Anal. Chem. 1995,67, 1074-1078
Separation of Chloride Isotopes by Capillary Electrophoresis Based on the Isotope Effect on Ion Mobility Charles A. Lucy* and Tracey L. McDonald
Department of Chemistry, The University of Calgary, 2500 University Drive N W, Calgaly, Albetfa, Canada T2N 1N4
Near baseline resolution is obtainedfor chloride isotopes using capillary zone electrophoresis. Ultrahigh resolution is achieved by adjusting the electroosmotic flow to be equal but opposite to the migration of the chloride ions. The separation was optimized with respect to electrolyte concentration, buffer composition, electrolyte pH, and applied voltage. Optimum separation was achievedusing an electrolyte composed of 5 mM chromate and 3 mM borate at pH 9.7 (25 "C) and an applied voltage of 20 kV. The observed chloride isotope ratio was 3.197, which agrees with the known value within the 95%coddence interval. The relative standard deviation was 2.0%. Elemental isotopic ratios are important in geochemical clocks and isotope tracer studies. A rapid increase in the use of inorganic mass spectrometry has resulted in recent years due to the introduction of inductively coupled plasma mass spectrometry UCP-MS). Furthermore, isotope dilution by ICP-MS and other mass spectroscopy techniques can be an extremely accurate and precise instrumental method of determination. As a result of the utility of isotopic measurements, mass spectrometric techniques are widely used for elemental analysis despite their high capital cost and complexity.' This paper explores the use of capillary zone electrophoresis as a simple and inexpensive alternative for the determination of isotopic ratios. In recent years, there have been a number of reports discussing separations of isotopic compounds by liquid chromatography and capillary electrophoresis. Isotopic fractionation has been observed due to the differences in the partition behavior of hydrogenated and deuterated compound^^-^ and due to ionization control, wherein isotopic substitution alters the dissociation of ionizable functionalities such as carboxylic acid^,^^^ phenols7 and amine^.^-^ In all of these cases, the separation factor (a)is less than 1.01, necessitating the use of high-efficiency techniques if baseline separation is to be achieved. In chromatography, sufficiently high efficiencies can be achieved only with use of long capillary columns or recycling of eluent with conventional col* FAX: 403-289-9488. INTERNET:
[email protected]. (1) Gijbels, R: Adams, F. In Inorganic Mass Spectrometry;Adams, F., Gijbels, R, Van Grieken, E., Eds.: Wiley: New York, 1988; Chapter 8. (2) Tanaka, N.; Thomton, E. R. J. Am. Chem. SOC.1976,98,1617. (3) Tanaka, N.; Thomton, E. R. J. Am. Chem. SOC.1977,99,7300. (4) Lockley,W. J. S. J. Chromatogr. 1989,483,413. (5) Tanaka, N.: Araki, M. J. Am. Chem. SOC.1985,107, 7780. (6) Terabe, S.; Yashima, T.: Tanaka, N.; Araki, M. Anal. Chem. 1988,60,1673. (7) Tanaka, N.; Hosoya, IC;Nomura, IC: Yoshimura, T.: Ohki, T.;Yamaoka, R.; Kimata, IC: Araki, M. Nature 1989,341, 727. (8) Bushey, M. M.: Jorgenson, J. W. Anal. Chem. 1989,61,491. (9) Gardner, W. S.; Herche, L. R.; St. John, P. A,; Seitzinger, S. P. Anal. Chem. 1991,63,1838. 1074 Analytical Chemistry, Vol. 67, No. 6, March 15, 7995
umns. In capillary electrophoresis, such isotopic separations have been achieved using capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC or MECC). A more challenging case is the separation of isotopically substituted species which do not possess an ionizable group. Lindemann proposed in 1921 that isotopes could be separated on the basis of differences in their electrophoretic mobilities.'O Subsequent classical electromigration experiments have given separation factors of 1.003 85 for 39Kt/41K+ and 1.0012-1.0026 for 35C1-/37C1-.12J3Similarly, variations in the 35C1-/37C1-ratio in 15 000-year-old groundwater were best fit assuming diffusion control with an isotopic d ~ s i o ratio n of l.OOl2.14 As per intuition, the lighter ion migrated more rapidly in each case. Recently, Avdalovic et al. observed a partial separation of the isotopes of chloride while performing CZE of inorganic anions.15 In this report we investigate the feasibility of performing elemental isotopic separations