Electroconcentration by using countercurrent due to pressurized flow

with electrodes, connecting tubes, and a pump for aspirating the sample solution is proposed. The sample solution is aspirated into small tubes (1.5-m...
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Anal. Chem. 1993, 65, 2882-2886

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Electroconcentration by Using Countercurrent due to Pressurized Flow and Electrophoretic Mobility Akihiro Hori, Takatoshi Matsumoto, and Yuji Nimura First Department of Surgery, School of Medicine, Nagoya University, Tsuruma-cho 65, Showa, Nagoya 466, Japan

Masakazu Ikedo, Hideki Okada, and Takao Tsuda* Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466, Japan

A pressurized flow and countermigration,due to electrophoretic mobility of a solute, are used for sample concentration. An apparatuscomposed of two rooms with electrodes, connecting tubes, and a pump for aspirating the sample solution is proposed. The sample solution is aspirated into small tubes (1.5-mm i.d. and 15 mm long), over whicha voltage is applied (300-800 V/cm). A solute of high electrophoretic mobility is found to be drawn back from the small tube. Naphthalenesulfonic acids, herring DNA, and magnesium ions are concentrated 10-47 times under pressurized flow (0.1-0.4mL/min). We call this method "countercurrent electroconcentration".

A

B

4

Figure 1. Schematic diagram for countercurrent electroconcentra-

INTRODUCTION There are several methods in which an electric field is used for sample concentration,lq Such as electrodeposition, cementation, electrodissolution, electrodialysis, electrodiffusion,' isotachophoresis,2 ele~trochromatography,~~~ and a method using an electrochemical cell under flow? These methods are summarized in ref 1. The methods proposed by Svintsova et aL5and some of the methods of electrodep~sition~~~ are performed under pressurized flow. Svintsova et al. use an electrochemical flowthrough cell which has two compartments, separated by an anion-exchange membrane, and positive and negative electrodes covered with cationic and anionic ion-exchange membranes, respectively. They apply an electric field across the direction of the flow of the sample solution. Negatively charged metal complexes in the solution are decomposed by the very acidic media in the anodic compartment of the cell, and the metal liberated from the complex is concentrated in the compartments.5 Fujinaga et al.s used a column packed with particles of silver metal, through which a sample solution flows. An electric field (its absolute value less than 1 V) is applied between the media and the silver particles. Metal ions are electricallydeposited on the silver during its passage through the particles and elution of its concentrated solution follows (1) Zolotov, Yu. A.; Kuz'n, N. N. Preconcentration of trace elements; Comprehensive Analytical Chemistry XXV; Elsevier: Amsterdam, 1990. (2) Dolnik, V.; Cobb, K. A.; Novotny, M. J . Microcolumn Sep. 1990, 2,127-131. (3) Tsuda, T.; Muramatsu, M. J. Chromatogr. 1990,515,645-652. (4) Soini, H.; Tsuda, T.; Novotny, M. J. Chromatogr. 1991,559,547558. (5) Svintaova, L. D.; Kalplin, A. A.; Rubinskaya, T. B.; Mordvinova, M. M. J. Anal. Chem. USSR 1991,46,119-122. (6) Fujinaga, T.; Nagai, T.; Okazaki, S.; Takagi, C. Nippon Kagaku Kaishi 1963,84, 941-942. QQQ3-27QQf 93IQ365-2882$04.QQ/Q

tion: (A and B) rooms made by polyethylene vial; (1) tube; (2 and 3) platinum wire for electrode; (4) pump for aspiration of solution.

after releasing the electric field. Fujinaga called this method electrolytic chromatography. Volland et al. used a cylindrical cathode of ultrapure graphite as an electrolysis cell, through which the electrolyte is cycled. The graphite cathode can be used directly for a sensitive determination of deposited elements by flameless atomic absorption spectrometry.7 Electric voltage is again applied across the flow. Tsuda and Muramatsu used a glass column packed with silica gel modified with octadecylsilane and applied a high electric voltage over the column. The sample solution was fed continuously into the column. Solute with enough electrophoretic mobility against the pressurized flow can be concentrated at the head part of the column. After releasing the voltage, the concentrate can be eluted.3 In this paper, we propose a new method for sample concentration under an electric field. A high voltage (greater than 300 V/cm) is applied along an open small tube and the solution, including ions, is continuously passed through the tube. We use a counterelectrophoretic migration of ions against pressurizedflow. We callthis method "countercurrent electroconcentration".

