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Anal. C h m . 1983, 55, 1608-1610
dye laser system, a few hundred ions per second. This enhancement significantly exceeds the expectations of rateequation calculations (6). This difference may result from non-Gaussian propagation of the experimental laser beam or uncertainties in the modeling parameters; further studies are in progress. The measured selectivity of the resonance ionization process is enhanced with CW ionization as well. Figure 2 shows a mass scan for an equal-atom lutetium/ytterbium sample, obtained through resonance ionization. In conventional thermal ionization, ytterbium isotopes would appear at masses 170 to 174; here they can be barely discerned above the scattered ion noise of the instrument, the result of low levels of thermal ionization by the sample filaments themselves. Discrimination factors approaching lo6 have been measured against 174Yb. This figure is limited not by the selectivity of the resonance ionization process but by the resolution of the mass spectrometer, which allows tailing of the 176Lupeak into the lower mass position. The selectivity of CW ionization is also demonstrated by the fidelity of the 175Lu/176Luratio in mixed lutetium/ytterbium samples, in spite of an isobaric 176Ybinterference. In general, a ratio within 5% of the accepted value of 37.6 was found with 2 min of measurement at each mass. Wavelength instability in the dye laser due to room thermal fluctuations caused a large part of the ratio uncertainty. Considering this along with atomic line shifts and hyperfine splitting differences, some degree of isotopic selectivity in resonance ionization is not unexpected. Actively stabilized dye lasers would be of benefit in overcoming this problem and improving the accuracy of the measurements. Single-frequency dye lasers might also allow exploitation of the isotope shift; isotopically selective ionization would reduce the problem of incomplete mass discrimination in the spectrometer for the measurement of large ratios.
These initial experiments with CW laser resonance ionization mass spectrometry have shown an increase in both the sensitivity and the selectivity relative to our previous pulsed laser experiments ( I ) . Application of this technique to routine analyses should be possible in the near future. Registry No. 175Lu,14391-25-4;176Lu,14452-47-2;lutetium, 7439-94-3;ytterbium, 7440-64-4. LITERATURE CITED Miller, C. M.; Nogar, N. S.;Gancarz, A. J.; §hields, W. R. Anal. Chem. 1982, 54, 2377-2378. Donohue, D. L.; Young, J. P.; Smith, D. H. I n t . J . Mass Specfrom. Ion Phys. l982?43, 293-307. (3) Young, J. P.; Donohue, D. L. Anal. Chem. 1983, 55, 88-91. (4) Donohue, D. L.; Young, J. P. Anal. Chem. 1983,55, 378-379. (5) Hurst, G. S.;Nayfeh, M. H.; Young, J. P. Phys. Rev. A 1977, 75, 2283-2292. Miller, C. M.; Nogar, N. 8. Anal. Chem. 1983,55,481-488. Inghram, M. G.; Chupka, W. A. Rev. Sci. Instrum. 1953, 2 4 , 518-520. Catanzaro, E. J.; Murphy, T. J.; Garner, E. L.; Shields, W. R. J . Res. Natl. Bur. Stand., Sect. A 1086, 7 0 , 453-458. Shields, W. R. NBS Tech. Note ( U . S . )1066, No. 277, 1-16. Martin, W. C.; Zalubas, R.; Hagen, L. "Atomic Energy Levels-The Rare Earth Elements": National Bureau of Standards: Washington, DC, 1978; pp 398-403. (11) Keller, R. A.; Engleman, R., Jr.;'Zalewski, E. F. J . Opt. SOC. Am. 197% 69, 738-742. (12) Keller, R. A.; Zalewski, E. F. Appl. Opt. 1980, 19, 3301-3305.
C. M. Miller* N. 8 . Nogar Groups INC-7 and CHM-2 Los Alamos National Laboratory Los Alamos, New Mexico 87545
RECEIVED for review March 11,1983. Accepted May 3,1983. Los Alamos National Laboratory is operated by the University of California for the United States Department of Energy under Contract W-7405-ENG-36.