by CZE using natural abundance chloride as the test system. EXPERIMENTAL SECTION Apparatus. All experiments were performed on a P/ACE 2100 (Beckman Instruments, Fullerton, CA) with the electrodes in the reversed configuration (injection at cathode, detection at anode). System control and data collection at 5 Hz were performed using System Gold software (Beckman) on a 386 microcomputer. The internal diameter of the capillary (Polymicro Technologies, Phoenix, AZ) was 75 pm, with a total length (LJ of 47 cm and a length from injection to detection (Ld) of 40 cm. Indirect detection with chromate at 254 nm was used throughout. Reagents. All solutions were prepared with distilled and deionized water (Bamstead Type D4700 NANOpure deionization system) and analytical grade reagents and were filtered through 0.45 pm nylon syringe filters prior to use. Chromate buffers were prepared and studied over a range of concentrations from 5 to 15 mM and a pH range from 8.0 to 10.1. Borate (3.0 mM) was added to the electrolyte in later experiments to increase the buffering capacity. Sodium hydroxide and sulfuric acid were used to adjust the buffer pH. Sample anion solutions were prepared at concen(10) Lindemann, F. A. Proc. R. SOC.London 1921,A99,102. (11) Brewer, A. IC; Madorsky, S. L.; Taylor, J. IC; Dibeler, V. H.; Bradt, P.; Parham, 0. L.: Britten, R. J.; Red. J. G., Jr. J. Res. Natl. Bur. Stand. 1947, 38,137. (12) Madorsky, S. L.; Straus, S. J Res. Natl. Bur. Stand. 1947,38,185. (13) Konstantinov, B. P.: Bakulin, E. A. R u s . J. Phys. Chem. 1965,39,315. (14) Desaulniers, D. E.; Kaufmann, R. S.; Cherry, J. A,; Bentley, H. W. Geochim. Cosmochim. Acta 1986,50, 1757. (15) Avdalovic, N.: Pohl, C. A: Rocklin, R. D.: Stillian, J. R. Anal. Chem. 1993, 65,1470. 0003-2700/95/0367-1074$9.00/0 0 1995 American Chemical Society
trations of 10 and 20 ppm. Isotopically pure Na35C1 (99%) was purchased from Cambridge Isotope Laboratories and used as received. Procedures. New capillaries were pretreated for 10 min with 0.1 M NaOH. Before each run, the capillary was rinsed with buffer at high pressure (20 psi) for 5 min. Samples were introduced onto the capillary using a 4 s low-pressure (0.5 psi) hydrodynamic injection, corresponding to the injection of 2.6 nL of sample. Buffer pH was adjusted prior to analysis of each batch of samples. After each run, the capillary was rinsed 2 min at high pressure with 0.1 M NaOH, 2 min with distilled water, 1.5 min with 0.01 M HzS04, and finally 1.5 min with distilled water. All rinses and capillary pretreatments were thermostated to 25 "C. Buffer reservoirs were changed after each run to ensure reproducible separation conditions. RESULTS AND DISCUSSION Terabe et a1.6 have provided an excellent discussion of the factors which govern ultrahigh resolution CZE. Therefore, only a brief discussion of these factors will be given herein. In capillary electrophoresis, the resolution between two bands is expressed as:16,17
where N is the average number of theoretical plates, Av is the difference in the zone velocities, and i j is the average zone velocity. In untreated capillaries, electromigration will be accompanied by electroosmotic flow. Thus, zone velocity is a function of the intrinsic mobility of the ion, p,, and the electroosmotic flow coefficient, peoP
where V is the applied voltage and Lt is the total length of the capillary. The efficiency, N, in capillary electrophoresis is primarily governed by longitudinal diffusion, although other factors can be ~ignifcant.'~The efficiency based on longitudinal diffusion alone is given by
(3) where Ld is the length from the capillary inlet to the detector, D is the diffusion coefficient, and t~ is the migration time of the ion to the detector. The migration time is related to the zone velocity by
t
Ld
--
M-
Y
Combining the expressions above yields the relationship
(5) where Meis the difference in the intrinsic mobilities of the two (16)Giddings, J. C. Sep. Sci. 1969,4, 181. (17)Ewing, A G.;Wallingford, R A; Olefirowicz, T. M. Anal. Chem. 1989,61, 29%
(18)Jorgenson, J. W.; Lukacs, K. D. Science 1983,222, 266. (19) Grossman, P. D. In Capillary Electrophoresis: 7heoy andPractice; Grossman, P. D.,Colbum, J. C., Eds.; Academic Press: San Diego, CA, 1992;Chapter 1.