THEORY The velocity of the pressurized flow and migration due to electrophoretic mobility can be controlled by the pump (4 in Figure 1)and the applied voltage, respectively. These two flows are usually in opposite directions. The sample solution in room A is aspirated into an open tube. The solute, which is assumed to have a negative valance, is also aspirated into the open tube. Without applied voltage at both ends of the (7) Volland, G.; Tschopel, P.; Tolgh, G. Anal. Chim. Acta 1977,90, 15-23.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993

tube, the zone front of the solute forms the profile suggested byTaylor.8 Eachsolute has a localvelocity, v(pres),, in which y stands for an axis of cross-sectionaldirection (perpendicular to the column axis x ) . Most of the local velocity is in the region of u(pres) Et26, where u(pres) and 6 are mean preasurized flow velocity and its standard deviation, respectively. With applied voltage along the tube, the solute having a negative charge moves with its electrophoretic mobility, u(mob), toward the positive electrode. We assume there is a plug flow profile of the zone front due to mobility. When the absolute velocity of the solute due to electrophoretic mobility is larger than the absolute value of the flow velocity due to pressurized flow, the solutewill be concentrated in room A, shown in Figure 1. Namely, the charged solute for concentration will stay in room A because the velocity of migration due to electrophoretic mobility is fast enough to come out from the tube after the solute has been aspirated into the tube. The flow profile of pressurized flow is parabolic, and the profile of migration of the solute due to electrophoretic mobility is assumed to be plug. The flow velocity due to mobility, u(mob), depends on the potential gradient. The apparent local flow velocity, u(app),, and the mean apparent flow velocity of a solute, u(app), are given by u(pres), = u(pres) f Au,

(1)

u(app), = u(pres), - u(mob)

(2)

u(app) = u(pres) - u(mob) (3) The positive sign of each velocity means that its flow direction is from the column inlet to the outlet. The value of Au, is given by u(pres), - u(pres) and depends on the mean velocity of the pressurized flow, diffusion coefficient, and tube radiusSa The maximum and minimum apparent flow velocities of the solute are given as follows: u(app),,

= u(pres)

+ 26 - u(mob)

(4)

= u(pres) - 26 - u(mob) (5) If u(app), is given a negative value, most of the solute may be drawn back into the reservoir of room A, and consequently, the solute is concentrated in room A. Therefore if u(app),,

u(pres) + 26 < -u(mob) (6) most of the solute is forced to be drawn back to the room with the positive electrode. When the u(pres) is too rapid and the contribution of the diffusion coefficient to the front zone profile of a solute is minor, the u(pres), at the central position of the tube is twice the mean velocity of the pressurized flow. Equation 6 becomes 2u(pres) < -u(mob) (7) For concentration, the solute must have sufficient electrophoretic mobility against a pressurized flow. When the solute has enough mobility and eq 6 or 7 is attained, most of the solute can be concentrated in room A. As a consequence of eq 7, u(mob) must be a t least twice v(pres). EXPERIMENTAL SECTION A schematic diagram of an apparatus for countercurrent electroconcentrationis shown in Figure 1. The apparatus consists of two rooms (A and B) together, one or two small glass tubes which connect room A and room B, a pump (Microfeeder, Type

MF-2,AzumaDenki Kogya Co., Tokyo) for generating a constant pressurized suction flow in a small glass tube (1.7-mm i.d. and (8)Taylor, G.R o c . R. SOC.1953, 219A,186-203.

