Anal. Chem. 1994,66, 3997-4004
Concentration and Optical Measurement of Aqueous Analytes in an Organic Solvent Segmented Capillary under High Electric Field Hong J. Zheng and Purnendu K. Dasgupta’ Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409- 106 1
Concentration by solvent extraction is examined in a fused silica capillary under high electric field. An immiscible solvent segment, containing a sufficient concentration of a supporting electrolyte, can be introduced in an aqueous electrolyte-filled capillaryby electroosmotic suction. Followingthe introduction of the solvent segment, the capillary terminus is reintroduced into an aqueous electrolyte and the electric field is restored. The segment is carried along by the electroosmotic flow. Aside from any intrinsic conductivityof the saline immisciblesolvent, a thin aqueous film around it permits the electrical circuit to remain continuous. If ions or ionizable complex moieties (extractable as ion pairs) are initially present in the aqueous electrolyte, they are concentrated by extraction in the organic solvent and/or accumulate in the aqueous phase ahead of the interface. For the analyte extracted into the organic segment, the charge type and nature of the extracted ion and the applied voltage polarity determine whether the accumulation occurs on the front or the rear edge of the solvent segment. The peak analyte concentration in the organic segment is typically an order of magnitude greater than the initial concentration in the aqueous phase. If a similar experiment is conducted with the introductionand transport of an organicsolvent segment under gravity, no significant extraction signal is observed. Solvent extraction remains a powerful and widely used tool in analytical chemistry. Extraction of an ionic surfactant as an ion pair with a colored or fluorescent dye and theselective extraction of a metal as a neutral complex or as a ternary complex (a charged metal complex, paired with an oppositely charged hydrophobic ion) constitute typical examples. Many a prescribed official or standard method of analysis involves solvent extraction; a significant number have been automated. Automated solvent extraction schemes were first implemented in segmented continuous flow analyzers.* Karlberg and Thelander3 pioneered the introduction of such schemes in the simpler format of flow injection analysis (FIA). This and further developments have resulted in systems that require much smaller amounts of a sample relative to manual extractions in a separatory funnel. Nevertheless, tens, more commonly hundreds, of microliters of sample are still necesE-mail address:
[email protected]. (1) (a) Kolthoff, I. M.; Elving, P. J., Eds. A Treatise On Analytical Chemistry. Part 11: Analytical Chemistry of the Elements; Wiley: New York, 19611980 Vols. 1-17. (b) Horwitz, E. P., Ed. Soloent Extraction and Ion Exchange: Marcel-Dekker: New York, 1983-1992; Vols. 1-10. (c) Cheng, K. L.; Ueno, K.; Imamura, T. Handbook of Organic Analytical Reagents; CRC Press: Boca Raton, FL, 1982. (2) Wallace, V. Anal. Eiochem. 1967, 20, 411-418. (3) Karlberg, B.; Thelander, S. Anal. Cfiim. Acta 1978, 98, 1-7. 0003-2700/94/0366-3997$04.50/0
0 1994 American Chemical Society
sary. In a number of areas, it will be clearly desirable to improve upon this. In addition, for a variety of analytes, it will be desirable to lower the quantity of solvents used because of their hazard and limited future a~ailability.~ In 1981 Jorgenson and Lukacs5 demonstrated the extraordinary power of electrophoreticseparations in a fused silica capillary under high electric field. A great deal of interest has since been focused on microdeterminations in a capillary format. Increasingly, the scope of such schemes extend beyond electrophoretic separations. The electroosmotic flow (EOF) generated in a silica capillary provides a unique, highly controllable and reproducible mechanism for fluid propulsion. This has now been exploited in FIA,6 sequential injection analysis,’ and enzyme-mediated microanalysis.8 It was therefore of interest to us to examine if solvent extraction can be carried out in a capillary format. If electroosmosis is used in a configuration where a grounding joint electrically isolates the analytical system from the pumping fluid (which is nevertheless hydraulically coupled to the pumped liquid),6b there are few surprises. Electroosmosis in this case acts merely as an external pump. A more interesting configuration is where the entire analytical system is under a high electric field. It is not intuitive how poorly conductive immiscible organic solvent segments can be introduced and maintained in such a system. In conventional flow injection solvent extraction systems, the extraction efficiency is acutely dependent on intrasegment circulation pattern^.^ Even if organic solvent segments can be introduced by electroosmosis, it is not obvious how the flat velocity profile, characteristic of EOF in a capillary, will affect extraction efficiency. This paper describes results of experiments designed to accomplish organic solvent extraction in a capillary under high electric field. Several aspects of the behavior of such systems are unexpected and are discussed here.
EXPER I MENTAL SECT1ON Equipment. The general layout of the experimental setup is similar to that of a home-made instrument previously described.6a The instrument is equipped with two microprocessor addressable rotatable turrets on the source side that hold a number of vials. The two turrets have a fixed height (4) Noble, D. Anal. Chem. 1993.65, 693A-695A. (5) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1305. (6) (a) Liu, S.;Dasgupta, P. K. Anal. Cfiim.Acta 1992,268, 1-6. (b) Dasgupta, P. K.; Liu, S. Anal. Cfiem. 1994, 66, 551-556. (7) Liu, S.; Dasgupta, P. K. Talanta, in press. (8) (a) Bao, J.; Regnier, F. E. J . Cfiromatogr. 1992,608,217-224. (b) Harmon, B. J.; Patterson, D. H.; Regnier, F. E. Anal. Chem. 1993. 65, 2655-2662. (9) Nord, L.; Karlberg, B. Anal. Chim. Acta 1984, 164, 233-249.
