Comparison of Sample Introduction Methods for Continuous Chemical

Oct 10, 2014 - Two-dimensional electro-fluid-dynamic (2-D EFD) devices, in which both electric field and hydrodynamic pressure are used to drive the a...
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Comparison of Sample Introduction Methods for Continuous Chemical Purification in Two-Dimensional Electro-Fluid-Dynamic Devices Chang Liu†,§ and David D. Y. Chen*,†,‡ †

Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, Jiangsu 210046, China

‡

S Supporting Information *

ABSTRACT: Two-dimensional electro-fluid-dynamic (2-D EFD) devices, in which both electric field and hydrodynamic pressure are used to drive the analyte and fluid migration, enable chemical separation to proceed in two-dimensional channel networks instead of a onedimensional column and provide better control on the migration and distribution of analyte in complex channel networks. We have reported the use of a 2-D EFD device to continuously purify multiple components from complex samples (Liu et al. Anal. Chem. 2010, 82, 2182−2185 and Liu et al. Anal. Chem. 2011, 83, 8208−8214). A continuous solution stream containing a mixture can be separated into different channels, each containing a pure compound. In previous studies, the sample mixture was introduced into the device by applying an electric field, also known as electrokinetic sample introduction. The initial separation junction requires three separate voltages and one pressure source. In this study, we investigated the mass transfer at the separation junction when the hydrodynamic pressure is used to deliver the sample. The initial separation junction has two voltages and two pressure sources. Continuous chemical purification is demonstrated in EFD devices with different geometries, and the comparison of both sample introduction approaches indicates that hydrodynamic sample introduction is superior to electrokinetic sample introduction.

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ince it was introduced in 1981,1 capillary electrophoresis (CE) has been used to separate species that cannot be resolved by other methods. Because the separation takes place in free solution, the integrity of labile samples can often be preserved.2−5 A number of research groups have developed methods for performing fraction collection in CE.6,7 While effective, fraction collection produces only small amounts of purified sample components due to limitations associated with the small dimensions of the capillary columns. Therefore, a platform that is capable of purifying chemicals in a preparative fashion from complex mixtures is still needed for obtaining enough pure compound for structure determination and property characterization. We have developed a new generation of devices for continuous chemical purification, on the basis of the interactions of analyte with multiple types of driving forces in an electro-fluid-dynamic (EFD) system.8,9 The two-dimensional EFD devices, in which both electric field and hydrodynamic pressure are simultaneously utilized in 2-D channel networks to drive the mass transfer, provide better control of the analyte molecules by simply adjusting the magnitude of pressure in a preset electric field. The analytes can either migrate with the medium driven by nondiscriminative forces, such as pressure or electroosmosis, or migrate through the medium driven by discriminative forces from the applied electric field. These movements can exist simultaneously, giving © XXXX American Chemical Society

a net migration determined by the sum of the velocity vectors of each movement. Symmetrical Y-shaped microfluidic devices have been used to introduce individual samples from interconnecting channels, and the mixing process of samples introduced into these channels has been studied extensively.10 With the strategically applied electric field and hydrodynamic pressure, such mixing processes, which are spontaneous because of the increase in entropy, can be reversed on the device with the same channel geometry. A continuous solution stream containing a mixture of two components can be separated into two channels, each containing a pure compound.8 By increasing the geometry complexity, more complex samples can be processed. We have demonstrated the use of a multibranched two-dimensional electro-fluid-dynamic (2-D EFD) device, for continuously purifying multiple components.9 Each component in the introduced mixture can be directed to enter its specific collection channel, without any contamination. The predictable nature and ease of operation of this technique could lead to a new generation of purification devices to serve the needs of biomedical research and other commercial and academic activities. Received: September 4, 2014 Accepted: October 10, 2014

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main channel and lateral channels of both devices were 80 and 40 μm, respectively, in the photomask. The depth of all channels was 50 μm. Before introducing analytes into the EFD device, the channels were filled with BGE first. The analytes mixture, prepared from diluting the stock solution by 100 times, was then introduced into the device for separation. The electric potential at point B (2000 V) was provided by high-voltage power suppliers (SL150, Spellman High Voltage Electronics, Hauppauge, NY). The current in Channel AC was monitored during the experiment, which was kept as zero to ensure the electric field strength in this channel was zero. Two syringe pumps (Harvard Apparatus, Holliston, MA) were used to control the sample introduction speed in Channel AC, and the counter pressure at F, respectively. A Nikon Eclipse 80i microscope was used in this study, and fluorescence signals were recorded by a Photometrics Evolve camera (Tucson, AZ). When it was necessary to monitor the migration behaviors of two analytes simultaneously, the microscope was operated at two wavelengths using a MAG Biosystems dual-view objective (Optical in Sights, Tucson, AZ) with a 565 nm dichroic filter. A 540 nm filter was used for rhodamine 110, and a 600 nm filter was used for ethidium bromide detection. The optical bandpass filters were from Thorlabs (Newton, NJ), and their full width at half-maximum (fwhm) was 10 nm.