Surface-Modified Electrochemical Detector for Liquid Chromatography Sir: It i s a well-known fact that solid electrodes are very susceptible to poisoning by sample contaminants as well as by the products of electrochemical reactions (1,2). This is particularly true in complex biological matrices (such as serum, plasma, urine) containing a variety of proteins and other components which strongly adsorb on the electrode surface, thereby attenuating electrode processes that produce the observed signal (3). Further the presence of electroactive interferences in the sample can result in unacceptably high background signals. With the advent of liquid chromatography and flow injection analysis utilizing electrochemical detection (LCEC),these problems have become very critical in determining the reliability of the analysis. In most LCEC applications involving biological matrices the electroactive species of interest is extracted into a suitable solvent prior to chromatographic analysis. Alternatively, some samples such as urine can be diluted (150 dilution, for example) and directly analyzed without extraction (4). Even with large dilutions the electrodes would eventually be affected by the contaminants in such samples, resulting in the loss of sensitivity. A more reasonable approach would be to coat the working electrode surface with an inert film that prevents protein adsorption and a t the same time facilitates selective transport of the electroactive species of interest toward the electrode. Such physical modifications would eliminate the
electroactive interferences while retaining the heterogeneous electron transfer properties of the electrode. The latter feature is a significant advantage over using chemically modified electrodes in the LCEC applications. We have examined the effects of proteins on the response of a platinum LCEC detector coated with a cellulose acetate (CA) film, using HzOzas the electroactive species of interest. Comparison of these results with that of a bare platinum electrode clearly demonstrates that the CA film effectively prevents electrode poisoning arising from protein adsorption. In addition, the film selectively eliminates some electroactive interferences such as ascorbate from the electrode surface. Thus differential selectivity appears possible with such physically modified electrodes and the results of these preliminary findings are presented below. EXPERIMENTAL SECTION All solutions were made in deionized water distilled over KMn04 and filtered with 0.45 pM Millipore filter (Millipore Corp., Bedford, MA), and all reagents used were of analytical grade. Hydrogen peroxide (H20z, 30%) was purchased from Fisher Scientific Co., Fair Lawn, NJ, and was standardized by titrating with Ce4+solutions 89 described elsewhere (5). Working standards were made from the above solutions by appropriate dilution. The separate sets of H20zworking standards were made at the same concentrations, but containing 100 mg/dL of glucose, 2.0 mg/dL
0003-2700/83/0355-1808$01.50/00 1983 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
Table I. Effect of BSA. on the Sensitivi6y of Platinum LCEC Electrode to H,C), slope of calibration curve,a nA/nmol bare P t CA-coated Pt standard solutionb (i3.0nA/nmol) (rl.O nA/nmol) 100.7 58.6 %Oz 55.8 H,O, -+ 0.2%BSA 49.5 H,O, (repeat) 53.6 a The platinum working electrode was a t + 0.800 v vs. Ag/AgCl: mobile phase, 0.10 M PB, pH 7.4; flow rate, Standlard solutions 0.50 mL/min in the FIA mode. were made such that 20-pL injections contained 0.0281.40 nmol of H,O,.
50*
40-
0.
r
-
i 0.
5.