Analytical Chemistry, Vol. 66, No. 22,November 15, 1994 3997
difference, the lower one being at the same height as the destination vial, located after the detector. The electrodes are platinum, 0.25 mm indiameter. The high voltage electrode is affixed to the turret end of the capillary. Polyimide-coated silica capillaries (75 pm i.d., 350 pm 0.d.; Polymicro Technologies,Phoenix, AZ) wereused throughout. The turret end of the capillary can be moved by means of two independent electropneumatic actuators for dipping/lifting into any given vial in either turret. The individual vial that the capillary is dipped in can be changed by rotating the motor-driven turret. If the capillary is put into a vial on the higher turret, the fluid in that vial is introduced into the capillary by gravity. During the time voltage is applied, the capillary is dipped in a lower turret vial to avoid any concurrent hydrostatic flow. The capillaries used in these experiments were 50 cm long. The polyimide coating was removed for 1 cm at -40 cm from the turret end. The bare portion constituted the radial path detection cell for a ball lens equipped variable wavelength absorbance detector (LINEARUVIS 200, Spectra-Physics). The high voltage power supply (*30 kV, 300 pA maximum limits) was a model CZE lOOOR unit (Spellman Inc., Plainview, NY). All instrument functions were automated through a programmable microcontroller (LS-100, Minarik Electric, Los Angeles, CA). For data acquisition, vendor supplied software and a DAS-12 card (Analogic Corp., Wakefield, MA) incorporated in a 80386 class personal computer and/or a strip chart recorder (Omniscribe, B-2000, Houston, TX) were used. Except as stated, PC-based data acquisition was conducted at a sampling rate of 10 Hz. Reagents. A spectrophotometric grade of chloroform was used. Tetrabutylammonium perchlorate (TBAP) (Sachem, Austin, TX) was used as obtained, without drying. Unless otherwise stated, TBAP was dissolved in CHCl3 to attain a concentration of 0.1 g/mL. Other quaternary ammonium salts (QAS) were obtained from Aldrich. The pH and composition of an aqueous solution put in the capillary control the magnitude of the EOF generated. A solution of 2 mM NazB407 (pH 9.2) was chosen for this purpose because of the significant EOF generated by this solution. As extractable analytes, we studied anionic dyes such as Eosin Y (0-600 ppb), cationic dyes such as Crystal Violet (0-1 ppm), anionic chelates (all chelates contain an excess of the chelating agent) of metals such as Co2+ (0-800 ppb) complexed by 4-(2pyridy1azo)resorcinol (PAR, 10 ppm), neutral chelates such as Cu2+ (0-2 ppm) complexed by diethyldithiocarbamate (DEDC, 20 ppm), and cationic chelates such as Fe2+ (700 ppb) complexed by 1,lO-phenanthroline (0-Phen, 1 mM). The complexes were performed, and the desired amount (containing the excess reagent) was added to the borate solution. The concentrations stated above are the final concentrations in the borate solution. Because of the minute amount of the analyte added, there was no discernible change in the pH of the borate solution. Thespectra of the colored dyes/complexes were acquired on a Hewlett Packard 8451A diode array spectrophotometer,and the optimum measurement wavelength during the actual experiment was chosen on this basis. Protocol. The following steps were typically carried out: (i) Flush the capillary with borate solution containing analyte (0-50 pL) by gravity or applied pneumatic pressure from the source side; the destination side contains the undoctored borate
-
3998
Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
- O o 4 - , ' 100 ',
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'
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Flgure 1. (a) repeated introduction of organic segments, 10% tbap in CHCIBintroduced for 20 s each. (b) Detailed view of one segment.
solution in all experiments. (ii) Dip the capillary into a vial of CHCl3 containing dissolved TBAP and apply 15 kV for 10s. The chloroform segment is introduced into the capillary. (iii) Lift the capillary, rotate turret to a vial containing either (a) plain borate solution or (b) borate solution containing the analyte, dip capillary in vial, and apply +15 kV until the organic segment passes through the detector. In case b, steps i and ii can be repeated many times to perform replicate measurements.