In previous studies, there was a positive potential applied at the sample vial. The electric field driven flow overcame the counter-pressure and delivered the sample into the device. This type of sample introduction approach is known as electrokinetic injection. The quantity of sample introduced into the device is dependent on a number of parameters, including the electrophoretic mobilities of the analytes and the electroosmotic flow velocity in the channel. This method may be advantageous if the analyte of interest has a large electrophoretic mobility.11 Due to the direct contact of the electrode to the sample solution, the buffer depletion problem due to prolonged electrolysis, which is considered the major limiting factor for the analyte recovery rate in the chemical purification process of flow counterbalanced capillary electrophoresis (FCCE),12,13 has not been resolved. Pressure injection, or hydrodynamic injection, is an equally popular sample introduction method because of its simplicity. In this study, electric field and fluid field distributions were systematically studied in different 2-D EFD devices, when the sample was continuously introduced into the channel networks by hydrodynamic pressure. The results of the steady-state mass transfer study indicated that this hydrodynamic sample introduction method could be used for continuous chemical purification in 2-D EFD devices. The comparison of two sample introduction approaches showed that the hydrodynamic injection is superior to the electrokinetic injection by providing a better control of critical boundary conditions and faster sample processing rate and is more resistant to minor changes in electroosmotic flow (EOF). In addition, because no external electric potential is applied to the sample vial, the buffer depletion problem caused by electrolysis is avoided.

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RESULTS AND DISCUSSION We have introduced the utilization of symmetrical Y-shaped and multiple-branched 2-D EFD devices for continuous chemical purification of two or more analytes from a mixture.8,9 In those investigations, the electric field applied on the sample introduction channel overcame the effect of back pressure and delivered analytes into the device. In the current study, a hydrodynamic pressure applied at the sample inlet delivers the sample into the device, and the electric field strength in the sample introduction channel is kept at zero. In the discussion below, the directions of vectors are all along the channel length. Thus, these vectors are expressed as scalars, and the values are defined as positive when the vector direction is toward the intersection point C. The cross-sectional area ratio of lateral channels and the main channel is defined as α for 2-D EFD devices with both geometries shown in Figure 1 (SAC = SBC = αSCD for the symmetrical Y-shaped device and αSAC = SBC = αSCD for the multiple-branched device). The Electric Field and Hydrodynamic Fluid Field Distribution in 2-D EFD Devices. The conductivity of the solution in the device can be considered uniform if a relatively high concentration of buffer is used: σAC = σBC = σCD (1)

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EXPERIMENTAL SECTION Rhodamine 110 (Exciton, Dayton, OH) and ethidium bromide (Invitrogen, Eugene, OR) stock solutions were prepared at a concentration of 1 mg/mL in the background electrolyte (BGE, 160 mM borate, pH 9.0). The 2-D EFD devices shown in Figure 1 were fabricated with soda lime glass (Nanofilm, Westlake Village, CA) using standard photolithographic patterning and wet chemical etching methods to demonstrate the continuous chemical separation process.14 The width of the

where σ is the conductivity of the solution inside each channel. From Kirchhoff’s law, the net current at the intersection is zero. JAC SAC + JBC SBC + JCD SCD = 0

(2)

in which J is the current density in each channel and S represents the cross-sectional area of the respective channel. If Ohm’s law J = σE is used in eq 2, it becomes EAC SAC + EBC SBC + ECDSCD = 0

(3)

For different 2-D EFD devices, eq 3 can be rewritten according to the relationship of their channel cross-sectional area, which is αEAC + αEBC + ECD = 0 and EAC + αEBC + ECD = 0 for symmetrical Y-shaped and multiple-branched shaped devices, respectively. In the hydrodynamic sample introduction

Figure 1. 2-D EFD devices used in this paper: (a) symmetrical Yshaped device; (b) multiple-branched device. Two syringe pumps (illustrated by arrows) were used to control the sample introduction rate in Channel AC, and the counter pressure at F, respectively. Positive potentials were applied at collection vials. B