ions. As discussed above, the mobility di€ference between the isotopes of chloride is only 0.12%, with the lighter isotope migrating faster.12-14 Isotopic fractionation in geological groundwaters has been rationalized using the concept of ion filtration20 and erroneously Graham's law.l4J However, no rigorous explanation of the isotopic effect on the mobilities of isotopes has been made. Regardless of the cause, the difference in the intrinsic mobilities of the chloride isotopes is exceedingly small. Therefore, other factors within eq 5 must be exploited to achieve baseline separation of the chloride isotopes. Capillary Length. The effective length ratio of the capillary is defined as the length from injection to detection (Lb) divided by the total length of the capillary (U. Terabe et a1.6 have demonstrated that the effective length ratio of a capillary can have a significant impact on the resolution achieved. In the system used herein, the capillary length ratio is 0.85, thus minimizing the effect of the effective length ratio without necessitating unduly long separation times. Efficiency. The dependence of the resolution on applied voltage and diffusion coefficient in eq 5 arises from the assumption that longitudinal diffusion is the primary source of band broadening (eq 3). When indirect detection is used, this assumption is often invalid. With indirect detection,low concentrationsof buffer are required for sensitive When the buffer concentration is low, the sample ion can contribute significantly to the conductivity of the sample zone. This may lead to differences in the conductivity between the sample zone and the surrounding buffer, resulting in additional band broadening and peak asymmetry.19323124 The conductivity difference broadening is minimized by matching the electrophoretic mobility of the buffer to that of the analyte. Chloride is a highly mobile anion (equivalent conductance, A, of 76.31 x m2s/moP5);therefore, the buffer anion must also possess a high mobility. Chromate (A(1/2Cr042-) = 85 x low4 mWm01~~) has been found to be an effective electrolyte anion for the determination of high-mobility inorganic anions such as chloride.26 At the buffer pH values used in our studies (PH > 8), chromate is '96% ionized (pKa = 6.4g27). Therefore, the mobility of chromate is effectively constant at all pH values studied. However, the conductivity of the sample zone was still higher than the surrounding buffer due to the higher mobility of chloride, resulting in minor peak tailing. To determine if chromate was indeed an appropriate electrolyte buffer for the separation of the isotopes of chloride, the efficiency of the 35Cl- peak was monitored for chromate buffers from 5 to 15 mM at a constant pH of 8.0. Additional band broadening (Hnonidead, over and above that due to longitudinal diffusion, is determined by (20) Phillips, F. M.; Bentley, H. W. Geochim. Cosmochim. Acta 1987,51, 683. (21)Senftle, F. E.; Bracken, J. T. Geochim. Cosmochim. Acta 1955,7, 61. (22)Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991,63, 275A. (23)Mikkers, F.E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M.]. Chromatogr. 1979,169,11. (24)Hjerth, S.Electrophoresis 1990,1 1 , 665. (25)Vanysek, P. In Handbook ofChembty and Physics, 1st Student Edition; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1988; p D-105. (26)Romano, J.; Jandik, P.; Jones, W. R; Jackson, P. E. J. Chromatogr. 1991, 546, 411. (27)Handbook of Chembtv and Physics, 1st Student Edition; Weast, R C., Ed.; CRC Press: Boca Raton, FL, 1988; p D-103.
Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1075
Table 1. Effect of Chromate Concentration on the Nonlongitudinal Diffusion Band Broadening.
[chromate],M 5.0 7.5 10.0 12.5 15.0 5.0 7.5 10.0 12.5 15.0
min Hobs, pm A. Chromate Alone
tM,
Table 2. Effect of pH on the Nonlongitudinal Diffuslon Band Broadening.
Hnonideah pmb
PH
8.4 2.5 1.4 2.2 2.8
8.9 9.1 9.3 9.5 9.7 9.9 10.1
12.9 8.2 6.4 6.1 5.8
16.2 7.5 5.3 5.9 6.3 B. Chromate Plus 3.0 mM Borate 13.0 12.1 10.1 7.2 6.3 4.8 5.3 4.9 5.1 4.8
4.2 1.0 1.0 1.7 1.7
Experimental conditions: total capillary length, 47 cm; internal diameter, 75 pm; temperature, 25.0 "C; pH, 8.0; sample, 10 ppm 35Cl-; injection,4 s, 0.5 psi hydrodynamic performed in duplicate. Calculated from eq 6 using 2.03 x cm2/sZ8as the diffusion coefficient for chloride.
""0
Ld
The plate heights due to longitudinal diffusion were calculated using eq 3, the observed migration times, and a diffusion coefficient for chloride of 2.03 x cm2/s.28 The nonideal plate heights observed for 5-15 mM chromate buffers (PH 8.0) are shown in Table 1, along with the observed migration times and observed plate heights. Initially, as the chromate concentration was increased from 5 to 10 mM, the band broadening from sources other than longitudinal diffusion decreased. The increased buffer concentration decreases the effect of the fixed solute concentration on the conductivity of the migrating sample zone. Above 10 mM chromate, the nonideal plate height increases, presumably due to Joule heating within the ca~illary.'~ An additional factor which would affect the efficiency observed from these runs is alteration in the pH of the reservoir vials as a result of electrolysis. At pH 8.0, the chromate is 96%ionized; thus, the electrolyte solution has little buffering capacity. After a 15 min run at 20 kV, the inlet vial pH increased an average of 0.2, while the outlet vial had decreased by 0.2 pH units. The variation in pH along the capillary would not alter the electrophoretic mobilities of chloride and chromate significantly. However, the pH gradient would alter the localized ionization of the silanols, thus altering the localized 5 potential, which in turn governs the localized electroosmotic flow. Studies have demonstrated that variations in the localized electroosmotic flow along a capillary induce a hydrodynamic flow that causes additional band broadening.2y130Thus, it is necessary to suppress the pH gradient caused by electrolysis in order to achieve maximum efficiency. To increase the buffering capacity of the electrolyte buffer, 3.0 mM borate was added to the chromate. Table 1 shows that the addition of borate to the electrolyte decreased the band broadening arising from sources other than the longitudinal diffusion. Therefore, all further studies were performed in chromate solutions containing 3.0 mM borate buffer. Lower borate concentrations did not adequately control pH, and higher concentrations (28) Cussler, E. L. Diffusion: mass transfer in fluid systems; Cambridge University Press: New York, 1984; p 147. (29) Chien, R-L.; Helmer, J. C. Anal. Chem. 1991,63, 1354. (30)Towns,J. K; Regnier, F. E. Anal. Chem. 1992,64, 2473.
1076 Analytical Chemistry, Vol. 67, No. 6, March 15, 7995
tM,
min
8.52 f 0.08 9.59 f 0.05 10.4 i 0.4 10.6 f 0.3 13.4 f 0.1 15.0 f 0.7 11.9c
Hobs!
pm
6.5 7.3 7.8 8.2 9.8 10.0 10.2
Hnonideah
pmb
1.4 1.5 1.5 1.7 1.6 0.9 3.0
Experimental conditions: total capillary length, 47 cm; internal diameter, 75 pm; temperature, 25.0 "C; buffer, 10.0 mM chromate and 3.0 mM borate; sample, 10 ppm 35C1-; injection, 4 s, 0.5 psi hydrodynamic erformed in duplicate. Calculated from eq 6 using 2.03 x 10-5 cm2/s2 as the diffusion coefficient for chloride. Value for only a single run.
