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Anal. Chem. 1984, 56, 111-113
in the spectrum of lasalocid, which is assigned to (M - H + Na)Na+. With monensin spotted on a TLC plate, the sensitivity of detection was established as well below 0.1 pg. A major concern was the possible interference related to the components in the TLC adsorbent and the masking tape. However, no background interference was observed in the mass range of interest based on blank tests of both fluorescent and nonfluorescent TLC plates and direct tests of the masking tape. Also, contamination of the ion source by the TLC adsorbent, i.e., silica gel, was not observed. However, after the depletion of the FAB matrix liquid, the flake of adsorbent from plastic-backed TLC sheets appeared to remain intact while that from glass TLC plates tended to disintegrate. For this reason we favor the use of plastic-backed TLC sheets. In conclusion, we have demonstrated a simple and rapid method for directly obtaining FAB spectra of TLC fractions which circumvents the problems associated with the previously cited methods. ACKNOWLEDGMENT We wish to thank D. B. Borders and M. T. Lee of Lederle Laboratories, American Cyanamid Company, for providing the coccidiostats and the TLC separation procedure. LITERATURE CITED (1) Deverse, F. T.; Gipsteln, E.; Lesoine, L. G. Instrum. News 1967, 78, 16.
(2) Down, G. J.; Gwyn, S.A. J . Chromatogr. 1975, 703, 208-210. (3) Kraft, R.; Otto, A,; Makower, A,; Etzold, G. Anal. Biochem. 1981, 713, 193-196. (4) Nilsson, C. A.; Norstrom, A.; Andersson, K. J . Chromatogr. 1972, 73, 270-273. (5) Rlx, M. J.; Webster, B. R.; Wright, I. C. Chem. Ind. (London) 1969, 452. ( 6 ) Clemett, C. J. Anal. Chem. 1971, 4 3 , 490. (7) Clarke, R. L. Chem. Ind. (London) 1971, 7434-1435. (8) Koehler, M. Chromatographia 1975, 8, 665-669. (9) Dekker, D. J . Chromatogr. 1979, 768, 508-511. (IO) Unger, S.E.; Vincze, A,; Cooks, R. G.; Chrisman, R.; Rothman, L. D. Anal. Chem. 1981. 5 3 , 976-981. (11) U S . Patent 3896661, 1975. (12) Ramaley, L.; Vaughan, M. A,; Jamieson, W. D.; Burnett, N. H. 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI, June 6-11, 1982. (13) Barber, M.; Bordoli, R. S.;Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54, 645A-657A.
Ted T. Chang* Jackson 0.Lay, Jr. Rudolph J. France1 Chemical Research Division American Cyanamid Company P.O. Box 60 1937 West Main Street Stamford, Connecticut 06904 RECEIVED for review June 20,1983. Accepted August 18,1983.
Electrokinetic Separations with Micellar Solutions and Open-Tubular Capillaries Sir: The applicability of the solubilization by micelles to chromatography as a distribution process has briefly been discussed by Nakagawa ( I ) . The point of his discussion may be summarized as follows: Micelles of an ionic surfactant can migrate in an aqueous solution by electrophoresis. When a solubilizate is added into a micellar solution, some portion of the solubilizate may be solubilized into the micelle. Thus the solubilization by micelles can constitute a mechanism of retention in chromatography. The distribution ratio of a solubilizate will increase with an increase of the micellar concentration but will be constant regardless of the concentration of the solubilizate. If a solubilizate is soluble in an aqueous solution to a certain extent, the distribution equilibrium is considered to be established very rapidly because, e.g., the stay time of benzene in the micelle of sodium dodecyl sulfate has been estimated to be less than lo-* s (2). The electrokinetic separation method described in this paper may be classified as a type of liquid-liquid partition chromatography requiring no solid support to hold the stationary liquid phase, although micelles are