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Table 1. Electric Field and Fluid Velocity Field Distribution in Different Sample Introduction Modes of Symmetrical Y-Shaped and Multiple-Branched 2-D EFD Devices device symmetrical Y-shaped devices method

electrokinetic sample introduction

electric field

αEAC + αEBC + ECD = 0 fluid field

αvf , AC + αvf , BC + vf , CD = 0

multiple-branched devices

hydrodynamic sample introduction

electrokinetic sample introduction

⎧ αEBC + ECD = 0 ⎨ ⎩ EAC = 0 ⎪

EAC + αEBC + ECD = 0

αvinj + αvf , BC + vf , CD = 0

vf , AC + αvf , BC + vf , CD = 0

⎪

⎪

⎪

(4)

for both kinds of 2-D EFD devices. The fluid in the EFD device is assumed to be incompressible, so the fluid velocity in intersecting channels has the relationship of vf , ACSAC + vf , BCSBC + vf , CDSCD = 0

(5)

Equation 5 can be rewritten according to the cross-sectional area relationships of the channels, resulting in αvf,AC + αvf,BC + vf,CD = 0 and vf,AC + αvf,BC + vf,CD = 0 for symmetrical Y-shaped and multiple-branched devices, respectively. In the hydrodynamic sample introduction mode, the syringe pump delivers analytes toward the intersection point at a fixed net fluid velocity vinj in the injection channel AC. Therefore, the hydrodynamic fluid field distribution relationships can be written as αvinj + αvf,BC + vf,CD = 0 and vinj + αvf,BC + vf,CD = 0 for the two types of 2-D EFD devices, respectively. The results discussed in this section are summarized in Table 1. Migration Behavior of an Analyte in 2-D EFD Devices. The steady-state velocity of a charged particle moving in the channel can be written as v = vep + veo + vp = μep E + μeo E + vp = μep E + vf

⎧ αEBC + ECD = 0 ⎨ ⎩ EAC = 0 ⎪

⎪

vinj + αvf , BC + vf , CD = 0

electric field, from injection Vial A, through the intersecting point C, and enter the Channel CD. In the hydrodynamic sample introduction mode, the applied pressure delivers the analyte mixture into the device at the velocity vinj, and each analyte can have only three possible mass transfer pathways in the device. When the counter pressure is high, the analyte is pushed into the lateral channel BC at the intersection point. As the magnitude of the counter pressure decreases, the component can migrate into either channel BC or channel CD. When the pressure is very low, all the analytes migrate along the direction of electric field and the analyte at point C follows the migration pathway of A−C−D. The magnitudes of the fluid velocity in the lateral channel BC at critical boundary conditions are EBCμep + vinj and EBCμep for the symmetrical Y-shaped device and EBCμep + (1/α)vinj and EBCμep for the multiple-branched device. (See the Supporting Information for the detailed derivation.) The migration behavior of analytes in EFD devices with different geometries in the hydrodynamic sample introduction mode were demonstrated by fluorescent dyes, as illustrated in Figure 2, and the critical boundary conditions (CBCs) were shown as well. With the comparison of the electrokinetic

mode, the electric field strength in Channel AC is zero. The electric field distribution changes to ⎧ αEBC + ECD = 0 ⎨ ⎩ EAC = 0

hydrodynamic sample introduction

(6)

where electrophoretic velocity (vep) is discriminative and determined by its electrophoretic mobility (μep), which is an intrinsic property for a particular analyte. On the other hand, the electroosmotic velocity (veo) and pressure-induced velocity (vp) are nondiscriminative and affect all components equally. The direction of the steady-state migration velocity (v) reverses at the critical boundary condition (CBC) when the discriminative and nondiscriminative velocities have the same magnitude and reversed directions. We have reported that the analyte has four possible mass transfer pathways in 2-D EFD devices according to the various combinations of electric field and counter-pressure, when the sample is introduced into the device electrokinetically.8,9 When the pressure applied at F is high, the steady-state velocity of the analyte in either channel has the same direction as the pressure, and the analyte is forced to stay at Vial A and does not migrate into Channel AC or any other channels. As the magnitude of applied pressure decreases, the steady-state velocity of the analyte reverses in Channel AC first, making the analyte migrate through point C to the collection vial B. If the pressure is further reduced, the analyte at point C may migrate into both Channel CB and Channel CD. When the pressure is reduced even more, all analytes migrate along the direction of the

