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Flgure 1. Voltammograms (500 mV/s; 21’) at HMDE in buffer 5 X lo-‘ M in cobalt(II1)and containing BSA. Solution stirred for 10 min, stirring stopped, and potential of -1.05 V applied for 30 s. Voltammogram run anodically to 0 f 0.1 V and after 2 s cathodically. Molar concentration BSA: Curve(1): 5 X lo-’‘; (2) lo-”; (3) 2.5 X lo-”; (4) 5 X lo-’’; (5) lo-’’. Curve (6): blank with Co(II1)
rate of 20 mV f s undoubtedly are due to the instability of the oxidized “active Co” (vide infra). The time involved in the scanning between 0 and -1.4 V at a scanning rate of 20 mV/s is 70 s, during which time virtually complete inactivation of the oxidized “active cobalt” occurs. The scanning rate of our polarograph is 16 mV s and no i, is observed under the above conditions when scanning is done with this polarograph. In our work, we have always deposited “active cobalt” a t -1.05 V which was found to be the optimum voltage. Actually, the potential range of deposition of “active Co” was found to be between -0.9 and -1.15 V. Values of i, increased with increasing time of activation at -1.05 V. The optimum time was 3 min, after which i, decreased. A t potentials outside the range a t which it can be formed and at open circuit, “active cobalt” was found to be very unstable. This instability probably accounts for the fact that there is an optimum time of deposition of “active Co” a t -1.05 V. The oxidized form of active cobalt at 0 f 0.2 V was found to be highly unstable, even a t the potentials a t which it is formed. A preliminary study has been made of the valence of the “active cobalt” and its oxidized form. The number of microcoulombs (&) involved in the deposition a t -1.05 V was found from the current-time curve recorded with an oscilloscope, while the number associated with the oxidation was estimated from the area occupied by the anodic wave. The ratio pC at -1.05 V/pC anodic obtained in 3 experiments was
2.9 f 0.1 with Co(II1) and 1.9 f 0.15 with Co(I1). Tentatively we conclude that the “active cobalt” is uncharged and that the oxidized form has a charge of +l. It is clear from Figure 1 and the various factors affecting i, that the HMDE is ideal for the detection and estimation of ultratraces of BSA (and undoubtedly other proteins and low molecular weight thiols and disulfides). For example, after having placed the HMDE for 2 h in a stirred buffer which was 5 X lo-’* M in BSA (0.04 pg in 100 mL), i, was 82 - 20 = 62 pA (blank was 20 PA). The water used as solvent for ultratrace determinations must be highly purified to eliminate subtraces of organic impurities which are adsorbed on the HMDE and which also can yield a current similar to i,. With highly purified water as solvent, even 0.01 pg BSA in 100 mL can be determined. In conclusion it may be stated that a few experiments have been made with a buffer like in Figure 1 which was M in cysteine and 5 x lo-* M in Co(II1). In one experiment, the HMDE was kept in the solution for 40 s at potentials varying between -0.98 and -1.20 V, then 2 s at -0.1 V and scanned cathodically. A catalytic hydrogen current was observed similar to that with BSA. The optimum potential for deposition of “active Co” was -1.16 V and the peak current (at -1.45 V) 124 PA. When activation occurred at -1.05, -1.10, -1.14, -1.18, and -1.20 V, i, was 11, 58, 108, 96, and 0 pA respectively. Cystine in a concentration of 5 x lo-’ M behaved like 1 X lo4 M cysteine. A more detailed study of the new catalytic current observed with BSA and particularly with low molecular weight thiols and disulfides, as well as studies of the stability of “active cobalt” and its anodically formed oxidation product, are now being made.
LITERATURE CITED (1) B. A. Kuznetsov, “Blolcglcal Aspects of Electrochemistry”,Proceedings of the 1st International Symposium, Rome, 1971, p 381. (2) M. Senda, T. Ikeda, and H. Klnoshlta, Bloelecfrocbem. Bloenergeflcs, 3, 253 (1976). (3) I. M. Kolthoff, H. Sawamoto, and S. Klhara, J . Electroanal. Cbem., In
press. (4) R. BrdlEka, Collect. Czech. Cbem. Commun., 8, 366 (1936). (5) P. Anzenbacher and V. Kalous, Collecf.Czech. Chem. Commun., 38, 2418 (1973).
