LOOP A and LOOP B, each with dimensions of 200 μm i.d. × 365 μm o.d. × 100 cm length), were incorporated into the valve for housing different eluents; the eluents could be easily replenished as the valve was set at the other position (with respect to the position shown in Figure 1). The two outlets from the valve were connected via two 75 μm i.d. × 15 cm long capillaries to a microtee, and another 75 μm i.d. × 10 cm long capillary was used to connect the microtee to a nanoliter injector (V; VICI, Valco Instruments), followed by a packed capillary column (C; Waters, Atlantis dC18 NanoEase column, 75 μm i.d. × 100 mm length). A linear UVIS 200 absorbance detector (D; Spectra-Physics, Fremont, CA) was set at 210 nm for absorbance monitoring. The absorbance signal was acquired using an NI multifunctional DAQ card (6062E, National Instruments, Austin, TX), and the data were processed with an in-house-written LabView program. The bi-Egg was controlled by a gradient high-voltage power supply, which was constructed in our laboratory using a programmable linear dc power supply (GWINSTEK GPD2303S, Global Test Supply, Wilmington, NC) and two highvoltage power supplies (0−30 kV, EMCO model 4300, Sutter Creek, CA). The voltages applied to the EOPs were controlled through the USB port of a computer using a LabView (National Instruments, Figure S2 in the Supporting Information) program. Protein Digestion. Tryptic digests of cytochrome c and BSA were used to evaluate the performance of the nanoflow gradient generator and the miniaturized HPLC system. Cytochrome c or BSA was first dissolved in 50 mM ammonium bicarbonate with 30% ACN (v/v) to a concentration of 10 mg/ mL for each protein. Trypsin was added at a substrate-toenzyme ratio of 100:1 to digest the proteins for 8 h at 37 °C. Formic acid (1%, v/v) was added to the solution to terminate the digestion. The digests were concentrated in a vacuum concentrator to a volume of ∼25 μL and stored at −20 °C. The mixture was rediluted to a final concentration of 10 mg of

Figure 2. SEM images of typical negative monoliths prepared by the conventional (a, b) and pressure-assisted (c, d) preparation methods. The scale bar is 10 μm for (a) and (c) and 1 μm for (b) and (d).

scanning electron microscopy (SEM) images of the monoliths prepared with and without pressure. Smaller pores and lower porosities were formed with pressure, compared to what we reported previously.10 Using the same approach as described in ref 10, we estimated the effective pore diameters and porosities of the monoliths prepared in this work to be around 500 nm and 36.3%. In the presence of this pressure, we could prepare the monolith reproducibly (∼5% in terms of maximum pressure and flow rate generation), and we could tune the pore size by adjusting the porogen quantities (1-propanol and 1,4-butanediol) (Figure S3 in the Supporting Information). For the time being, however, we have not had a chance to study this effect systematically, and we do not understand the mechanism completely. Presumably, it was due to the suppression of microbubble formation under pressure. It is also possible that pressure reduced the monolith microfracturing caused by polymerization shrinking. Theoretical Consideration of the Bi-Egg. When an EOP is utilized, the actual pump pressure (P) is smaller than the maximum pump pressure (Pmax), because the flow rate (Q) is greater than 0. According to our previous reports,7,10 the actual pressure output of an EOP can be expressed by

P = Pmax −

Pmax Q Q max

(1)

For a specific monolithic EOP, the maximum pump pressure (Pmax) and the maximum pump rate (Qmax = Qeo, the overall electroosmotic flow in the monolith) can be expressed by 32ημeo Pmax = V (2) d2 and 12216

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Figure 3. Pump characterization: (a) linear relationship between the maximum flow rate and the applied voltage, (b) linear relationship between the maximum pump pressure and the applied voltage, (c) pump pressure output as a function of the flow rate under an applied voltage of 10 kV. For all panels, the pump solution was 3 mM sodium acetate at pH 5.0.

Q max =

mπd 2μeo 4L

V

C=

(3)

where η represents the viscosity of the pump solution, μeo the electroosmotic mobility, d the effective pore diameter, L the length of the monolith, m the number of equivalent pores, and V the voltage applied across the monolith. Therefore, the actual pump pressure can be expressed by

P = αV − β Q

[MA]Q a + [MB]Q b Q0

= [MA] −

Qb Q0

([MA] − [MB]) (5a)

and C=

[MA]Q a + [MB]Q b Q0

(4)

= [MB] −

where α = 32ημeo/d2 and β = 128ηL/mπd4, and these constants can be obtained experimentally after Pmax and Qmax are measured under any given voltage (V0): α = Pmax/V0 and β = Pmax/Qmax. In the HPLC system as configured in Figure 1, the eluent flow rate through the column (Q0) equals the sum of the flow rates of EOP A (Qa) and EOP B (Qb); i.e., Q0 = Qa + Qb. Because the pressure drops across all connection capillaries can be neglected, the pressure on the column (P0) equals the pressure on EOP A or EOP B; i.e., P0 = Pa = Pb. When MA and MB are mixed, the eluent concentration can be expressed by