Figure 2. Mass migration pathways of analyte in 2-D EFD devices and critical boundary conditions: (a) symmetrical Y-shaped device; (b) multiple-branched device. C

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sample introduction mode,8,9 the differences between −vf,BC values are independent of the electric field strength and are only determined by the sample introduction speed. This property provides a more convenient approach to control the migration behavior of the analyte in the EFD device, which will be discussed in more detail in the following. Effects of Changing Controlling Parameters on Critical Boundary Conditions. In this study, the critical boundary value (CBV) is defined as the value of −vf,BC at the critical boundary condition (CBC). As discussed in the previous part, the fluid velocity in Channel BC at CBCs is crucial to the migration behavior manipulation of the analyte in the 2-D EFD device. By changing the controlling parameters of the device, e.g., syringe pump controlling fluid velocity or applied electric potential, CBVs can be adjusted to the appropriate value, making the components follow the proper migration pathways. In the electrokinetic sample introduction mode, CBVs are dependent on the electric field strength at the specific channel.8,9 As described in eq 3, it is impossible to change the electric field strength in only one channel by simply adjusting only one electric potential, while leaving other critical boundary conditions unchanged (see Supporting Information for details). This complex relationship makes it difficult to control the analyte migration behavior. In the hydrodynamic sample introduction mode, the sample mixture is introduced into the EFD device by a hydrodynamic pressure, and the electric field is only dependent on the electrical potential applied at point B. On the basis of the critical boundary conditions illustrated in Figure 2, the change of the sample introduction rate does not affect the second CBV (Y), at which the steady-state velocity of the analyte reverses at Channel BC. In contrast, the first CBV (X), where the analyte changes the migration direction in Channel CD, is moved by the magnitude of Δvinj for the symmetrical Y-shaped EFD device (Figure S1, Supporting Information), and (1/α)Δvinj for the multiple-branched EFD device. Therefore, controlling the sample introduction speed provides a convenient approach to adjust the difference between two CBVs, while the second CBV remains unchanged. Adjusting the potential applied at point B is another way to control the CBV values in the hydrodynamic sample introduction mode. Because the difference between the two CBVs is only dependent on the sample introduction peed, the change of the electric field strength moves the two CBVs with the same magnitude of ΔEBCμep for both symmetrical Y-shaped and multiple-branched 2-D EFD devices (Figure S2, Supporting Information). Combining these two approaches is a convenient and powerful approach to regulate the position of the two CBCs. The position of the second CBC (Y), at which the steady-state migration velocity of the analyte reverses at channel BC, can be manipulated by adjusting the electric potential at the point B. The position of the first CBC (X) can then be set by controlling the difference between the two CBCs, by way of changing the sample introduction rate. Sample Processing Rate. On the basis of the discussion above, the CBVs are dependent on the electrophoretic mobility of the analyte. Therefore, different components may have distinctive migration pathways at certain electric field and hydrodynamic pressure conditions, and they can be directed into their specific collection locations. In order to achieve continuous purification, the applied electric potential and counter-pressure make the slower migration component follow

the pathway A−C−B, while all of the faster migrating components go through the main channel CD at the intersection C and have a migration pathway of A−C−D. Therefore, for the symmetrical Y-shaped EFD device, the magnitude of the net fluid velocity in Channel BC needs to be within the range of: EBC μep , slow + vinj < −vf , BC < EBC μep , fast

(7)

Consequently, as long as the sample introduction speed is kept in the range of vinj < EBC (μep , fast − μep , slow )

(8)

The magnitude of the fluid velocity in Channel BC can be arranged into the range indicated in eq 7 to achieve continuous purifications. Because of the existence of the maximum sample introduction speed, the minimum sample mixture processing time can be calculated: t=

Vtot Vtot > vinjSAC EBC (μep , fast − μep , slow )SAC

(9)

in which t is the time required to process the sample mixture with the volume of Vtot. Similarly, for the multiple-branched EFD device, the magnitude of the fluid velocity at channel BC when continuous purification occurs is EBC μep , slow +

1 vinj = −vf , BC < EBC μep , fast α

(10)

The requirement of the sample introduction speed is vinj < αEBC (μep , fast − μep , slow )

(11)

and the minimum sample processing time is t=

Vtot Vtot > αvinj(μep , fast − μep , slow )SAC vinjSAC

(12)

The continuous chemical purification processes in different EFD devices were demonstrated in Figure 3. It is indicated that some analyte may enter the unintended channels initially. However, since the steady-state flow defines the net migration

