Anal.
Chem. 1980, 6 1 , 979-981
profile, forcing confluent mixing of viscous streams. The coiled reactors are also useful in the mixing process, although not of the same magnitude as the knotted reactors. It is apparent that as the degree of secondary flow increases, the efficiency for mixing of viscous solutions increases in even greater proportion, and so for mixing very viscous solutions, the knotted reactor is very effective. Only 3.40 s was required to mix the solution of 7 = 5.4 CPwith the diluent, whereas 6.10 and 9.92 s were required for neutralization in the 0.524- and 4.17-cm coiled reactors, respectively. A mixing time of 40 s was required for mixing in the straight line reactor. Some applications may require mixing of two viscous streams. Therefore, an experiment in which both streams had the same viscosity was performed by using a coiled reactor. The data are plotted in Figure 10. The mixing time increases dramatically with 7 for the lower viscosities and then apparently levels off. The encouraging result in this experiment is that the coiled reactor using a 30-30 tee will mix two streams of high viscosity with quite good efficiency, if necessary. Under our experimental conditions, the two reactors, coiled and knotted, offer a distinct advantage over straight and curved reactors when mixing two solutions, one with a high viscosity (e.g., blood serum). It is obvious that mixing is promoted in the coiled and knotted reactor by secondary flow, and that either would be useful when faced with sample pretreatment in which one must dilute a highly viscous sample stream.
ACKNOWLEDGMENT The authors thank David A. Whitman for many helpful discussions.
079
LITERATURE CITED Stewart, J. W. B.; Ruzicka, J.; Bergamin Fo, H.; Zagatto, E. A. G. Anal. Chim. Acta 1978, 8 1 , 371. Ruzicka, J.; Stewart, J. W. B.; Zagatto, E. A. G. Anal. Chim. 1976, 81. 387. Stewart, J. W. B.; Ruzicka, J. Anal. Chim. Acta 1978, 8 2 , 137. Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1978, 99, 9. Frei, R. W. Chemical DerivatlzatlonIn Analytical Chemistry. I n Modern Analytcal Chemistry; Frel, R. W., Lawrence, J. F., Eds.; Plenum Press: New York, 1981; Vd. 1. p 211. Silfwerbrandlindh, C.; Nord, L.; Danielsson, L. G.; Ingman, F. Anal. Chim. Acta 1984, 160, 11. Ruzicka, J.; Hansen. E. H. Flow Inbtlon Ana!&, 2nd ed.;Wiiey-Interscience: New York. 1988. Mardsen, A. 8.; Tyson, F. F. Anal. Proc. 1988, 2 5 , 89. Zagatto, E. A. G.; Reis, B. F.; Maitineili, M.; Krug, F. J.; Bergamin F., H.; Gine. M. F. Anal. Chim. Acta 1987, 198, 153. Tjissen, R. Anal. Chim. Acta 1980. 114, 71. Neue, U.;Engelhardt, H. Chromatographia 1982, 15, 403. Ruzlcka, J.; Hansen, E. H. Anal. Chim. Acta 1984, 181, 1. Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1980, 114, 19. Reijn. J. M.; Van der Linden, W. E.; Poppe, H. Anal. Chim. Acta 1881, 123, 229. Crowe, C. D.; Levln, H. W.; Betterme, D.; Wade, A. P. Anal. Chim. Acta 1987, 194, 49. Whitman, D. E.; Christian. 0. D. 42nd Northwest Regbnai Meeting of the American Chemical Society, Beiiingham, WA, June 17-19, 1987. Huber, J. F. K.; Jonker, K. M.; Poppe, H. Anal. Chem. 1980, 52, 2. Berry, R. S.; Rice, S. A.; Ross, J. physlcal Chemistry. John Wlley & Sons: New York, 1980; p 1104. Golay, M. J. E. J . Chromtogr. 1979, 186. 341. Giddings, J. C. Dynamics of Chromatography, Part I , Princlph and Theory; Dekker: New York, 1985. CRC Handbook of Chemhtry and physics, 64th ed.;Weest, R. C., Ed.; CRC Press: Boca Raton, FL. 1983; p F-48. Pfeffer, J., unpublished results. Hungerford, J. M. Doctoral Dissertation, University of Washington. Hungerford, J. M.; Christlan, G. D.; Ruzicka. J.; Giddings, J. C. Ana/. Chem. 1985, 57, 1794.
RECEIVED for review July 14, 1988. Resubmitted February 3, 1989. Accepted February 3, 1989.