+
RESULTS AND DISCUSSION Introductionof an Organic Solvent Segment. It is obvious that a segment of pure organic solvent can be introduced into a capillary filled with an aqueous electrolyte by gravity. However, when the electric field is reestablished after reinsertion of the capillary into an aqueous electrolyte, an open circuit results within tensof seconds. The high resistance of the CHC13segment causes most of the voltage to be applied across this part. The circuit is seemingly disconnected by vapor pockets formed, whether due to Joule heating alone or a thermal runaway condition brought about by the dielectric breakdown of the wet solvent. Ebullition in the organic segment can be discerned immediately prior to circuit disconnection. Incorporation of a QAS in the Organic Solvent. QAS compounds are routinely used as supporting electrolytes in nonaqueous solventsI0 and as phase transfer catalysts.' We found that if a QAS is incorporated in the CHC13 in sufficient amounts, organic solvent introduction can be markedly improved. Different concentrations of TBAP were dissolved in CHC13. Concentrations 55 w/v% were ineffective. A concentration of 7.5% TBAP did prevent the occurrence of an open circuit, but the reproducibility of the results was limited. At a TBAP concentration of 10% (0.29 M), highly reproducible introduction of CHC13 segments is observed. An example is shown in Figure la. A single segment is shown magnified in Figure 1b. For the data shown, the reproducibility of migration time (from injection of CHC13 to detection) is 0.51%, and the reproducibility of the peak width is 1.72% (20 s introduction) when conducted in a given capillary. (10) Adams, R. N. Electrochemistry at Solid Electrodes; Marcel-Dekker: New York, 1969. (11) Masson, D.; Magdassi, S.;Sasson, Y. J . Org. Chem. 1990, 55, 2714-2719.
30 3
0
0 0 0
0 0 0
i
0 0
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0
4
0
0
Field Strength, V/cm Figure 2. Current-voltage relationship for 10% TBAP in CHCb in a 50 cm long, 75 pm bore capillary.
Continued increases in TBAP concentration does not improve reproducibility further. The migration time is marginally reduced at higher TBAP concentrations. Among other common QAS substances, tetraethylammonium and tetra(n-propyljammonium perchlorate do not dissolve in CHC13 at sufficiently high concentration to permit reproducible introduction of organic segments. However, reproducible organic solvent introduction was observed with other tetrabutylammonium salts such as bromide, bisulfate and iodide, and tetrapentylammonium or tetrahexylammonium bromide, all at a concentration of 0.29 M each in CHC13. The migration time of the segment was not markedly dependent on the specific salt. As is well known, individual silica capillaries show some variations in EOF. The rate of CHC13 introduction appears to be simply governed by the EOF in the capillary, Le., the segment is introduced by electroosmotic suction. With the 2 mM borax electrolyte used in this work, the EOF as well as the CHC13 introduction rate was 12 f 1.5 nL/s at a field strength of 300 V/cm. For a 75-pm capillary and a 10-s introduction period typically used, this results in a 27 f 3 mm long segment. Electrical Conductivity and Electroosmotic Flow. The incorporation of a QAS into a solvent like CHCl3 markedly increases its electrical conductivity. If a 75-pm bore 50-cm capillary is completely filled with a CHC13 solution of 0.29 M TBAP, the current-voltage plot is linear only at low electric field strengths (Figure 2). It is possible that at higher electric field strengths the current increases more than linearly due to Joule heating. The dissipation of the heat generated is much less efficient in CIICl3 relative to that in water; the thermal conductivity of CHC13 is only one-sixth that of water. In any case, it is noteworthy that at an electric field strength of 300 V/cm (the typical value used in this work), the steadystate current through a 0.29 M solution of TBAP is significantly greater than that through 2 mM aqueous Na,B407 (10.4 vs 4.3 PA). Although the TBAP solution in CHC13 permitted a significant flow of current, there was no significant EOF as
evidenced from an injected neutral marker (diphenylthiocarbazone in CHC13 or a short segment of an aqueous electrolyte). According to theSmoluchowski equation,12EOF is directly dependent on the lpotential of the surface, on the dielectric constant (e) of the fluid, and inversely on the viscosity (7) of the fluid. Even in the unlikely case that the {potential of a silica surface is the same when filled with an aqueous borate electrolyte vs a solution of TBAP in CHC13, the e / q ratio for CHCl3 is nearly an order of magnitude less than that of water. This should result in a proportionately lower EOF. No detector response was observed after -60 min of neutral marker introduction (this is -2OX the migration time of an organic segment introduced into the aqueous borate electrolyte). Electrical Continuity in the System. It may seem at first sight that the continuity of the electric circuit in the present system is maintained because the organic solvent segment containing the dissolved QAS is quite conductive. However, unlike electrical continuity between two metals connected in series, the charge carriers in an aqueous borate solution and that in a QAS-doped CHC13 solution are different. To have current conduction in an organic solvent segmented aqueous electrolyte, it would be necessary that the cationic and anionic constituents of the aqueous phase can respectively migrate from the anode and cathode side into the organic segment. Further, either these ions will then have to migrate through the organic segment or a proportionate amount of the TBA+ and the C104- ions can leave from the correspondingly opposite end of the organic segment. Because of the relatively low polarizabilities of Na+ and B(OH)4- ions, their entry into the organic solvent does not appear very probable. The key role of QAS in the present system may be that of a phase transfer agent: it lowers the interfacial energy sufficiently to permit the existence of a thin aqueous film between the organic segment and the silica wall. Available evidence supports this contention. Two silica capillary segments were connected with a polyvinyl chloride (PVC) tubing, and several millimeters of the PVC tube was left internally exposed at the joint. With a purely aqueous electrolyte, the observed current and EOF were both stable under a high electric field. When a QAS-containing CHC13 segment was injected, current flowed until the organic segment reached the joint. We surmise that as soon as the organic segment reached the hydrophobic PVC surface there was no affinity for the wall to maintain an aqueous film and the aqueous film surrounding the organic segment disappeared. In the absence of the film, an open circuit soon occurred as most of the voltage was dissipated across the organic segment, and dielectric breakdown probably led to overheating and bubble formation. Output Characteristics. In the absence of an extractable analyte in the aqueous carrier, the injected organic segment is registered as a flat-bottomed rectangular negative pulse by the detector, as shown in Figure lb. Negative absorbance relative to the aqueous electrolyte is observed. This may have two reasons. Light losses from interfacial reflection and beam divergence are reduced with CHCL as the intervening medium. Chloroform has a much closer refractive index (1.44) to silica (12) Smoluchowski, M. V. Physik. Z.1905,6, 529-531.