would contribute further to peak asymmetry due to conductivity differences. Varying the pH of the 10.0 mM chromate/3.0 mM borate electrolyte buffer from 8.9 to 9.9 did not increase the nonlongitudinal diffusion band broadening (Hnonideal), as shown in Table 2. At pH 10.1, the nonideal plate height increased, suggesting that the increased ionization of borate has resulted in Joule heating. within the capillary. Further studies were restricted to the pH values less than 10 in order to maintain maximum efficiency. Increasing the applied voltage will increase efficiency by allowing less time for longitudinal diffusion to occur (eq 3). However, with 10.0 mM chromate/3.0 mM borate @H 9.7), applied voltages greater than 20 kV resulted in a decrease in the efficiency (N), presumably due to Joule heating. Apparent Mobility. Of these factors which affect the resolution (eq 5), the apparent mobility (iie pea)is the most powerful.6 If the electroosmotic coefficient be,,)is approximately equal in magnitude but opposite in direction to the mean electrophoretic mobility (iie),extremely high resolutions can be achieved, albeit at the expense of large separation times.31 It is this approach which will be used to separate the isotopes of chloride. The electroosmotic flow coefficient is given byly
+
(7) where (J* is the surface charge density, 7 is the viscosity coefficient, and K - ~is the double layer thickness given by32
where EO is the permittivity of vacuum, E is the dielectric constant, k is the Boltzmann constant, T is the absolute temperature, e is the electric charge, no is the electrolyte concentration in the bulk solution, and z is the ionic charge for the binary electrolyte, in which z+ = -2-. From eq 7, it is apparent that the electroosmotic flow can be altered by changing the surface charge, the double layer thickness, or the viscosity.1g Only the first two approaches are used in this work. The surface charge on the capillary walls is a function of the protolysis of the silanol groups and the dissociative and nondissociative adsorption from Thus, while the (31) Jorgenson, J. W.; Lukacs, K D.Anal. Chem. 1981,53, 1298. (32) Schwer, C.; Kenndler, E. Anal. Chem. 1991,63, 1801.
0.003
I
1
O.OO0
9.0
8.4
9.2
0.6
0.8
10.0
10.0
10.2
Tlme (mlnutea)
0.0°3
10.4
10.6
10.8
11.0
Tlme (mlnutea)
r-----l
0.WO 11.0
11.2
11.4
11.6
11.8
12.0
13.0
13.2
13.4
Time (minutes)
13.6
13.8
141
Tlme (mlnutes)
Figure 1. Effect of pH on the isotopic separation of natural abundance chloride. Experimental conditions: buffer, 10 mM chromate; applied voltage, 20 kV; temperature, 25.0 "C;and sample, 10 ppm natural abundance chloride.
acid dissociation constant for the silanols has been determined to be 5.3,32the electroosmotic flow varies with pH over a much wider range (PH 2-11) than expected if only protolysis were The effect of varying the buffer pH on the separation of the isotopes of chloride is shown in Figure 1. Increasing the pH increases the electroosmotic flow, which in turn decreases the apparent mobility (ue peJ. This has the net effect of increasing the resolution. The pH could not be increased above pH 9.7, as behavior becomes irreproducible. The electroosmotic flow can also be increased by increasing the thickness of the double layer (eq 7). A simple means of increasing the double layer thickness is to decrease the electrolyte concentration in the bulk solution (no in eq 8). The borate concentration could not be reduced, as it was required for pH control. Decreasing the concentration of chromate to 5.0 mM increased the electroosmotic flow such that a buffer pH of 9.2 yielded baseline separation of the isotopes of chloride (Figure 2). During the runs at pH 9.2, a baseline drift was observed. This drift has been subtracted from the raw data for Figures 2 and 3. The peaks were identified on the basis of separate injections of isotopically pure 35Cl-. As expected, the low apparent mobility required to achieve this separation resulted in long analysis times. Isotopic Separation. Attempts to reduce the analysis time by increasing the applied voltage yielded the results presented in Figure 3. Distinct improvements were observed in the resolution of the peaks. However, increased baseline noise was observed at the higher voltages. Therefore, all quantitative work was performed under the conditions of Figure 2. The isotopic ratio for the chloride peaks was determined using the expression
+
35
40
45
50
Time (min) Figure 2. Isotopic elemental analysis of chloride by capillary electrophoresis. Experimental conditions: capillary, 75 pm i.d. x 47 cm long; electrolyte, 5 mM chromate and 3 mM borate at pH 9.2; temperature, 25.0 "C;sample, 20 ppm natural abundance chloride; and applied voltage, 20 kV.