Electrodes Modified with a Film of Phosphatidylcholine: Electrochemistry inside a Lipid Layer Orlando J. Garcia, Pablo A. Quintela, and Angel E. Kaifer* Chemistry Department, University of Miami, Coral Gables, Florida 33124 Prellmlnary results on the behavior of glassy carbon electrades modlfled by cast layers of phosphatldylchdlne(PC) are presented. Redox-actlve amphlphlles are extracted from a Contacting aqueous solutlon Into the cast llpld layer where they undergo electrochemical reactlons wlth the underlylng electrode surface. Once the llpld layer Is loaded wlth electroactlve amphlphlllc materlal, the electrode can be transferred to pure supporting electrolyte solutions wlth retentlon of the electroactlvlty. I n contrast, hydrophlllc electroactlve substrates are kept away from the electrode surface by the PC layer largely preventlng the observatlon of electron transfer reactions wlth the electrode.
In this communication we report the peculiar electrochemical behavior of electrode surfaces covered by a layer of phosphatidylcholine (PC).Redox-active amphiphiles readily partition into this lipid layer from a contacting aqueous solution and undergo electrochemical reactions at the electrode surface. In contrast, more hydrophilic versions of these electroactive compounds are rejected by the lipid layer preventing their electron transfer reactions with the electrode. Several groups are actively investigating the properties of electrode surfaces derivatized with hydrophobic materials. A 0003-2700/89/0381-0979$01.50/0
common approach is based on the self-assembly of electroinactive (1-5) or electroactive (6-12) amphiphiles at the eledrode-solution interface. An alternative methodology uses Langmuir-Blodgett techniques to transfer monolayers (7, 13-18) or multilayers (19) from the air-solution interface to a substrate that is then employed as an electrode. The effects of cast layers of palmitic acid on the electrochemical behavior of several substrates at the underlying electrode were recently reported by Tanaka et al. (20). In their work, these authors noted that the hydrophobic layers hindered substantially the observation of electrochemistry of hydrophilic species. We reasoned that a PC layer could behave as a selective barrier (membrane) on the electrode and perhaps incorporate redox-active lipophilic substrates. We present here preliminary results that clearly indicate both features using the following redox-active substrates.
2 Br-
I R = C,H,, , X = BrR = CH3 , X - = I-
O 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
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A
i
-a r
-1.0
I
,
c
I
v
0.0
POTENTIAL
d o '
(V vs
r
01s
SSCE>
Voltammetric response of GC/PC electrodes (8.9 X lo-' mol/cm2)at 50 mV/s. Other condtons are as follows: (A) contacting solution, 1.0 mM 1 + 50 mM phosphate buffer (pH = 7); scans were recorded every 5 min; first scan is labeled 1; (e) contacting solution, 1.O mM 2 + 50 mM phosphate buffer (pH = 7); scans were recorded every 5 min; (C) contacting solution, 50 mM phosphate buffer (pH = 7); loading was performed by previous immersion of the electrode during 45 min in a solution containing 1.0 rnM 3 + 50 mM phosphate buffer (pH = 7); (D) same as in C but the loading step was done in a solution containing 1.0 mM 4 + 50 mM phosphate buffer (pH = 7). Flgure 1.
EXPERIMENTAL SECTION Materials. Compound 1 was synthesized as described elsewhere (21). 2 was purchased from Aldrich and recrystallized from methanol. 3 and 4 were synthesized according to a published procedure (22) by treatment of [(dimethylamino)methyl]ferrocene (Aldrich) with heptyl bromide or methyl iodide, respectively. Egg yolk PC was purchased from Sigma as a 100 mg/mL solution in chloroform. Distilled water was further purified by passage through a Barnstead Nanopure four-cartridge system. All other reagents were of the best commercial quality available. Electrode Modification. The PC/chloroform solution from Sigma was 10-fold diluted with chloroform to yield a 10 mg of PC/mL solution. A carefully measured volume of this solution was deposited on the tip of a GC electrode (BioanalyticalSystems) and the chloroform allowed to evaporate. The evaporation was usually conducted while slowly rotating the electrode (100 rpm) to increase the homogeneity of the resulting cast lipid layer. Equipment. The electrochemical instrumentation has already been described (21).
RESULTS AND DISCUSSION Figure 1A shows the time dependence of the cyclic voltammograms obtained on a glassy carbon (GC) electrode covered with a cast PC film (8.9 X mol/cm2) immersed in a 1.0 mM solution of 1 in 50 mM phosphate buffer (pH = 7.0). The viologen reduction waves clearly increase with time, thus revealing the gradual incorporation (loading) of 1 into the lipid layer covering the electrode. Once a certain degree of loading is attained the working electrode can be transferred to a solution containing only supporting electrolyte with retention of the viologen waves. A similar electrode contacting a 1.0 mM solution of methylviologen (2) in the same buffer medium gives the voltammograms recorded in Figure 1B. This shows that the less lipophilic compound 2 does not partition favorably into the lipid layer and, therefore, its reduction at the electrode surface is almost completely suppressed by the presence of the PC film.