Analytical Chemistty, Vol. 66,No. 22, November 15, 1994
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7.5
j
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1
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6.5
+J
c
!!
5.5
5.5
3
0 4.5
4.5
3.5
3.5
0
10
Time, min Figure 3. Current-time profile observed with the introductionof CHCl3 containing (a) 10 and (b) 20% w/v TBAP. The segment is introduced just before the current trace starts going down, and the maximum current occurs just as the segment leaves the capillary.
(1 -46)than does water (1.33). The amplitude of the negative pulse is typically in the range of 25-30 mAU for the capillary dimensions used, but the exact value is critically dependent on the precise mounting of the capillary in the detector. The magnitude of the negative signal is significantly larger than what can be accounted for by reduced interfacial light loss. Also, this factor cannot produce a negative absorbance if there is an aqueous film of significant thickness surrounding the CHC13 segment. The negative signal must therefore be due to the lensing effect of the CHCl3 segment. At each edge of the negative pulse, small positive excursions can occasionally be seen (not visible in Figure lb). This can be caused by some irregularity at the interface or, more likely, by change of the refractive index of the aqueous solution immediately adjacent to the organic segment due to the leakage of the QAS dissolved in the organic solvent. Leakage of QAS from the Organic Segment. Temporal Current Profiles. Manual extraction experiments with a known amount of TBAP distributed between CHC13 and water indicate a distribution constant of 150, as determined by conductometry of the aqueous extract. Some leakageof TBAP into the aqueous electrolyte in the capillary system is therefore inevitable. The temporal current profiles, at two different TBAP concentrations (Figure 3) indicate that this indeed occurs. A reasonably quantitative picture can be obtained as follows. The measured specific conductance of a 2 mM Na2B407 solution is 300 pS/cm. Neglecting any surface currents, the current through a 50 cm long 75 pm bore (cross section 4.4 X cm2) capillary is calculated to be -4 pA for an applied voltage of 15 kV, in good agreement with the initial current of -4.3 pA observed in Figure 3. The current initially decreases with the introduction of the organic segment. A minimum current of -3.6 pA is observed (Figure 3a); this represents an increase in the resistance of the capillary from the initial value by a factor of 1.2. Considering that the organic segment is 2.7 f 0.3 long in a 50 cm capillary, it is readily computed that the ratio of the specific resistance of the organic zone and the aqueous phase is 4.8 f 0.6. The field
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
strengths in the organic vs aqueous zones are expected to be in the same ratio. However, as soon as the organic segment is introduced, QAS begins to leak from it and increases the conductance of the adjacent aqueous phase. With further downstream movement of the segment, more of the aqueous phase is contaminated by leaked out QAS, the ionic constituents of which also continue to migrate further into the aqueous phase by electromigration, diffusion, and hydrodynamic mixing. The field strengths in the aqueous vs the organic zone was experimentally determined by measuring the voltage drop across a bifilar wire probel3 (38 pm diameter nickel conductors spaced 40 pm apart) interposed radially in the capillary through a PVC joint. This ratio was measured to be 5.1 f 0.5 when CHC13 segments containing 10% TBAP were introduced, in good agreement with the expectations above. The temporal current profiles in Figure 3 indicate that the as the leakage of QAS continues from the segment, past the initial minimum, the current continues to increase, reaching a maximum at the point the segment leaves the capillary. How much QAS leaks from the segment into the aqueous phase? An estimate is possible from the observed maximum current. Consider that the maximum observed current in Figure 3b is -8.3 PA, -4 pA higher than the initial value. From the known equivalent conductance of TBA+ (1 9.1) and C104- (67.9), it can be computed that this excess current can be produced if 3.4 mM TBAP (equivalent to 7.4 nmol, 2.5 pg) is uniformly distributed in the aqueous phase in addition to the borate. The value for the aqueous phase TBAP concentration in equilibrium with 0.58 M TBAP in CHC13 (as used in Figure 3b) is 3.9 mM, so a leakage equivalent to 3-4 mM TBAP is not unreasonable. Of course, the aqueous TBAP concentration is doubtless higher in the immediate neighborhood of the segment and must decline toward the anode vial where fresh borate electrolyte is being introduced. It is noteworthy that the peak current is lower when the QAS loading is lower (Figure 3a vs 3b), suggesting that the total leakage into the aqueous phase is dependent on the QAS concentration in the organic phase. There should be less leakage of the QAS as the hydrophobicity of the QAS is increased. With 0.29 M tetraoctylammonium bromide (TOAB), considerably more hydrophobic than TBAP, it was found that the current decreases after the introduction of the organic segment as with TBAP but does not increase again, suggesting that little or no leakage of TOAB occurs. Mechanism of Current Conduction. In the present system, current conduction most likely occurs via the thin aqueous film surrounding the organic segment. For the moment, we assume that the charge transfer through the QAS-containing organic solvent itself is negligible relative to that through the film surrounding it. It has already been noted that the experimentally measured field strength in the organic zone is 4-8 f 0.6 times that in the bulk aqueous phase. Should we assume a 7.5 pm thick aqueous film (surrounding a 60 pm diameter organic solvent), we can readily compute that the film has 20% of the cross section of the capillary bore and should therefore lead to a field strength that is five times greater over this zone than the bulk aqueous electrolyte. (13) Kar,
S.;Dasgupta, P.K.; Liu, H.; Hwang, H. Anal. Chem. 1994,66, 2537-
2543.