Qa Q0

([MB] − [MA]) (5b)

where [MA] and [MB] are the eluent concentrations in MA and MB. Combining eqs 4 and 5, we have Va =

β Q ([MB] − C) P0 + a 0 αa αa([MB] − [MA])

(6a)

β Q ([MA] − C) P0 + b 0 αb αb([MA] − [MB])

(6b)

and Vb = 12217

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profiles followed the programmed profiles closely. Technically speaking, we can obtain any desired gradient profiles (see Figure S4 in the Supporting Information as an example) by programming Va and Vb properly. Integration of the Bi-Egg into a Miniaturized HPLC System for Peptide Separation. After the bi-Egg was characterized, we integrated it into a miniaturized nanoflow HPLC system as presented in Figure 1 and utilized the HPLC system for separations of peptides from trypsin-digested cytochrome c and BSA. Figure 5 presents the performance comparisons between the bi-Egg LC and Agilent 1200 HPLC systems under similar conditions. As can be seen, most of the peptides were well resolved in both systems. We could identify ∼30 peaks in the chromatograms of cytochrome c (Figure 5A

These are fundamental equations that guide us to programming the voltages on EOPs for achieving desired gradient profiles C (a time-dependent function). Details of how to apply these equations to create a desired gradient are provided in Figure S2 in the Supporting Information. Performance of the Bi-Egg. It is recommended that every EOP should be characterized after being prepared. Figure 3 presents the characterization of the EOPs used in this work. As expected, the maximum flow rate and pressure increase with the voltage applied to the EOP; good linear relationships were obtained (R2 > 0.99, Figure 3a,b). When a high voltage of 10 kV was applied, the EOP could generate a maximum flow rate of 450 nL/min and a maximum output pressure of 5600 psi, which was about 3× higher than that of our previous EOPs.10,16 On the basis of these results, we computed αa = 558.2 psi/kV and βa = 14.0 psi·min/nL for EOP A and αb = 582.6 psi/kV and βb = 14.7 psi·min/nL for EOP B. After measurement of the maximum pressure and maximum flow rate under a given voltage, we obtained the working curve (Figure 3c) of the EOP. We then determined the separation pressure (P0) to be ∼1500 psi under a preset flow rate (Q0) of 200 nL/min. We also set [MA] = 2% ACN and [MB] = 90% ACN, both in 0.1% TFA. Va and Vb were programmed according to eqs 6a and 6b. In this experiment, when Va varied from 7.5 to 4.2 kV and Vb changed from 2.8 to 6.1 kV, the gradient profile changed from 5% ACN to 65% ACN. Figure 4 presents two bi-Egg-produced gradient profiles (solid lines). These profiles are very smooth and linear,

Figure 4. Typical gradient profiles by the bi-Egg. The gradient strength varied from 5% to 65% ACN in 30 min (black lines) or in 60 min (red lines). The solid lines represent the experimentally measured values, while the dashed lines represent the programmed gradient profiles according to eqs 6a and 6b.

comparable to those generated by a commercial Agilent 1200 HPLC system. [Note: When we said we used an Agilent 1200 HPLC, we meant that we used an Agilent gradient generation system. To reduce the gradient delay time at 200 nL/min, we actually utilized the gradient generation system in conjunction with a flow splitter, and the flow after the splitter was connected to the injection valve and capillary column, which are identical to those used in the bi-Egg LC system.] The dashed lines display the calculated results from eqs 6a and 6b without considering diffusion and dispersion. The experimental data matched the calculated results well, and importantly, the actual

Figure 5. Performance comparison between the bi-Egg LC and Agilent 1200 systems: (A) typical chromatograms for trypsin digests of cytochrome c (10 mg/mL), (B) typical chromatograms for trypsin digests of BSA (10 mg/mL). The eluent contained a constant 0.1% TFA and a gradient concentration of ACN in water. The ACN concentration was increased from 10% to 35% in 50 min for cytochrome c and from 10% to 40% in 60 min for BSA. The flow rates were all about 200 nL/min. The separation column was a Waters Atlantis dC18, 3.0 μm, 75 μm i.d. × 100 mm. The injection volume was 15 nL, and the detection wavelength was 210 nm. Sample injections were effected at 1 min for the bi-Egg LC system (5 min for the Agilent 1200 system) after the gradient pumps had started. 12218

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003). L.Z. is grateful for the financial support of the National Natural Science Foundation of China (Grant 21105040) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant 20110211120010).