The voltammetric results obtained with PC-covered GC (GC/PC) electrodes contacting solutions of 1 are also remarkable because both redox steps (V2+/V+and V+/V) are clearly discernible and the observed half-wave reduction potentials (-0.50 and -0.84 V vs SSCE) suggest a rather hydrophobic environment (23,24). The reduced forms (V+ and V) of lipophilic viologens, such as 1, typically precipitate on the electrode surface which complicates the electrochemical behavior (21, 23, 24). These precipitation effects are not observed on GC/PC electrodes suggesting that the lipid layer provides a matrix where the electrochemistry of lipophilic viologens can be studied free from these complications. The loading of ferrocene derivatives 3 and 4 was also investigated. However, cycling of a GC/PC electrode through the oxidation potential of 3 does not lead t o an increase of the observed voltammetric waves because the oxidation of 3 generates a dication that appears to have a very low partition coefficient into the lipid film. Nonetheless, 3 can be loaded into the lipid layer without electrochemical cycling. The cyclic voltammogram of Figure 1C shows the response of a GC/PC electrode in pure 50 mM phosphate buffer after 45 min of loading in a 1.0 mM solution of 3 in the same buffer medium. Figure 1D shows the voltammogram corresponding to the same experiment but this time using compound 4 as the electroactive probe. Both of these voltammograms were recorded 15 min after the electrode was transferred from the loading solution to the pure buffer solution. In excellent correspondence with the viologen results, the less lipophilic compound 4 partitions poorly into the lipid layer resulting in very low currents for the 4+/4 couple. In analogy with methods devised in the investigation of Nafion-modified electrodes (Z), the electroactive amphiphile was also dissolved in the lipid/chloroform solution used to cast the lipid films on the GC electrodes. This procedure eliminates the need to load the redox-active surfactants in the lipid film. Electrodes prepared in this way show stable voltammetric waves when immersed in pure buffer solutions. For instance, Figure 2A shows the voltammogram recorded on a GC electrode covered by 8.9 X mol/cm2 of PC and 8.9 X mol/cm2 of 1 after 20 min in pure buffer solution. The behavior of an electrode covered by 8.9 X mol/cm2 of PC and 8.9 X mol/cm2 of 1 after 3 h in the 50 mM phosphate buffer solution is shown in Figure 2B. Both electrodes were found to give stable voltammetric waves during several hours with little or no loss of 1 from the lipid layer. The two redox couples in the voltammogram of Figure 2A exhibit peak-to-peak splittings below 59 mV as expected for low coverage (equivalent to about three monolayers of 1) of the electrode surface. This was not observed in the electrode of Figure 2B as it would be anticipated for larger coverages. Control experiments indicate that hydrophilic redox species, such as R U ( N & ) ~ ~or+Fe(CN)64-,give very low voltammetric currents at GC/PC electrodes (the peak currents decrease by a factor of 16 for Fe(CN);- and 25 for Ru(NH&~+as compared to those recorded with the bare GC electrode) in good agreement with Tanaka's observations (20). We have shown here that these electrodes are capable of extracting amphiphilic electroactive materials from solution. The resulting electroactive lipid layer is stable for long periods of time (hours to days depending on the system) in supporting electrolyte solutions. Surfactant complexing agents (21) in the solution, like a-cyclodextrin, do not affect much the electrochemical behavior of these electrodes. On the other hand, the lipid layer is disrupted (solubilized) by the presence of large concentrations of micelle-forming surfactants (for instance, 0.1 M SDS). A present difficulty of GC/PC electrodes is the low mechanical stability of the lipid layer. Thus, care must be exercised in handling these electrodes to keep the lipid layer
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
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electrodes covered with Langmuir-Blodgett (7,14,15,19) or self-assembled (6-8) monolayers. In contrast to this, the permeation of ions through biological membranes is usually very unfavorable in the absence of ionophores or active, open channels. Thus, we are currently investigating the processes involved in charge (and ion) transport across the lipid layers on these electrodes. Registry No. 1,114094-51-8; 1 (V+ form), 119638-18-5; 1 (V form), 119638-19-6; 2,3240-78-6; 3, 119638-20-9;4,12086-40-7; C,7440-44-0. I
1
I
I
B
1
1
POTENTIAL
I
1
( V vs SSCE)
Flgure 2. Voltammetric response (10 mV/s) in 50 mM phosphate buffer (pH = 7) of GC electrodes modified by casting a mixture of PC and 1: (A) cast surface coverages, 8.9 X lo-' mol/cm2 PC and 8.9 x 1 0 - l ~d / c m 2 I. Voltammogram recorded after 20 min of e b o d e immersion in the buffer solution; (6)cast surface coverages, 8.9 X 10" md/cm2 PC and 8.9 X lo4 mol/cm21. Voltammogram recorded after 3 h of electrode immersion in the buffer solution.