Figure 3. Demonstrations of continuous chemical purification process in different 2-D EFD devices: (a) symmetrical Y-shaped device; (b) multiple-branched device. The volumetric flow rate for the sample introduction was controlled as 0.100 μL/min for both cases. The flow rate of the syringe pump at point F was controlled at 0.320 μL/min for the symmetrical Y-shaped device and 0.600 μL/min for the multiplebranched device. The electric potential at point B was mentioned at 2000 V for the purification on both devices. D

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1 (EAC + EBC )(μeo + μep , slow ) < |vp , AC| = −vp , AC 2

behavior away from the immediate vicinity of the channel intersections, the molecules are forced to go back to the proper channel if the channels are long enough. The “proof-reading” mechanism described in the previous study is still valid in the hydrodynamic sample introduction mode.9 Comparison of Hydrodynamic and Electrokinetic Sample Introduction. We have described that the hydrodynamic sample introduction provides a more convenient approach to control the position of critical boundary conditions. Here, we theoretically compare the sample processing speed and the resistance to the fluctuating electroosmotic flow (EOF) and studied which sample introduction method provides better operability in the continuous chemical purification process. In the following discussion, we took the symmetrical Y-shaped EFD device as an example to make the comparison. Because of the continuous nature of the purification process, the amount of the sample mixture injected into the device during a time period equals the amount of the analytes processed and collected during that time. Therefore, the sample processing speed can be described by the sample introduction rate. In the hydrodynamic sample introduction mode, the injection rate for every analyte is the same, which is vinj. As discussed in the previous part, the maximum sample introduction rate to achieve the sample continuous purification is vinj < EBC(μep,fast − μep,slow) (eq 8). For the electrokinetic sample introduction method, the rate of delivering components into the EFD device is analyte dependent, which is vinj = EAC (μeo + μep ) + vp , AC

< EBC (μeo + μep , fast )

The sample processing rate for the mixture is limited by the sample introduction rate of the slower component, which is EAC (μeo + μep , slow ) − EBC (μeo + μep , fast ) < vinj , slow
(EAC − EBC )(μeo + μep )

1 (EAC − EBC )(μeo + μep , slow ) 2

Assuming that the electric fields in the lateral channel have the same strength in both hydrodynamic and electrokinetic sample introduction modes, the difference in the maximum sample introduction rate between these two modes is

If the counter-pressure is relatively high, the analyte is forced at the injection point A, and the sample introduction rate is negative. When the counter-pressure is decreasing and the analyte has the migration path of A−C−B, the magnitude of the pressure-induced velocity in the injection Channel AC is in the range of 1/2(EAC + EBC)(μeo + μep) < |vp,AC| = −vp,AC < EAC(μeo + μep), and the range of the sample introduction rate of the analyte in this migration situation is 0 < vinj
EBC during the purification process in the symmetrical Y-shaped device, EAC + (1/(2α))ECD does not equal zero. Therefore, the minor changes in EOF can cause the fluctuation for the sample introduction rate. As described in eq 17, for the electrokinetic introduction method, the magnitude of the pressure-induced velocity in lateral channels is kept in the range of 1/2(EAC + EBC)(μeo + μep,slow) < |vp,AC| = −vp,AC < EBC(μeo + μep,fast). During the continuous chemical purification process, the sample introduction rate for the faster moving component (vinj,fast = EAC(μeo + μep,fast) + vp,AC) is in the range of

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CONCLUSIONS Mass transport in different 2-D EFD devices was studied when the sample was introduced into the device by a hydrodynamic pressure. The continuous purification processes were successfully demonstrated in both a symmetrical Y-shaped device and a multiple-branched device. The comparison of two sample introduction methods was carried out systematically. The hydrodynamic sample introduction approach was determined to be superior to the electrokinetic method because it can provide faster sample processing and be more resistant to variations caused by minor changes in EOF. In addition, it can avoid the buffer depletion problem in the sample vial.

(EAC − EBC )(μeo + μep , fast ) < vinj , fast < EAC (μeo + μep , fast ) −

1 (EAC + EBC )(μeo + μep , slow ) 2

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(22)

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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EAC (μeo + μep , slow ) − EBC (μeo + μep , fast ) < vinj , slow 1 (EAC − EBC )(μeo + μep , slow ) 2

ASSOCIATED CONTENT

S Supporting Information *

and for the slower migration component, the range of the sample introduction rate (vinj,slow = EAC(μeo + μep,slow) + vp,AC) is