or Figure S5a,b in the Supporting Information) and ∼60 peaks in the chromatograms of BSA (Figure 5B or Figure S5c,d). More detailed performance comparisons are provided in Figure S5. However, we did see differences between the two systems. For example, resolutions for some peaks (b vs b′, c vs c′, e vs e′, g vs g′, and h vs h′) were higher while resolutions for some other peaks (a vs a′, d vs d′, and f vs f′) were lower from the biEgg LC system than those from the Agilent 1200 system. Also, peptides were eluted out faster by the bi-Egg LC system than by the Agilent 1200 system. There are two major reasons for this: (1) The gradient delay time for the bi-Egg LC system was short (5 min) of the Agilent HPLC system. Because sample injections were effected on the basis of these delay times, the actual gradient for the separation in the bi-Egg LC system could be steeper than that of the Agilent 1200 system. (2) The bi-Egg HPLC system uses a constant pressure source, while the Agilent HPLC system uses a constant flow rate source. Because the viscosity decreased with increasing [ACN], the flow rate of the bi-Egg LC system increased with time in this experiment. [Note: The flow rate of the bi-Egg LC system changed less than 5% when the solution being delivered had a constant composition.]

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CONCLUSIONS We have developed a new pressure-assisted protocol to prepare monoliths for EOP construction; this process has allowed us to produce more efficient, reliable, and reproducible EOPs compared with the ambient-pressure preparation method. We have successfully developed a binary EOP gradient generator for a miniaturized nanoflow HPLC system; the gradient generator can be built at a cost of a few hundred dollars, compared to tens of thousands of dollars for a commercial nanoflow gradient generator. We have developed equations for tuning the gradient profiles. As long as the EOPs are properly characterized, any desired gradient profile can be obtained automatically by adjusting the voltages applied to the EOPs via a computer program. We have utilized the miniaturized nanoflow HPLC system for separating complex peptide samples and obtained comparable resolutions and peak capacities with respect to those obtained from a commercial HPLC system.

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REFERENCES

(1) Kutter, J. P. J. Chromatogr., A 2012, 1221, 72−82. (2) Desmet, G.; Eeltink, S. Anal. Chem. 2013, 85, 543−556. (3) Gauthier, G. L.; Grimm, R. Drug Discovery Today: Technol. 2006, 3, 59−66. (4) Laser, D. J.; Santiago, J. G. J. Micromech. Microeng. 2004, 14, R35−R64. (5) Wang, X. Y.; Wang, S. L.; Gendhar, B.; Cheng, C.; Byun, C. K.; Li, G. B.; Zhao, M. P.; Liu, S. R. Trends Anal. Chem. 2009, 28, 64−74. (6) Wang, X. Y.; Chen, C.; Wang, S. L.; Liu, S. R. Microfluid. Nanofluid. 2009, 6, 145−162. (7) He, C. Y.; Lu, J. J.; Jia, Z. J.; Wang, W.; Wang, X. Y.; Dasgupta, P. K.; Liu, S. R. Anal. Chem. 2011, 83, 2430−2433. (8) He, C. Y.; Zhu, Z. F.; Gu, C. Y.; Lu, J.; Liu, S. R. J. Chromatogr., A 2012, 1227, 253−258. (9) Wang, W.; Gu, C. Y.; Lynch, K. B.; Lu, J. J.; Zhang, Z. Y.; Pu, Q. S.; Liu, S. R. Anal. Chem. 2014, 86, 1958−964. (10) Gu, C. Y.; Jia, Z. J.; Zhu, Z. F.; He, C. Y.; Wang, W.; Morgan, A.; Lu, J. J.; Liu, S. R. Anal. Chem. 2012, 84, 9609−9614. (11) Nikitas, P.; Pappa-Louisi, A. J. Chromatogr., A 2009, 1216, 1737−1755. (12) Kaliszan, R.; Wiczling, P. Trends Anal. Chem. 2011, 30, 1372− 1381. (13) Cappiello, A.; Famiglini, G.; Fiorucci, C.; Mangani, F.; Palma, P.; Siviero, A. Anal. Chem. 2003, 75, 1173−1179. (14) Deguchi, K.; Ito, S.; Yoshioka, S.; Ogata, I.; Takeda, A. Anal. Chem. 2004, 76, 1524−1528. (15) Brennen, R. A.; Yin, H. F.; Killeen, K. P. Anal. Chem. 2007, 79, 9302−9309. (16) Chen, A. P.; Lynch, K. B.; Wang, X. C.; Lu, J. J.; Gu, C. Y.; Liu, S. R. Anal. Chim. Acta 2014, 844, 90−98. (17) Byun, C. K.; Wang, X. Y.; Pu, Q. S.; Liu, S. R. Anal. Chem. 2007, 79, 3862−3866.

ASSOCIATED CONTENT

S Supporting Information *

Figures showing application of pressure to the polymerizing solution, the programming voltage on the EOP for gradient generation, the effect of the porogen ratio on the maximum flow rate and maximum pressure, generation of the gradient profile, and a performance comparison between the bi-Egg and Agilent HPLC systems. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 405-325-6111. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially sponsored by the Department of Energy (Grant DE-SC0006351) and the Oklahoma Center for the Advancement of Science and Technology (Grant AR1112219

dx.doi.org/10.1021/ac503223r | Anal. Chem. 2014, 86, 12214−12219