considered to be a pseudophase. It should be noted that this technique is distinctly different from the reversed-phase liquid chromatography with micellar mobile phase (3): In the latter, the separation is based on distribution processes among three phases, stationary bonded phase, micelle, and water, and micelles migrate with water as an aqueous pseudophase. This paper presents the results of some preliminary studies on electrokinetic separation with micellar solution in opentubular capillaries, in which use was made of the technique of free zone electrophoresis in open-tubular capillaries ( 4 , 5 ) . EXPERIMENTAL SECTION Apparatus. Electrokinetic separation was performed in mi0003-2700/84/0356-0111$01.50/0
crobore vitreous silica tubing, 650 or 900 mm long, 0.05 mm i.d. (Scientific Glass Engineering Inc.), with a Model HSR-24P regulated high-voltagedc power supply (MatsusadaPrecision Devices, Otsu, Japan) delivering +3 to +25 kV. Each end of the capillary tube was dipped in a small glass beaker containing a surfactant solution covered with a silicone-rubber stopper having two small-bore holes, one for a platinum electrode and the other for the capillary tube. The electric current was monitored between the negative electrode and the negative terminal of the power supply with an ammeter throughout the operation. Detection was carried out by on-column measurement of UV absorption through a slit of 0.05 mm X 0.75 mm, the long axis of which was placed parallel to the column axis at a position 150 mm from the negative end of the tube. The polymer coating of the vitreous silica tubing was partly burned out at the detection point of the tube to make an on-column UV cell. A Jasco UVIDEC-100-11 spectrophotometricdetector (Tokyo,Japan) was used with minor modification to obtain a higher amplifier gain and a shorter response time than the conventional one. Reagent. Sodium dodecyl sulfate (SDS) of protein-research grade purchased from Nakarai Chemicals (Kyoto, Japan) was used as it was received. Water was purified with a Milli-Q system. Other reagents were of analytical-reagent grade and were used without further purification. Borate-phosphate buffer solution, pH 7.0 was prepared by mixing a 0.025 M sodium tetraborate solution and a 0.05 M sodium dihydrogen phosphate solution in an appropriate ratio t o indicate pH 7.0. An SDS solution was prepared by dissolving 1 mmol of SDS in 20 mL of the boratephosphate buffer solution followed by filtration of the solution through a membrane filter of 0.5-fim pore size. Procedure. A capillary tube was filled with an SDS solution by use of a microsyringe and 1.5 mL each of the same SDS solution was introduced in two beakers placed at the same level. For the sample injection, the positive end of the tube was moved into a vessel containing a sample solution and the level of the sample solution was raised about 4 cm higher than that of the SDS 0 1963 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL.
56, NO. 1, JANUARY 1984
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Flgure 1. Electrokinetic separation of phenols with an SDS solution: (1)water, (2) acetylacetone, (3)phenol, (4) o-cresol, (5)m-cresol, (6) p-cresol, (7) o-chlorophenol, (8)m-chlorophenol, (9)p-chlorophenol, (10) 2,6-xylenol,(11) 2,3-xylenol,(12) 2,5-xylenol,(13) 3,4-xylenol,(14) 3,5-xylenol, (15) 2,4-xylenol, (16) p-ethylphenol;micellar solution, 1 mmol of SDS in 20 mL of borate-phosphate buffer, pH 7.0; current, 28 p A ; detection wavelength, 270 nm; temperature, ca. 25 OC.
solution to allow the sample solution to flow downward into the capillary tube. After 5 to 90 s depending on the desired amount of injection, the end of the tube was returned to the beaker and a high voltage was applied.