(9) where A35 and A37 are the areas determined for the two isotopes of chloride after fast Fourier transform VFT) smoothing and baseline subtraction of the data, and 7s5 and 137are the first (33) Pure Appl. Chem. 1991,63, 991. (34) Fujiwara, S.; Honda, S. Anal. Chem. 1986,58, 1811. (35) Fujiwara, S.; Honda, S. Anal. Chem. 1987,59, 2773. (36) Dose, E. V.; Guiochon, G.Anal. Chem. 1991,63, 1154. (37) Fassett, J. D.; Paulsen, P. J. Anal. Chem. 1989,61,643.4.
Analytical Chemistry, Vol. 67, No. 6,March 15, 1995
lO?7
I 0.004 "."""
V = 2 0 kV
I
0
u
9 0.003 c 2 0.002
9 0.001
36
38
40
42 44 46 Time (mtn)
40
50
0.005
V-30kV
0.004 .. P)
0
f
0.003 ..
c 2 0.002.. 2 0.001 .. O T
24
26
28
30 32 Time (min)
34
36
38
Figure 3. Effect of applied voltage on the isotopic separation of chloride. Experimental conditions: Capillary, 75 pn i.d. x 47 cm long; electrolyte, 5 mM chromate and 3 mM borate at pH 9.2; temperature, 25.0 "C;and sample, 20 ppm natural abundance chloride.
statistical moments (centers of gravity) of the two peaks, respectively. The center of gravity 0)replaces the traditional migration
1078 Analytical Chemistry, Vol. 67,No. 6,March 15, 1995
time of the peak maximum in eq 9 because of the peak asymmetry caused by the conductivity difference between the sample zone and the surrounding buffer, as discussed above.19,23,24 The degree of peak asymmetry is greater for the more abundant isotope, as would be expected. The isotopic ratio observed for natural abundance chloride for quadruplicate injections was 3.197, with a standard deviation of 0.065, which agrees with the known value of 3.12733within the 95%confidence interval. The relative standard deviation of these measurements is 2.0%. The high relative standard deviation observed in this work is primarily due to the use of indirect absorbance for detection. In absorbance spectroscopy, the detector performance is shot-noise limited. Thus, with the high background absorbance needed for indirect detection less light reaches the photomultiplier, and so the baseline noise increases22 Previous studies utilizing internal standards with direct detection for quantification in capillary electrophoresis have demonstrated precisions (lo) of 0.6-2.0%.34-36Thus, in order to achieve the 0.25-0.50% relative standard deviations required for isotope dilution application^?^ direct detection will have to be used in conjunction with ultrahigh resolution capillary electrophoresis. Once this improved precision is achieved, capillary electrophoresis will be an attractive alternative to inductively coupled plasma and thermal ionization mass spectrometric techniques in some a p plications due to its 6-10 fold lower capital cost and the lower level of expertise required to operate the instrument. ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada and the University of Calgary. The Undergraduate Student Travel Award provided to T.L.M. by the Analytical Division of the Canadian Society for Chemistry is gratefully acknowledged. This work was presented at the 77th Canadian Chemistry Conference in Winnipeg, MB, June 1994. Received for review August 31, 1994. Accepted December
21, 1994.@ AC940877E @Abstractpublished in Advance ACS Abstracts, February 1, 1995.