I. M. Kolthoff* Sorin Kihara School of Chemistry University of Minnesota Minneapolis, Minnesota 55455
RECEIVED for review June 28,1977. Accepted August 30,1977. This research was supported by Public Health Service Grant CA16466-03 from the National Cancer Institute.
Determination of Catecholamines in Urine by Reverse-Phase Liquid Chromatography with Electrochemical Detection Sir: The objectives of our laboratory require the development of highly specific and sensitive procedures for the determination of tyrosine and tryptophan metabolites in biological fluids and tissue homogenates. Liquid chromatography with electrochemical detection has proved to be a useful tool in this regard (1) and progress in this area has been recently reviewed (2, 3). A highly effective procedure for
simultaneous assay of urinary catecholamines has been published (4). In this earlier work, a pellicular cation-exchange stationary phase was used in combination with amperometric detection at a carbon paste electrode. The present brief report describes an improved sample isolation procedure. In addition, the use of a microparticle reverse phase column modified by an anionic detergent (5) provides greater seANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
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lectivity and sensitivity while reducing the time for each determination.
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EXPERIMENTAL Apparatus. The chromatographic system was similar to that described previously ( I ) except that all stainless steel components were used. A 30-cm microparticle reverse phase CiBcolumn (Waters Associates, Milford, Mass. 01757) was used with a 100-gL rotary sampling valve (Rheodyne, 2809 Tenth Street, Berkeley, Calif., 94710; Model 70-10). A thin-layer amperometric detector (Bioanalytical Systems Inc., Model LC-10) was operated at +0.720 V vs. a Ag/AgCl reference electrode. Reagents. (1)Hydrochloric acid, 6 M; (2) ammonium sulfate, 2 M; (3) sulfuric acid, 0.7 M; (4) disodium ethylenediamine tetraacetate (EDTA), 10% (w/v); (5) thioglycolic acid, 4% (w/v); (6) phosphate buffer, pH 7: dissolve 4.32 g of sodium hydrogen phosphate, 1.18 g of potassium dihydrogen phosphate, and 10.0 g of disodium EDTA in distilled water and dilute to 1 L; (7) Tris buffer, 3 M, pH 8.6;(8) acid washed aluminum oxide (Bioanalytical Systems, Model AAO); (9) acetic acid, 1.0 M; (10) citric acid, 0.1 M; (11)sodium hydrogen phosphate, 0.1 M; (12) mobile phase: combine 300 mL of 0.1 M citric acid and 160 mL of 0.1 M Na2HP04and pass through a 0.22 pm average pore size filter (Millipore Corporation, Ashby Road, Bedford, Mass. 01730, Model XX1004700). Dissolve 0.010 g of sodium octylsulfate in the above solution. Because of the variability of commercial reverse-phase columns and their tendency to change properties with extensive use, it may be necessary to adjust the mobile phase composition to obtain optimum results for any given column; (13) cationexchange isolation columns (BioRad Laboratories, Richmond, Calif. 94804, Cat. No. 1892202). Spent resin may be recycled by washing with successive volumes of 3 M hydrochloric acid, 3 M sodium hydroxide, 3 M acetic acid, 1.0 M ammonium acetate (pH 6.51, and 0.1 M ammonium acetate (pH 6.5). The washing steps are carried out with gentle manual agitation. A magnetic stirrer is not recommended. The pH is adjusted to 6.5 during the last wash if necessary. The extraction columns (BioRad) are reloaded with resin, capped, and stored at room temperature prior to use; (14) catecholamine standard solutions-norepinephrine, epinephrine, and dopamine solutions at 100 mg/L (as the free base) in 0.01 M hydrochloric acid. These solutions are kept refrigerated; (15) standard urine pool-approximately 0.5 L of a urine pool from healthy individuals is acidified to pH 3 with 6 M hydrochloric acid. Add 1 mL each of 10% EDTA and 4% thioglycolic acid. Determine the concentration of each of the catecholamines in the urine pool by standard addition; (16) internal standard-3,4-dihydroxybenzylamine (Aldrich Chemical Co.) 10 mg/L. Procedure. Transfer aliquots (ca. 20 mL) of freshly voided urine into glass scintillation vials containing 150 pL of both 10% EDTA and 47'0 thioglycolic acid solutions to each vial. Store the specimens at -35 "C prior to analysis for a period not to exceed 2 weeks. Transfer 5 mL of urine in a 20-mL beaker and add 50 gL of the internal standard solution and 15 mL of phosphate buffer (pH 7) containing 1% EDTA. Pour this solution onto the cation-exchange extraction columns. With each group of ten specimens, analyze at least one aliquot of the urine pool standard. Allow the urine to drain completely