10. TIME
of ascorbic acid, and 200 mg/dL of bovine serum albumin (BSA, RIA grade, Sigma Chemical Co., St. Louis, MO). The glucose and ascorbate concentrations in the above solutions are approximately the same all that found ?n normal human plasma samples (6). Cellulose acetate (39.8% acetyl content) was purchased from Aldrich Chemical Co., M[ilwaukee, WI. Normal Control Seruim (NCS, product code 1180, Lot no. 027x01) was obtained from Ortho Diagnostics Inc., Raritan, NJ, in a freeze-dried powder form and was dissolved in 25.0 mL of distilled water according to manufacturer’s recommendations. This control s e m was composed of human serum and nonprotein constituents with no preservatives or stabilizers added. In all experiments the WCS was freshly made from the freeze-dried material stored at 2-8 “C. Normal human serum samples (pooled) were also used in these experiments and were obtained from the Department of Clinical Pathology, College of Medicine, University of Arizona, Tucson, AZ. The liquid chromatographic system consisted of a SP 8700 Solvent Delivery System (Spectra-Physics,Smta Clara, CA), pulse damper column, injector (Altex Model 210) and a LC-4B electrochemical detector (Biormalytical Systems, West Lafayette, IN) with a platinum working electrode and a Ag/AgC1 reference. AU experiments were run in a flow-injection mode with no column between the injector and the detector. Peak currents were monitored on the LC-4B whereas the peak areas were measured with a Hewlett-Packard 3390A recording integrator. The mobile phase consisted of 0.10 h4 phosphate buffer (PB), pH 7.4, containing 2.0 mM EDTA. In the latter stapes 0.05% BSA was included in the mobile phase exposing the CA film-coated electrode to protein continuously. Physical Modification of the Platinum Electrode with Cellulose Acetate Coating. The platinum working electrode (TL-lOA,Bioanalytical Systems, West Lafayette, IN) was cleaned by washing with acetone or methanol and distilled water and then cycling in 1 F H2S04frorn -0.800 V to +0.800 V (Ag/AgCl reference) several times. The cycling was stopped at i-0.800 V, and the platinum working electrode was washed with distilled water and wiped carefully with Lint-free lens cloth. The Teflon gasket with the flow channel was replaced on the Kel-F block according to manufacturers instructions. Exactly 25 PI,of CA solution (2% solution in a 1:l mixture of cyclohexanone and acetone) was pipetted over the platinum electrode and was carefully spread with a brush to cover the entire channel inclluding the platinum electrode surface. The solvents evaporated in 15-30 min leaving a uniform film of CA over the electrode suirface and the entire channel. The upper Kel-F block was carefully replaced taking extreme precaution to prevent moving the gasket. Such movements will cause breaks in the film allowing leaks that would expose the working electrode to proteins and other contaminanta in the mobile phase or the sample. The asseimbled electrode was then equilibrated with tho mobile phase flowing at 0.1 mL/min overnight and at tan applied potential of +.800 V vs. Ag/AgCl.
RESULTS AND DISCUSSION The response of the platinum LCEC detector to H2Q2under various conditions is summarized in Table I. The bare electrode response to hydrogen peroxide in the absence of proteins shows good response but the addition of 0.2% BSA
I609
r
15.
20.
25.
30.
IN MINUTES
Figure 1. Response of the bare platinum electrode to H,Op standard containing 0.2% BSA. Peak currents were measured in the flow injection mode by injecting 0.028 nmol of H202at 1-min intervals. The platinum electrode was held at +0.800 V vs. Ag/AgCI reference. Mobile phase was phosphate buffer (0.10 M), pH 7.4;flow rate was 0.5 mL/mln.