Considering that the amount of leaked QAS in the aqueous film surrounding the organic solvent is probably much greater than that in the bulk aqueous phase, the specific conductance of this film is likely much higher than that of the bulk aqueous medium in the capillary. Therefore, the actual aqueous film thickness may be significantly smaller than 7.5 pm for the observed field strength ratio. Present SystemCompared to FIA-Based Solvent Extraction. Before the results of the present “extraction” experiments are considered, the difference of the present experiments with typical FIA-based solvent extraction needs to be noted. In FIA, the sample is injected, typically mixed with appropriate reagents, and then segmented by a multitude of small (ca. 1 mm long) solvent ~ e g m e n t s . 1If ~ a single, relatively long, solvent segment is introduced into a sample (plus reagent) filled conduit, as in the present experiments, a reasonable “extraction efficiency” can only be observed if the extractable material is adsorbed on the conduit ~ a l 1 s . lIt~ should be obvious that the extractant can otherwise “see” only a very limited amount of sample. With the typical extracted species being hydrophobic, such adsorption is common with PTFE tubing typically used in FIA. However, this is not the case for a hydrophilic silica wall, with the exception that cationic species may be adsorbed on the negatively charged wall. For any other analyte, adsorptive accumulation on the wall cannot be invoked. Extraction of Cationic Solutes: Crystal Violet and Tris-( 1,lO-phenanthroline) Iron(I1). Crystal Violet (N,N,N’,N’,N”,N”-hexamethyl p,p’,p”-triaminotriphenylcarbinol, hereinafter abbreviated CV) is a strongly hydrophobic triphenylmethane dye. Most commonly, the dye is supplied as the hydrochloride that can exist in two tautomeric forms: one in which the chlorine is covalently bonded to the central carbon atom of the triphenylmethane moiety (Ph3CC1) and another in which chloride and the triphenylmethane carbonium ions exist as distinct ionic entities (Ph3C+and C1-).’6 While CV is soluble in water, it adsorbs tenaciously on most surfaces and is readily extracted by polar to modestly polar organic solvents. Figure 4a shows the extraction signal obtained when the capillary is initially filled with a solution containing 230 ppb CV in the borate electrolyte and a 2.7 cm long CHC13 segment (heretofore it is implicit that the CHC13 segment is 0.29 M in TBAP) is passed through it. Figure 4a depicts two distinct traces: the solid line corresponds to a wavelength of 600 nm, close to the absorption maximum of CV, and the dashed line corresponds to a wavelength of 720 nm where CV has essentially no absorption and can therefore be treated as the reference wavelength. This type of two wavelength absorption data was used throughout this work. The detector used in this work could only monitor one wavelength at a time; the two traces such as those in Figure 4a were obtained in sequential experiments. In the absence of the analyte such as CV, the two wavelength traces were virtually identical. In cases where a minor difference existed (due presumably to the different refractive indices at the two different wavelengths), one trace was (14) Ruzicka, J.; Hansen, E. H. Flow Injecfion Analysis, 2nd ed.; Wiley: New
York, 1988.
-2E-0034
Flgure 4. Extraction signal for 230 ppb Crystal Violet. (a) The two traces represent two different monitoring wavelengths, as indicated, one (the dashed trace) being used as a reference. (b) The net signal obtained from the difference of the two traces. The boundaries of the organic segment are marked in each case.