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17 mm long or 1.5" i.d. and 15mm long),two electrodes which are placed in each room, and a high-voltagedirect-currentpower supply (dc 1200, V, Type 11-B, Toyo Roshi Co., Tokyo). The electrode for room B is insertad into the small hole at the side of the polyethylene vial and then is sealed with epoxy glue. The small tubes are connected together with two vials (rooms A and B) by using epoxy glue. The solution containing a solute for concentration is kept in room A at the beginning, and solution is added into room A during operation by using a dropwise syringe method if necessary. Reagents of guaranteed grade (WakoPure Chemical Industries, Ltd., Osaka) and deoxyribonucleic acid (DNA, type IV from herring sperm, Sigma Chemical Co., St. Louis, MO) were used as purchased. Operation. The operational procedure is as follows: The system is filled with the sample solution in which a solute for concentration is included in 1-2 mM ammonium acetate buffer. Electrovoltage (300-1100 V) is applied under a pressurized flow of suction, and pressurized flow of (100-400 rWmin) is sent by the operation of the microfeeder. The pressurized flow is directed from room A and room B, and the flow of the solute due to electrophoreticmobility is toward room A. Consequently, the solute is forced to return to room A. The concentrate remaining in room A (finalsolution)is subjected to the analysis by capillary zone electrophoresis (capillary electrophoresis, Model Isco 3850, Nikkaki Co., Tokyo and a homemade instrument) for organic ions and flame atomic spectrometry (type SAS727, Seiko Instruments, Tokyo) for magnesium ion. Analytical Conditions. The analytical conditions for capillary zone electrophoresis (CZE) are as follows: column, 100-pm inner diameter and 30 cm long (the effective length of capillary from inlet to detector is 15cm); applied voltage, 11kV; detection, UV 254 nm; medium, 8 mM phosphate buffer (pH 8) containing 0.5 % ethylene glycol. The analytical conditionsfor aflame atomic spectrometer for magnesium ion determination are as follows: 285.2 nm; slit 3; 12 mm above burner; head holocathode lamp, lOmA, highvoltage, 310V;sampleflowrate, 29s/2mL;acetylene, 3.8 L/min; air 17 L/min.

RESULTS AND DISCUSSION Typical Examples of Countercurrent Electroconcentration. The predicted enhancement, V(ratio), is given as follows V(ratio) = V(used)/ V(final) (8) where V(used) is the volume of the solution used for concentration and V(final) is the volume of the final solution kept in room A after the run. When V(used) is 10 mL, the final volume, V(final), of 1.0 and 0.2 mL means that their predicted enhancements are 10 and 50 times, respectively. A concentration enhancement, E, is given as follows: E = (concentration of a solute in the final solution)/(concentration of a solute in an original solution). The values of these concentrations are calculated mostly from the peak areas of solutes in the electropherogram. In Figures 2 and 3, the predicted enhancements for 2,6naphthalenedisulfonic acid are 10 and 50, and their concentration enhancements are 9.7 and 47, respectively. The mobility of 2,6-naphthalenedisulfonicacid (solute 2) is an estimated 7.6 X 10-4cm2 V-1 s-1 from its elution time obtained by capillary zone electrophoresis. The applied voltage used is 600 V along a small tube (1.7-mm i.d. and 17 mm long), u(mob) for solute 2 is -18.2 cm/min, and u(pres) is set a t 5.5 cm/min to satisfying eq 6. Therefore, u(app) for solute 2 is -12.7 cm/min. Negative linear flow velocity means that the flow is from reservoir B to A in Figure 1. Herring DNA of 1 0 - 2 g/L is concentrated 9.6 times using two small tubes (1.5-mm i.d. and 15 mm long) when applied voltage, u(pres), and V(ratio) are 600 V, 7.6 cm/min, and 10, respectively, as shown in Figure 4. As the mobility for DNA is estimated to be 9.8 X 10-4 cm*V-1 s-1 from its elution time in CZE, u(mob) and u(app) are -23.5 and -15.9 cm/s, respectively.

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B A

C

I

Concentration of 2,8naphthalenedIsulfonlcacM, 9.7 times. Capnlary zone electropherogramsof A 4 are 0.01 mM samplesolution, the concentrated solution, and 0.1 mM standard solution, respecthrely. Sample volume treated, 10 mL; flnal volume concentrated, 1.0 mL. Flgure 2.

c3

liin

C

Concentration of 2,&naphthalenedlsuifonlc acM. 47 tlmes. Experimental conditions are same as Figure 2 except that the flnal volume concentrated was 0.2 mL. Figure 3.

a

H Imln

Concentration of herring DNA. A and B are orlglnal and concentrated solutlons, respecthrely. Magnesium ion (1o-BM in 1 mM ammonium acetate buffer) is also concentrated 10.3 times by this method when applied voltage, u(mob), u(pres),and V(ratio) are 1100V, -15.5,+7.6, and 20, respectively. The u(mob) for magnesium ions is Flgure 4.