and then wash the columns with 10 mL of distilled water, followed (after complete drainage) by 1.5 mL of 0.7 M sulfuric acid. Elute the catecholamines into a 5-mL conical reaction vial with 4 mL of 2 M ammonium sulfate. Add 100 gL of 4% thioglycolic acid, 500 pL of 3 M pH 8.6 Tris buffer, and 100 mg of aluminum oxide to each vial. Cap and immediately agitate each vial for 10 min using a reciprocal shaker. Allow the alumina to settle and then carefully aspirate the liquid without disturbing the adsorbent. Wash the alumina with distilled water. Remove the wash by aspiration and then add 500 pL of 1 M acetic acid. Recap the vials and shake for 10 min. After the alumina settles, transfer the eluate into a 0.22-pm filter tube mounted in a centrifuge (Bioanalytical Systems, Model CF-1). Inject the centrifugally filtered sample on the chromatographic column. Calculate the actual catecholamine concentrations by comparing the peak heights (divided by the internal standard) for the urine specimens to those for the calibrated urine pool. 2110
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
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Flgure 1. Effect of sodium chloride concentration on the relative recovery of dopamine from cation-exchange isolation columns
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Figure 2. Chromatogram of norepinephrine (NE), epinephrine (EPJ), and dopamine @A) isolated from human urine by ion exchange followed by adsorption on alumina. Concentrations in the urine were NE: 60 pg/L, E: 22 pgIL, DA: 510 pglL
RESULTS AND DISCUSSION The procedure described above offers advantages over the previous method (4)in every respect. The overall absolute analytical recovery was found to be 51 f 170for distilled water standards,the calibrated urine pool, and urine specimens. Salt (NaCl) concentrations up to 0.4 mol/L did not affect the recovery as illustrated in Figure 1. The major loss of recovery was encountered in the alumina extraction step which was only 65 % efficient. The method is linear over the range 1-1000 pg/L and detection limits for quantitation a t the 10% re1 std dev level are 1pg/L which is well below the concentration encountered in human urine specimens. The justification for this work was the improved speed, sensitivity, and selectivity which could be expected from microparticle reverse-phase chromatography (6). Comparison of Figure 2 with the figure in our original paper ( 4 ) will reveal the spectacular nature of this improvement. It was necessary to alter the sample preparation to eliminate acidic and neutral catechols which are retained on the CI8 column but not on the ion-exchange column employed in the earlier work. The effectiveness of this new procedure is indicated by the negligible response due to nonretained components. The high resolution of the microparticle CI8 column made it possible to use an internal standard without increasing the time required for an individual run and with minimum likelihood of interferences. The precision for repetitive determination of dopamine was evaluated with (re1 std dev = 1.7%;N = 6) and without (re1std dev = 7.270;N = 6) the internal standard. I t is now easily possible to fully process 50 urine specimens in a single working day. Although only a limited population of male researchers has been studied with this new method,
the results are statistically indistinguishable from those obtained on the same population using our earlier procedure ( 4 ) and are in general agreement with the literature ( 4 , 7). The method described here is notable for its combined sensitivity, selectivity, and speed when compared with all previously published reports. The advantage of amperometric detection is dramatically evident when comparison is made with a recent paper in which a UV absorption detector was used (7) to quantitate norepinephrine and dopamine (epinephrine could not be measured at normal levels) in over 100 mL of urine.
ACKNOWLEDGMENT We thank Ronald E. Shoup and Lawrence J. Felice for their helpful suggestions and assistance.
LITERATURE CITED (1) P. T. Kissinger, L. J. Felice, R. M. Riggin, L. A. Pachla, and D. C. Wenke, Ciin. Chem. ( Winston-Salem, N . C . ) ,20, 992 (1974).
(2) P. T. Klsslnger, Anal. Chem., 49, A447 (1977). (3) R . E. Shoup and P. T. Kissinger, Clln. Chem. ( Winston-Salem, N.C.), 2 3 , 1268 (1977). (4) P. T. Kissinger, R. M. Riggin, R. L. Aicorn, and L. D. Rau, Biochem. M e d . , 13, 299 (1975). (5) P. T. Kissinger, Anal. Chem., 49, 883 (1977) and references therein. (6) J. H. Knox and G. R . Laird, J . Chromatogr., 122, 17 (1978). (7) L. D. Meli and A. B. Gustafson, Ciin. Chem. (Winston-Salem, N . C . ) , 23, 473 (1977).