normalized with respect to the other, using data obtained in the absence of the analyte. The same normalization factor was then used to process the data obtained in the presence of the analyte. Similarly, the occurrence of the organic segment in the two sets of data at the two different wavelengths were synchronized as best as possible using software written inhouse. After normalization and synchronization, subtraction of the data obtained at the reference wavelength from that obtained at the analyte absorption wavelength produced the net analyte signal. The net analyte signal for the experiment in Figure 4a is shown in Figure 4b. It is apparent from Figures 4a,b that CV is concentrated at the front edge of the organic segment. For the triaminotriphenylmethane dyes, it is the cationic quinonimine form, with the positive charge delocalized among the three imine nitrogens at the terminal end of the molecule, that is believed to be responsible for the visible absorption.I5 The distribution of theabsorbing species within theorganic segment suggests that significant polarization occurs in the organic segment due to the high electric field such that the cationic constituents are dominantly distributed near the cathodic edge of the segment. That the distribution is not due to the hydrodynamics of the extraction process is proven by the following experiment. When CV is extracted into TBAP containing CHCl3 in a test tube experiment and a short segment of this dye containing CHC13 solution is introduced into an aqueous borate filled capillary, the color due to CV is initially uniformly distributed in the segment. When high voltage is applied and the segment is eluted through the detector, CV is again found to be concentrated at the front edge, similar to the distribution observed in the standard extraction experiment. With specialized equipment,I7 presumably the distribution can be directly imaged in a small capillary. We performed an experiment in a thin wall larger bore glass capillary (ca. 300 pm) without any external protective coating such that both the liquid-liquid interface and the color due to CV could be
(15) Lindgren, C. C.; Dasgupta, P. K. Talanta 1992, 39, 101-111.
(16) Dasgupta, P. K.; Decesare, K. B.; Ullrey, J. C. Anal. Chem. 1980, 52, 19121922.
(17) (a) Kuhr, W. G.; Licklider, L.; Amankwa, L. Anal. Chem. 1993,65,277-282. (b) Taylor, J. A.; Yeung, E. S . Anal. Chem. 1993, 65, 2928-2932.
Analytical Chemistty, Vol. 66, No. 22,November 15, 1994
4001
3 1E-002
6 0
6
5E-003
20
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e
6
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1
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4
-4E-002
150
160
170
Time,
180
s
- 1E-002
100
110
120
130
Time, s
Flgure 5. Net extraction signal for 700 ppb Fe(ePhen)s2+.
Flgure 6. Net extraction signal for 200 ppb Eosin Y.
readily observed under a low magnification microscope. Even at modest field strengths (50-100 V/cm), the CV accumulates so fast at the cathode edge of the organic segment that a depletion zone develops immediately behind the concentrated band at the edge. (A video record of such an experiment is available from the authors on request.) The dye is concentrated in a very narrow band at the edge: in experiments conducted after most of the work reported in this paper was completed, it was discovered that the analyte absorption peak at the interface appears more than three times higher if the data are acquired at a rate of 50 Hz rather than at 10 Hz. No further gains occur at higher sampling rates, the detector rise time becomes the limiting factor. Consequently, it is appropriate to point out that the data relating to the absorbance right at the interface reported in this paper are lower limits rather than true values. The extraction signal intensity for CV decreases with increasing TBAP content. With 20% TBAP in CHC13, the signal is only 59% of that with 10% TBAP. Incontrast toCV, tris(1,lO-phenanthroline)iron(I1) [Fe(oPhen)32+] is not extracted into CHC13 unless a hydrophobic anion is added to form an ion pair. Figure 5 shows the net extraction signal for a solution containing 700 ppb (Fe(oPhen)32+). The analyte and the reference signals were acquired at 5 10 and 620 nm, respectively. It is obvious that, in marked contrast with CV, ( F e ( ~ - P h e n ) is ~ ~concentrated + at the rear edge of the organic segment. Also in contrast with CV, if the analyte is preextracted into TBAP containing CHCl3 and the segment is eluted under high electric field, no preferential redistribution to one edge of the segment is observed, Le., the signal remains a rectangular negative pulse, save that the amplitude is smaller than that of blank CHC13. Also in contrast to CV, in this and all the following experiments with other analytes, no significant dependence of the intensity of the extraction signal was observed upon increasing the TBAP content of the CHC13 beyond 10%. Extraction of Anionic Solutes: Eosin Y and Cobalt 4-(2pyridy1azo)resorcinolate. Eosin Y is an intensely colored carboxylic acid dye that is present in the anionic form in a pH 9 medium. The stoichiometry of the cobalt 4-(2-pyridylazo)-
resorcinolate chelate (Co-PAR) has not been fully elucidated. At high pH, the phenolic O H group of the ligand is ionized, giving a chelate with a greater negative charge or, in the presence of excess ligand, a chelate with a greater ligand to metal ratio.lb Neither Eosin Y nor Co-PAR is extracted into CHCl3 unless a hydrophobic cation such as TBA+ is concurrently present for ion pairing. The net extraction signals observed with both thesespecies are similar and areillustrated for Eosin Y in Figure 6 using 500 and 620 nm as analytical and reference wavelengths, respectively. Note that an accumulation of the analyte occurs both in the front edge of the organic segment and in the aqueous phase, a small distance ahead of the phase interface. This behavior is quite reproducible. The observed peak height precision for a series of injections of 200,400,600, and 800 ppb Co-PAR was 1.1,2.0, 3.0, and 0.8%, respectively, in relative standard deviation. Extractionof a Neutral Solute: Bis(diethy1dithiocarbamate) Copper(I1). Sodium diethyldithiocarbamate (DEDC) is a colorless water-soluble complexing agent that forms a yellowbrown precipitate with Cu(I1) in water. At concentrations lower than -2 mg L-l Cu, precipitation does not occur, but the chelate formed can be extracted into CHC13 (with or without TBAP being present) and a sensitive measurement can be made by absorptiometry at 440 nm. In the present system, extraction signals from the Cu(DEDC)2 experiment resembled the case for anionic solutes such as Eosin Y. Analytical Potential: Calibration Behavior and Degree of Concentration Achieved. Although the present study is largely focused on the phenomenology rather than analytical utility, it is useful to examine how the output signal changes as a function of analyte concentration. For the present purposes, we merely plotted the height of the signal at the analytical wavelength above the prevailing baseline (Le., its maximum positive excursion) adjacent to the negative absorbance signal for the organic segment. For CV, Eosin Y, Co-PAR, and Cu(DEDC)2 over the respective ranges of 0-1000, 0-300, 0-800, and 0-1000 ppb, the respective linear r2 values were 0.9904, 0.9935, 0.9999, and 0.9892. In all cases, a finite positive Y-intercept was observed. In some cases, notably with Co-PAR, appreciable extraction of the PAR ligand leads to a significant blank even in the absence of any Co.