to the standards results in about 50% loss in sensitivity. In fact, the “poisoning” of the electrode takes place very rapidly and one can observe the decrease in peak currents when successive aliquots (20 pL) of a H20zstandard containing 0.2% BSA were injected at, 1-min intervals, as shown in Figure 1. The peak currents decrease rapidly in the first 10 min and continue to decrease thereafter at a slower rate. At this stage, 20-pL aliquots of the same standard without BSA were injected (not shown in Figure 1)but the peak currents did not increase to the original level. Evidently this loss of sensitivity is due to the irreversible adsorption of BSA on the platinum surface and it must be removed by a cleaning procedure similar to that described in the Experimental Section. The CA-coated platinum electrode shows very minimal decrease in the observed slope when BSA is present in the standards (Table I). Obviously the CA film appears to prevent BSA adsorption on the electrode surface and this is not surprising since the protein is too large (molecular weight = 60000) to diffuse through the film. These observations are consistent with the diffusivity data (7,10, 13) which clearly show that only small molecules (HzQz,0,)and ions (Na+, Cl-, etc.) can diffuse rapidly through CA films. Even in the absence of BSA in HzOz standards, the slope measured for CA-coated platinum electrode response (Table I) was considerably less than what was measured for the bare electrode. This loss of sensitivity is, however, due to diffusional constraints imposed by the CA film on the transport of HzQz toward the electrode. For example, 0.028 nmol of HzOz yields a 30 nA signal (Figure 1) with an uncoated platinum electrode, whereas the CA-coated platinum response was about 65 nA for 1.40 nmol of H202 (Figure 2). This translates into approximately 20-fold attenuation of the signal by the CA film cast on the platinum surface. Considering the fact that the diffusivity of HzOz in CA membranes (7) is approximately 1.5-2.0 orders of magnitude smaller, the observed attenuation is not unreasonable. In any case, such a decrease in sensitivity is not a matter of concern since H202 can still be detected a t picomolar-to-nanomolar levels with signal-to-noise ratios well above 100. At these levels, electrochemical detection is far superior to the existing spectrophotometric methods for HzOzdetermination (8). Table I1 shows the response of the CA-coated electrode to H,Oz, glucose, and ascorbate, and the latter two compounds show very small peak currents. Glucose is not electroactive whereas ascorbate can oxidize at the bare platinum electrode under the present conditions (2,3). Both compounds, however, have low diffusivity in the CA film (3, thus accounting
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
I
I
Figure 2. Response of the cellulose acetate coated platlnum electrode to H,O, before and after exposure to serum samples. Peak currents and areas were measured in all cases and the experimental condffions were the same as those given in Figure 1: (A) 1.40 nmol of H202;(B) 20-pL injections of Normal Control Serum (undiluted),after standing at 4 “C in the dark €or 35 h; (C) 20-pL injections of pooled human serum (undiluted). The pooled serum was used 6 h after collection.
Table 11. Response of a Platinum LCEC Detector (CA Coated) to H,O, in the Presence of Glucose and Ascorbatea standard solutions
HzO, H,O,
+ glucosec
glucose ascorbate (fresh)d
peak current in nA (k1.5 nA) 54.5 55.6 2.2 2.0
a All experimental conditions were the same as those given in Table I. In all solutions H,O, concentration M so that a 2O-pL aliquot would was 7.05 X Glucose concentracorrespond to 1.40 nmol of W,O,. Ascorbate concentration = 2.0 mg/ tion = 100 mg/dL. dL.
for the negligible peak currents observed. Therefore it appears that such physical modification of the electrode not only prevents electrode “poisoning” but also selectively eliminates certain electroactive interferences found in complex biological matrices. In fact, such membranes (cellulose acetate and polycarbonate) (9) have been used previously to isolate electrodes from reducing agents which interfere with HzOz detection and to protect electrical probes (10-18). However, to our knowledge no such application has yet been reported in the LCEC literature. To further investigate the utility of the protecting film, the CA-coated platinum electrode was exposed to serum samples. The response of the electrode to HzOzwas measured before and after injecting 20-pL aliquots of the serum into the mobile phase, and the results are shown in Figure 2. Several interesting features can be observed: (a) Current response of the platinum to HzOzwas practically unchanged by the exposure to serum samples, as shown by the identical peak currents measured. The integrated peak areas agreed within 3-5%. Clearly the ”poisoning” effect appears to have been eliminated. (b) When pooled serum samples were injected, negligible currents (2-3 nA) were observed indicating that practically no electroactive species were diffusing across the CA film toward the electrode. However, this does not imply that such species are absent in serum samples. A more reasonable explanation is that the mass transport properties of these species are poor in CA films as indicated above. (c) Freshly made control serum samples (NCS) showed substantial peaks (not shown in Figure 2) of some unknown electroactive species which diffused through CA film rapidly.