RESULTS AND DISCUSSION The chromatogram shown in Figure 1illustrates the high resolution obtained by electrokinetic separation with micellar solution. The applied voltage between both ends of 900 mm tube was ca. 25 kV. The separation performed in the 750 mm portion from the injection end to the detector cell was recorded by the on-column detection technique. Fourteen phenol derivatives injected as a water solution were completely resolved within 19 min. The injected amount of each phenol was estimated to be 0.7-1 ng and the total injection volume about 12 nL. Theoretical plate numbers calculated from the chromatogram were 210000 for phenol, 260000 to 350000 for cresols and chlorophenols,and 300000 to 400000 for xylenols and p-ethylphenol, corresponding to plate height equivalent to a theoretical plate of 1.9-3.6 pm. Lower plate numbers observed for the peaks at shorter retention times may be attributable to the adverse effect of the large sample volume. It has been reported that the electroosmotic flow is much stronger than the electrophoretic migration of an ion in the case of electrophoresis in open-tubular glass capillaries (5). Similar results were observed in this study: The SDS solution as a whole was carried from the positive electrode to the negative one and negatively charged micelles of SDS also migrated toward the negative electrode as opposed to the electrophoretic attraction. This means that every sample injected at the positive end of the tube can be detected at the negative side of the tube. When a cationic surfactant such as cetyltrimethylammonium bromide was employed instead of an anionic one, the situation was reversed and hence the inversion of polarities of electrodes was needed. The volume flow by electroosmosisin a narrow cylindrical capillary increases linearly with the applied electric field and also with the current (6). When the electrokinetic radius KU is larger than 50, where K is the reciprocal of the Debye length and a is the radius of the capillary tube, the velocity profile has been calculated to be flat in the range 0 5 r 5 0 . 9 ~by Rice and Whitehead (6),where r is the point distance from the axis. The value KU is estimated much larger than 50 under the conditions employed in this study. Therefore, the plug-shape flow of electroosmosis is one of the reasons for the high efficiency attained in this study. The linear relationship between the electroosmotic migration velocity vE0 and the current was
always recognized. However, the plot of the velocity vE0 vs. the total applied voltage showed a positive deviation from linearity at higher voltages, although the actual strength of the applied field in the tube was not measured. The retention parameters in electrokinetic separation are different from those in the conventional elution chromatography, because the retention time of any sample, if it is electrically neutral, should fall between the retention times of an insolubilized solute and a micelle itself in this method. Two assumptions are made for simplicity of the discussions below. One is that solute molecules are electrically neutral under the separation conditions. The other is that the electroosmotic velocity is larger than the electrophoretic velocity of a micelle and that their migrating directions are opposite. A solute which is not solubilized by micelles at all should migrate with the same velocity as the electroosmoticflow uE0 and be eluted first at the retention time to. On the other hand, a solute which is completely solubilized with micelles should migrate with the same velocity as that of a micelle vMC and be eluted last at the retention time tMC. The velocity U M C is the difference between vEO and the electrophoretic velocity of a micelle uEP, or vMC = vEO - uEp. The retention time of an ordinary sample should depend on the capacity factor k', which is given by the ratio of the total moles of the solute in the micelle nMC to those in the aqueous phase nw, or k' = n ~ c / n w .The retention time tR should appear in the range to ItR 5 tMc. The R value, the fraction of the solute in the aqueous phase, is given by
R=
"S
- "MC
uEO - vMC
(1)
where us is the migration velocity of the solute. Now, the R value can be related to k' by (7)
R=- 1 1
+ k'
Inserting the relationship, uEO = L/to, uMC = L/tMc,and us = L/tR, where L is the tube length from the injection end to the detector cell, into eq 1,followed by combination with eq 2 gives k' =
tR
-
t o 0 - (~R/~Mc))
(3)
The term (1 - ( t R / t ~ c )comes ) from the retention behavior characteristic of electrokinetic separations. When tMCbecomes infinite, eq 3 is equivalent to the well-known equation for conventional chromatography. The chromatogram shown in Figure 2 clearly reflects the situation described above. Methanol was chosen as an insolubilized solute to measure to and Sudan I11 to determine ~ M C .The capacity factors of the solutes in Figure 2 are 0,0.49, 1.28,2.27,3.08,and infinity in the order of elution. A capacity factor of infinity means the solute will not be eluted by traditional chromatography and it also means that the solute is totally associated with the micelle in this case. The linear decrease of k' with the increase of the current or UEO was observed although slopes of the plots k'vs. VEO were different among solutes. The reason for this dependence remains to be clarified but can probably be found in the increase of the solution temperature by Joule heating with increasing applied voltage and/or the possible change in the physical property of a micelle by the strong external electric field. Electrokinetic separations with micellar solutions in open-tubular capillaries have been proved to be a high-resolution chromatographicmethod. It is limited to an analytical application because of a small sample size at present, but the zone electrophoretic technique with some kinds of stabilizing media widely utilized in the field of electrophoresis may be
Anal. Chem. 1984, 56,113-116
113
micellar solutions would be useful for studying chemistry of micelles as well as for analytical purposes. Further extensive investigations are being continued to develop the possibilities of this technique.
ACKNOWLEDGMENT We thank T. Nakagawa who has proposed the application of solubilization by micelles to chromatography and H. Jizomoto for their helpful suggestions and discussions.