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It is useful to compare the absorbance signal for any given analyte obtained above relative to that of the original analyte (with, for example, aqueous borate as the zero reference). At the sub parts per million concentrations used, for most of the analytes, a direct absorbance measurement in the capillary leads to values with significant uncertainties. The following steps were therefore taken: (a) Construct a Beer’s law plot for the analyte covering the concentration range used for extraction and beyond, in a conventional 1 cm path length cell using a calibrated spectrophotometer. (b) Measure the absorbance of the analyte solutions in the capillary using concentrations at the high end of those used in (a) and thereby compute the effective pathlength of the capillary. (c) Calculate the magnification observed for the extraction signal relative to that of the original analyte from the observed signal and the data obtained in (a) and (b). With 10% TBAP in CHC13, the magnification factor for CV, Eosin Y, Co-PAR, and Cu(DEDC)2 was computed to be 21, 14, 6.3, and 17, respectively. In our experiments, the capillary is filled with the analyte, followed by the passage of the extractant segment. It is of interest to calculate how much of the analyte in the capillary is accounted for in the total net signal in both the organic and the aqueous phase near the interface (e.g., as in Figure 6b). This is not directly related to the peak intensities because the distribution of the analytes as well as the peak width is different in each case. The area under the net analyte signal was integrated and divided by the measured flow rate and converted to mass units using molar absorptivity values determined in (a). For CV and Eosin Y, 34 and 37% respectively, were present in the extraction zone. Mechanism of Extraction into Organic Phase and Concentration in Aqueous Phase near the Interface. The anatomy of the net analyte signal is obviously distinctly different in the three cases of CV, Fe(0-Phen)3~+,and Eosin Y/Co-PAR/ Cu(DEDC)2. In order to understand the behavior, we need to consider how extraction into the organic segment occurs in the first place. One possibility, akin to what happens in FIA, is that analytes adsorb on the capillary wall and are desorbed by the extractant. The dependence of the extraction signal upon how much solution is passed through thecapillary before the organic segment is introduced (followed by plain borate electrolyte) was determined for both CV and Eosin Y. The results for Eosin Y and CV are shown in Figure 7. For Eosin Y, the maximum signal is reached by the time the capillary is approximately half filled with the analyte (total volume of capillary 2.2 pL). Qualitatively, this behavior is independent of the analyte concentration and therefore suggests that significant analyte adsorption is not involved. In contrast, for CV the signal continues to increase up to a rinse volume of at least 3 pL. Qualitatively, this behavior was found to be the same at other CV test concentrations. This suggests that adsorption is involved for CV; this might have been expected from its cationic nature and its extreme affinity for surface adsorption. Only brief experiments have been conducted with Fe(0-Phen)3~+;these results indicate that adsorption on the surface may be important for Fe(o-Phen)32+ also. While surface adsorption and uptake may be important for some of the analytes above, it should not be construed that
0.02
0.00 0
2000
4000
L
0.02 0 0.00
2000
4000
Rinse Volume (ni)
Figure 7. Dependence of the extractionsignal height upon how much analytesolution has flowed through the capillary prior to the introduction of the organic segment. The capillary volume is 2200 nL. (a) Eosin Y and (b) Crystal Vlolet as test analytes.