estimated from its mobility, 4.7 X 10-4cm2 V-15-1 measured in 0.7 M formic acid.@ The values of concentration enhancements obtained are 90-95% of the predicted enhancements except in the case of magnesium ions. The concentration enhancement for magnesium ions is nearly half of the predicted enhancement. As ita u(mob) isjust equal to twice u(prea),a portion of magnesium ions located in the central part in the glass tube might be carried with u(pres) into reservoir B in Figure 1. There are several factors which characterize the preconcentration method. They are concentration times, period of the operation, loss of desired solute by adsorption during the procedure,the possibilityof contaminationof other substrates, etc. The present method is based on a very simple principle, and the apparatus used for concentration has a simple structure. Therefore, there are not many adsorptive sites on the wall. To obtain alarge concentration enhancement,it is necessary to keep V(final)small enough. If we are able to handle a very small finalvolume,the theoreticalconcentration ratio becomes very high. Stability of the Operation. To obtain a constant pressurized flow in the tube, the suction pump should be operated in a very stable condition. The microfeeder used has enough stability to perform the present experiment. As the inside part of the small tube has the highest electric resistance in the apparatus, Joule heat is generated mostly in the tube due to the large potential gradient (from 300 to 800 V/cm). The heat should be dissipated from the outside surface of the tube. For this purpose, part of the tube is cooled by immersing in a water bath when the electric current and applied voltage are 1 mA and 600VI respectively. Under 0.2-mA electriccurrent we did not encounter bubble formation in the small tubes even though the apparatus was operated without cooling. If a bubble formed in the tube, the electric current became unstable or stopped. Therefore it is essential to operate the apparatus without bubble formation. Although we just used one or two capillary tubes in these experiments, it is more desirable to use multitubes of narrow inner diameter or a flit having macrochannels. They are able to dissipate the heat generated in the tube more efficiently. The other advantageto using narrower inner diameter tubes is that the variation of u(pres), due to the y-position becomes relatively smaller, and the front zone broadening of a solute due to the pressurized flow also becomes smaller during the passage of the solution.* Chemical Reaction at Electrode. There is some possibility that solute and buffer will be chemically changed due to electron transfer and/or reaction at the electrodes. Water is electrolyzed and produces hydrogen and oxygen gases at the negative and positive electrodes,respectively. Therefore, the area around the electrode is very oxidative or reductive. The solute coming to this region may be reduced or oxidized at the negative or positive electrode, respectively. When we concentrated 0.1 mM 2,6-naphthalenedisulfonic acid (aqueous solution) to 10times and got its 1 mM aqueous solution, we noted the formation of byproducts (b and c in Figure 5). The electropherograms in Figure 5 are obtained by using large amounts of injected sample to detect byproducts. One of the two byproducts (b in Figure 5) is unstable and seems to decompose to the original solute (a in Figure 5) and a minor byproduct (c in Figure 5 ) according to the elapsed time. These byproducts might be formed by the reaction of the original solute and oxygen produced on the surface of the electrode or by the oxidation of the sulfonic acid group in the solute on the surface of the electrode. (9) Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1972,68,207-230.

a

a

-

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lmln

288S

Table I. Apparent Flow Velocities of the T h m Successive Runs for Selective Concentration a parent flow V(fmal) pressurized flow veP, (cm/min) (mL) veloc(cm/min) solute1 solute2 solute3 f m t run secondrun third run

0.7 1.5 1.0

+21.0 +11.5 +7.6

flow veloc due to mobility (cm/min)

+10.8 +1.3 -2.6

+2.7 -6.8

-8.6

-10.2

-18.3

-29.6

I

4I

H Ill"

C

A

2

D

E

I

2

Flgure 5. Variation of the reactlon byproducts In the final concentrated solution with elapsed time. The electropherograms obtalned are (A) lmmedlately after the concentration,(B) after 90 mln, and (C)after 220 mln.