4
LITERATURE CITED
2
(1) Nakagawa, T. Newsl., Dlv. Colloid Surf. Chem., Chem. SOC.Jpn. 1981, 8 , No. 3, 1. (2) Nakagawa, T.; Tori, K. Kolloid 2.Z.Polym. 1964, 194. 143-147. (3) Armstrong, D.W.; Nome. F. Anal. Chem. 1981, 53, 1662-1666. (4) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M. J. Chromatogr. 1979, 189, 11-20. (5) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (6) Rlce, C. L.; Whitehead, R. J. Phys. Chem. 1985, 69, 4017-4024. (7) Karger, B. L.; Snyder, L. R.; Horvath, C. “An Introduction to Separation Science”; Wiiey: New York, 1973; Chapter 5.
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Figure 2. Chromatogram by electrokineticseparatlon Indicating the total range of elution: (1) methanol, (2) phenol, (3) p-cresol, (4) 2,6xylenol, (5) p-ethylphenol, (6) Sudan 111; total tube length, 650 mm; tube length from the Injection end to the detector cell, 500 mm; total applied voltage, ca.20 kV; current, 33 P A detection wavelength, 220 nm. Other conditions are the same as In Figure 1. employed for preparative purposes in electrokinetic separations. The use of a surfactant solution in an aqueous organic solvent will expand the applicability of this method to water-insoluble compounds. Electrokinetic separations with
Shigeru Terabe* Koji Otsuka Kunimichi Ichikawa Akihiro Tsuchiya Teiichi Ando Department of Industrial Chemistry Faculty of Engineering Kyoto University Sakyo-ku, Kyoto 606, Japan
RECEIVED for review July 8, 1983. Accepted September 19, 1983.
Electrodeposition of Actinides in a Mixed Oxalate-Chloride Electrolyte Sir: The increasing number of analyses of environmental and biological samples along with a greater need for more sensitivity have made almost impossible demands upon electrodeposition procedures. Electrodeposition has virtually become a requirement for high-quality isotopic identification and quantification of the a-emitting actinides, often under the most severe conditions. Increased sensitivity requires larger samples, and this, short of extensive separations, means more impurities during deposition. Because most deposition procedures are extremely sensitive to hydrolytic losses, even microgram amounts of impurities can cause problems with yield and reaolution. However, by merely increasing the acidity immediately prior to the beginning of the deposition, losses have been substantially decreased, and the tolerance to impurities has been improved for all the actinides without the addition of hydrofluoric acid as previously required (I). Previously, the pH was adjusted by addition of ammonium hydroxide and hydrochloric acid to a pH of about 4 using methyl red indicator. To eliminate this pH adjustment and the possibility of local hydrolysis around the drops of ammonium hydroxide, the sample is dissolved in a preadjusted electrolyte. A final pH adjustment is made with hydrochloric acid to a pH of -1.6-1.8, the salmon pink end point of thymol blue indicator. To ensure complete dissolution of extremely
hydrolytic nuclides such as protactinium and thorium, the sample, in sodium acid sulfate crystals, is heated in a hydrochloric acid-sulfuric acid mixture to sulfuric acid fumes just before deposition. The above-named changes markedly improved the reliability of all of our actinide analyses.
EXPERIMENTAL SECTION Apparatus. Early depositions were made with an Eberbach electroanalysis apparatus modified to maintain a constant preset current, with the motor replaced to reduce the speed of the anode to about 30 rpm. Subsequent depositions were made with an apparatus designed and fabricated in our own laboratory with characteristics similar to the Eberbach apparatus. The 20-mm i.d. glass cells described in a prior publication (I) are no longer available and have been replaced with disposable polyethylene cells molded in our laboratory. The brass bases and polyethylene collars have been modified to accommodate the new cells. A water-coolingjacket is no longer required in the base as the brass holder on the apparatus is water cooled and serves as a heat sink as well as an electrical conductor. The platinum anode and stainless steel disks are the same as before. The stainless steel disks are boiled in 16 M nitric acid for about 5 min, rinsed with distilled water, blotted, and air-dried. The a-emitting nuclides were deposited from solutions of approximately 2 x lo4dpm and were counted for 10 min by alpha scintillation as described by Sill ( 2 ) . a-energy analyses were obtained with a 450-mm2solid-
This article not subject to U.S. Copyright. Published 1983 by the American Chemical Society