theelectric field merely acts as a motive force for the transport of an organic segment that desorbs the analyte from the wall. With all of the presently examined analytes, if the organic segment is moved by gravity to the detector after its initial introduction, no perceptible extraction signal is observed! (This does not mean that precisely zero extraction occurs; when there is no preferential distribution within the organic phase, any small amount of extraction will only decrease the amplitude of the rectangular pulse in a minor fashion that cannot be detected .) Electrophoretic Migration of Analyte into Organic Solvent Segment. In the cases where wall adsorption/desorption cannot be a major route to theobserved signal, direct movement of the analyte into the solvent segment needs to be considered. For a situation where the capillary is reinserted into the analyte solution after the introduction of the organic segment, analyte molecules are present both ahead of and behind the extracting segment. When the capillary is inserted into plain borate electrolyte after the introduction of the organic segment, there is no analyte behind the segment. Thus, before the solvent segment is removed from the system, some of the positive ions trailing it and some of the negative ions ahead of it will have an opportunity to enter the segment. Let us assume that the length of the capillary is L (cm), the EOF-induced bulk flow velocity is ueo (cm/s), and the electrophoretic velocities (magnitudes) of positive and negative ions respectively are u+ and u- (cm/s). For the first case, when the analyte ion is positive and is present in the solution behind the segment, the residence time of the segment in the capillary is Lf ueo, and the analyte ions up to a distance of Lu+/u, (cm) behind the segment will have an opportunity to enter the segment. In the second case, we are considering anionic analytes present ahead of the segment. As the segment moves forward, while the analytes anions near the vicinity of the segment approach it, the liquid containing the analyte ions continues to fall off the capillary at the far end. There exists some time t when the last of the analyte-containing liquid has just exited the capillary and all the analyte anions originally present in the liquid ahead of the capillary havehad an opportunity to enter the solvent segment. Thence, t is given by L/(ueo+ u-), and the liquid length from which the Analytical Chemistty, Vol. 66, No. 22, November 15, 1994
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anion has had an opportunity to enter thesegment is L u - / ( u e o + u-) (cm). This would account for the fact that for a nonadsorbed anionic analyte like Eosin Y, the signal reaches a plateau value when the capillary is less than completely filled. Thus, the amount of cationic or anionic analytes that can get into the solvent segment from behind and ahead of the solvent segment, respectively, by having a net positive approach velocity increases with increasing electrophoretic mobility of the ion. However, the aboveconsiderations must be tempered by the reality that the QAS in the segment continually leaks out, resulting in a higher ionic strength near the segment, especially behind the segment as it moves. The net result is that the electric field strength in the immediate vicinity of the segment (especially behind it) is lower than that in most of the aqueous phase in the capillary. The intrinsic EOF in this region is correspondingly lower, resulting in distortion of the otherwise flat flow profile. The overall bulk flow, however, remains the weighted average of the EOF in the capillary.'* Because of the lowered electric field in the immediate vicinity of the segment, the electrophoretic movement is considerably decreased, and even forcations, it becomes difficult to approach the segment. In fact, we have observed that regardless of the charge type or the nature of the analyte, there is little or no transfer of the analyte to the segment from the solution behind it, Le., the signal from the segment is essentially the same whether the solution behind the segment contains analyte or not. Anatomy of the Extraction Signals. The results depicted in Figures 4-6 are comprehensible in the following fashion. We believe that a compound such as CV exists in a charged or at least highly polarized form in the organic solvent such that no matter how it enters the segment it migrates to the cathodic edge. We presume that TBA+ and C104-are similarly distributed in the segment, toward the cathodic (front) and the anodic (rear) edge, respectively. This also accounts for the apparent decrease in the CV extraction signal when the TBAP concentration in the organic solvent is increased. Since the analyte obviously enters from the front of the segment, possibly through the surrounding film, an increase in theTBA+ concentration at this edge hinders both the uptake and the distribution of the CV cation. For Fe(o-Phen)32+,we envision that the analyte enters the surface film from the front and then enters the segment through (18) Burgi, D. S.;Chien, R. L. Anal. Chem. 1991, 63, 2042-2047
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the rear where C104- concentration is high. Once it enters the segment, however, the ion pair is very tightly bound and behaves overall essentially as an uncharged entity. Careful examination of Figure 5 will reveal that there is a slight buildup of the Fe(o-Phen)3*+ up to the front of the segment (presumably to the flow profiledistortion in thevicinity of the segment), but the analyte is largely present in the rear of the segment. Following its entry, presumably the Fe(o-Phen)32+C104redistributes by diffusion: this distribution is clearly more broad than that observed for CV. For Eosin Y,Co-PAR, and Cu(DEDC)2, the major analyte peakactually occurs ahead of the segment. For the negatively charged species, the analyte ahead of the segment is electrophoretically transported toward the segment until it is in the immediate vicinity of the segment. Here the field is reduced, resulting in both reduced electrophoretic movement and distortion of the bulk flow profile, causing analyte buildup. Why this happens also for the uncharged Cu(DEDC)2 moiety is not clear. It is possible that while Cu(DEDC)2 is the species that is extracted, in a pH 9 borate medium anionic forms are present in protic equilibrium and are responsible for the observed behavior.
CONCLUSIONS We have described here the interesting behavior of different and differently charged analyte molecules in biphasic systems under a high electric field. Some behavior, e.g., increase in analyte signals if a longer capillary is used for filling with the analyte, can be predicted. Our best understanding of the extraction phenomenon or analyte accumulation near the phase boundaries in the present system indicates that this is a complex process and involves electrophoretic migration of the analyte, ion pairing at interfaces, bulk EOF, and local changes in the EOF due to leakage of QAS from the extractant segment. The concentration of analytes at the edge of an immiscible solvent segment seems to have great analytical potential for exploitation in capillary systems; this will likely also occur in an appropriately designed system in which an aqueous segment is present in an immiscible conductive organic stream. The possibilities of performing selective migration of species across the organic segment, e.g., by incorporating a hydrophobic ionophore that selectively binds specific ions, is alluring as well. Received for review June 2, 1994. Accepted August 9, 1994." Abstract published in Aduance ACS Absrracfs, October 1, 1994.