But formation of the byproduct is not observed when we concentrate the 0.01 mM original solution by 10 times (thus the final solution concentrated is 0.1 mM of 2,g-naphthalenedisulfonic acid). Therefore we could avoid the formation of byproduda by keepingthe concentrationof the solute lower. This means that the possibility of chemical reaction of solutes may be decreased more than the first order of concentration of the solute. Effect of Buffer. The pH variation from the beginning to the end of the operation is around 0.2-1.0 without a buffer. The degree of variation of pH is dependent on the nature of the sample concentrated. The use of a buffer gives less pH variation and better constancy of electric current, because it suppresses the pH variation around the electrodes during operation. To maintain low electriccurrent during operation, it is better to use buffer composed of weak base and weak acid. A 1-2 mM ammonium acetate buffer was usually used for countercurrent electroconcentration except with DNA. The latter was analyzed without buffer. At higher buffer concentrations, such as 5-10 mM ammonium acetate, the current was increased more than 1.7 mA and the operation became unstable in some cases due to the formation of bubbles in the small tubes. The current value during the experiment using 1 mM ammonium acetate buffer and 600-V applied voltage is high at the beginning of the operation and then gradually decreases to half of the value at the end of the operation. In some cases, the current value becomes high again at the end of the operation due to the concentrated final solution. This is because the conductivity of the medium in reservoir A becomes higher due to the concentration enhancement of the solute compared to the beginning of the operation. These phenomena do not seem to have an effect on the system performance. Selective Concentration. For concentration, the solute should have enough counterflowvelocity due to mobility under an electric field. That is, the absolute value of u(mob) should be larger than the absolute value of u(pres). If the value of u(app) is not negative, the solute is not concentrated. And if the solute is neutral or its u(mob) has the same flow direction with u(pres), it passes through the tube. Therefore we can

(I_ Typlcal example of the selectbe concentration from the mlxture. Experlmental conditions are llsted In Table I. Capillary zone electropherograms of A-E are obtalned from the following Sample solutions: (A) orlglnal solution containing solutes 1,2, and 3 (see text); (B) first concentrate obtalned from orlglnal solution; (C) second concentrate; (D) third concentrate; (E) solution passed through the glass tube In third run. Figure 6.

choose appropriate experimental conditions for the selective concentration of a specified solute from the mixture. By use of these schemes, we select one solute for concentration from among other components. The typical experimental plan and results are shown in Table I and Figure 6, respectively. A 10-mL aliquot of a mixture of naphthalene mono-, di-, and trisulfonic acid is used, and their names are abbreviated to solutes 1, 2, and 3, respectively. Three successiveruns are performed. The u(pres) for first, second, and third runs is planned to be 21, 11.5, and 7.6 cm/min, respectively. As the applied voltage used is 600 V (400V/cm), u(mob) for solute 1,solute 2, and solute 3 are -10.2, -18.3, and -29.6 cm/min, respectively. The procedure for the selective concentration is as follows: After the first run, the solution concentrated in room A is taken up, and then the solution in room B is transferred into room A as the sample solution for the second run. The original sample solution, three concentrated solutions obtained from the first, second, and third runs, and the solution in room B after the third run are analyzed by capillary zone electrophoresis,which results are shown in Figure 6A-E, respectively. As in the first, run u(pres) is 21 cm/min and only solute 3 has negative apparent flow velocity. Namely, solute 3 will be carried to or remain in room A. The concentrated solute obtained from the first run was analyzed and is shown in Figure 6B. The result shows that predicted enhancement and concentration enhancement for solute 3 are 14.3 and 12.1, respectively. Solutes 1and 2 are also concentrated 2.8 and 6.0 times, respectively. After the second run for selective concentration of solute 2, the result in Figure 6C shows that the most concentrated solute is solute 2. The electrophero-

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gram in Figure 6D shows solute 1is concentrated 3.3 times,

and solutes 2 and 3 are half of the original concentration and none, respectively. The solution which is in room B after the third run does not contain any of the three solutes, as shown in Figure 6E, although there is a sharp peak which might be a byproduct from the buffer. The experimental results show that solutes 1,2, and 3 are most concentrated in each final solution of the first, second, and third runs. Solutes 1and 2 are also somehow concentrated in the first run, and the same phenomena are also encountered in other runs. These results might come from the difference of local apparent flow velocity of the solute. Therefore, if we use a tube of very narrow inner diameter, we might overcome this problem because the front zone profile will be flatter compared to a tube of relatively large inner diameter. We propose an apparatus for countercurrent electroconcentration. Using this apparatus, we can set the final volume to 0.2 mL. If V(fina1) is set to 0.1 or 0.05 mL, the volume of

the original solution need only be 1 or 0.5 mL for 10 times concentration, respectively. Therefore, the design of the apparatus is essential to rapid countercurrent electroconcentration. We are now working along this line.

ACKNOWLEDGMENT We thank Isao Kojima of this help with the determination of magnesium ion, and Japan Scientific Instrument Co., Ltd., for lending the capillary electrophoresis system, Isco 3850. Part of this research is supported by a fund from Ajinomoto co.

RECEIVED for review December 28, 1992. Accepted July 14,1993.' 0

Abstract published in Advance ACS Abstracts, September 1,1993.