If the samples were allowed to stand for 24-48 h, these peaks disappeared, indicating a slow decomposition of the unknown species in solution, even when stored a t 4 “C in the dark. Work to identify and/or eliminate these components is in progress. The above results clearly demonstrate the potential utility of the CA film in electrodes used in LCEC. The loss of sensitivity due to such modifications will of course depend on the electroactive species of interest and their mass transport properties across the film. At least in the case of HzOzthe problem of sensitivity appears to be of no concern. It is, however, fair to say that the electrode “poisoning” has been prevented and the selective elimination of unwanted electroactive species has been demonstrated. We believe that these developments would enable LCEC to accommodate the analyses of more complex matrices with minimal sample preparation, especially when a variety of coatings become available which allow selective diffusion of the species of interest. Work along these lines is in progress in our laboratory at present. ACRNO WLEDGMENT We thank Arnaud Apoteker for the useful discussions on the diffueivities of small molecules though membranes. The platinum LCEC detector was a generous gift from Bioanalytical Systems, West Lafayette, IN. We also thank Paul Finley from the College of Medicine, IJniversity of Arizona, Tucson, AZ, for providing the serum samples used in this study. Registry No. Cellulose acetate, 9004-35-7;hydrogen peroxide, 7722-84-1. LITERATURE CITED Adams, R. N. “Electrochemistryat Solid Electrodes”; Marcel Dekker: New York. 1969; Chapter 10. Lane, R. F.; Hubbard, A. T. Anal. Chem. 1978, 48. 1287-1293. Brezlna, M.; Zuman, P. “Polarography in Medicine and Biochemistry”; Interscience: New Yark, 1958; Chapter 30. Roston, D. A.; Kissinger, P. T. Anal. Chem. 1982, 54,424-434. Vogel, A. I . “A Text Book of Quantiiative Anaysis”; Wiley: New York, 1958; Chapter 3, pp 316-325. Tietz, N. W., Ed. ”Fundamentals of Clinical Chemistry”; W. E. Saunders Co.: Philadelphia, PA, 1976; Appendix: Table of Normal Values, pp 1206-1227. Apoteker, A., Department of Chemistry, Universlty of Arizona, Tucson, AZ, personal communication, 1983. Sellers, R. M. Analyst (London) 1980, 105,950-954. Instruction Manual for YSI Model 25 Oxidase Meter and Y S I 2510 Oxidase Probe”; Yellow Springs Instrument Co., Scientlfic Dlvlslon: Yellow Sprlngs, OH, 1975. t;onsdale, H. K.; Cross, B. P.; Graber, F. M.; Milstead, C. E. I n Permselectlve Membranes”; Rogers, C. E., Ed.; Marcel Dekker: New York, 1971; pp 167-187. Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1980, 52, 1126-1130. Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1979, 51,439-444. Coiton, C. K.; Smith, K. A.; Merriii, E. W.; Farrel, P. C. J . Homed. Mater. Res. 1971, 5 , 458-486. Evans, N. T. S.; Qulnton, T. H. Resplr. Physiol. 1978, 35,89-99. Clark, L. C.; Duggan, C. A,; Grooms, T. A.; Hart, L. M.; Moore, M. E. Clln. Chem. (Wlnston-Salem, N . C . ) 1981, 27(12), 1978-1982. Pace, S.J. Sens. Actuators 1981, 1 , 475-525. Johnson, J. M.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1982, 54, 1377-1383. Pinkerton, T. C.; Lawson, B. L. Clln. Chem. (Wlnston-Salem, N . C . ) 1982. 28 (9), 1946-1955.
G. Sittampalam G . S. Wilson*
Department of Chemistry University of Arizona Tucson, Arizona 85721 RECEIVED for review March 2, 1983. Accepted May 5, 1983. This work was supported in part by Grants PCM80-11555and CHE80-22286 from the National Science Foundation and by a Biomedical Research Support Grant from the University of Arizona.