intact. Note that compounds 2 and 4 hardly partition into the PC layer while both compounds have been demonstrated to partition readily into Nafion films (26,27). Good permeation into PC films seems to require amphiphilic character, i.e., the presence of a lipophilic tail in the molecular structure of the substrate, as exemplified by compounds 1 and 3. This is a unique property of this new type of modified electrodes that resembles closely the behavior of biological membranes. However, it should be pointed out that the nominally insulating lipid films of this work (at least equivalent to several bilayers) surprisingly allow the passage of enough ionic current to permit the electroactivity of the incorporated amphiphiles. A high degree of ionic permeation has also been observed in
LITERATURE CITED (1) Porter, M. D.; Bright, T. 6.; Ailara. D.L.;Chidsey, C. E. J. Am. Chem. Soc.1987, 109, 3559. (2) Finkiea, H. 0.;Robinson, L. R.; Blackburn, A.; Richter, 6.; Ailara, D.; Bright. T. Langmuir 1086, 2 , 239. (3) Sabatini, E.;Rubinstein, I.; Maoz, R.; Sagiv, J. J. Electraenel. Chem. 1987, 219. 385. (4) Sabatini, E.; Rubinstein, I. J. Phys. Chem. 1987, 9 1 , 8683. ( 5 ) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Neture 1988, 332, 426. (6) Facci. J. S. Langmuk 1087, 3 , 525. (7) Lee, C.-W.; Bard, A. J. J. Electroanal. Chem. 1088, 239, 441. (8) Dlaz, A.; Kalfer, A. E. J . Electroanal. Chem. 1088, 249, 333. (9) Miller, C. J.; Majda, M. J. Am. Chem. Soc. 1986, 108. 3118. (10) Miller, C. J.; WMrig. C. A.; Charych, D. H.; Majda, M. J . phvs. Chem. 1988, 92, 1928. (11) a s s , C. A.; Miller, C. J.; Ma@. M. J . phvs. Chem. 1988, 9 2 , 1937. (12) Miller, C. J.; Ma@. M. Anal. Chem. 1088, 60, 1168. (13) Park, H. G.; Aoki, K. A.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1985, 195, 157. (14) Daifuku. H.; Aoki, K.; Tokuda, K.; Matsuda. H. J. Electroanel. Chem. 1985, 163, 1. (15) Daifuku, H.; Yoshimira, I.; Hirata, I.; Aoki, K.; Tokuda. K.; Matsuda, H. J. Electroanel. Chem. 1986. 199, 47. (16) Aoki, K.; Tokuda, K.; Matsuda, H. J. Electroanel. Chem. 1088, 199, 69. (17) Matsuda, H.; Aoki, K.; Tokuda, K. J. Electroanel. Chem. 1987, 217, 1. (18) Facci, J. S.; Faicigno, P. A,; Gold. J. M. Langmulr 1986. 2 , 732. (19) Fujihira, M.; Araki. T. Bull. Chem. Soc. Jpn. 1988, 5 9 , 2375. (20) Tanaka, K.; Tamamushi, R. J. Electroanel. Chem. 1987, 236, 305. (21) Diaz. A.: Quinteia. P. A.: Schuette. J. M.: Kaifer. A. E. J. Phys. Chem. 1988, 92. 3537. Lombardo, A.; Bleber, T. I. J. Chem. Educ. 1989, 60, 1080. Kaifer, A. J. Am. Chem. Soc. 1986. 108, 8837. Lu, T.; Cotton, T. M.; Hurst, J. K.; Thompson, D. H. P. J . Electroanel. Chem. 1988, 246, 337. Martin, C. R.; Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1982. 104. 4817. (26) Geuiielio, J. 0.; Gosh, P. K.; Bard, A. J. J. Am. Chem. Soc. 1985, 107, 3027. (27) White, H. S.; Leddy, J.; Bard, A. J. J. Am. Chem. Soc. 1082, 104, 4811.
RECEIVED for review November 22,1988. Accepted January 25, 1989.