Ralph M. Riggin’ Peter T. Kissinger* Department of Chemistry Purdue University West Lafayette, Indiana 47907 Present address, Battelle Columbus Laboratories, 505 King Avenue, Columbus, Ohio 43201.
RECEIVED for review June 30,1977. Accepted August 12,1977. This work was sponsored by grants from the NIGMS and the Showalter Trust Fund.
Comments on Fluorescence Excitation Profiles in Flames Sir: In their article, Green et al. (1) have reported interesting observations on laser induced atomic fluorescence of the first resonance lines of Ba and Na in flames and have drawn several useful conclusions about the potentialities of tunable continuous-wave (CW) dye-laser sources in atomic fluorescence spectrometry. In one of their experiments they measured what they called “fluorescence line profiles” for various Na concentrations in an H2-02-Ar flame, while scanning the wavelength of the CW dye laser (output power = 300 mW, bandwidth = 0,003 nm) over the 589.0-nm Na line. The Na-doublet fluorescence was focused directly onto a photomultiplier tube with no intervening frequency-selective device. The profiles obtained were displayed in Figure 8. At low concentration, where self-absorption was negligible, the spectral width of the profile was roughly as expected from the laser bandwidth and the estimated Na absorption linewidth. At high concentrations where self-absorption is important (as demonstrated by the bending of the analytical curve shown in Figure 7), a much broader profile was observed, showing a d i p at t h e line center. The authors have interpreted this dip as a self-reversal effect and in this connection have stressed the value of line profile analysis. We wish to comment on their suggestion that the profile functions shown in Figure 8 are spectral profiles of the fluorescence line and that the dip found should be identified with the self-reversal dip of the fluorescence line. A broadening and even a self-reversal dip can be expected to show up at high concentrations when one wavelengthscans the fluorescence radiation with the aid of, for example, a Fabry-Perot interferometer at fixed wavelength of the exciting radiation. Self-reversal arises with resonance lines when the fluorescence radiation has to pass through a part of the Na-colored flame that is not irradiated by the source; it is quite analogous to the well known self-reversal dip found with a thermal resonance line in a flame with a relatively cooler outer layer (2, 3 ) . The profiles shown by Green et al., however, should not be called spectral profiles of fluorescence radiation. They are, in fact, “fluorescence excitation profiles” describing the fluorescence intensity (integrated over the whole spectral Na doublet) as a function of laser detuning a t constant laser power. (The latter term has been correctly used by the authors to describe their Ba fluorescence profiles in Figure 5 . )
The question that arises is why a dip in the fluorescence excitation profiles was found a t high concentrations. A possible, trivial explanation may be that the Na fluorescence from the very edge of the flame facing the laser was not focused on the photocathode. The fluorescence intensity detected will then drop when the laser is tuned close to the line center at such high Na concentrations that the penetration depth of the narrow-band laser radiation is small relative to the flame diameter. At the laser power stated, no appreciable saturation is expected to occur. Therefore, only a small fraction of the original laser power will penetrate into the observed part of the flame. The fluorescence radiation excited in this part may be further weakened because it has to travel through about half the flame diameter before escaping. We have corroborated this explanation by doing a similar experiment in which the whole laser-illuminated vapor cloud was seen by the detector. The fluorescence excitation profiles showed an extra broadening when the Na concentration was raised above the level, about 40 ppm, where self-absorption sets in. However, no dip was found even a t concentrations of about 1 or 2 orders of magnitude above this level. On the other hand, when a thin layer at the barely visible edge of the flame facing the laser was screened off from the detector, a dip showed up indeed. We also observed visually that the fluorescent spot in the flame shrank and retracted to this thin layer when the laser was tuned close to the line center at high concentrations. It is quite possible that in the experiments of Green et al. at high concentrations, the fluorescent spot lay outside the visible Na-colored flame, where absorbing Na atoms were still present but thermal excitation was, virtually, reduced to zero. The results of our experiments are shown in Figure 1. We used a n Na concentration of 400 ppm in water (curve a and b) sprayed into a stoichiometric H2-02-Arflame of 1 atm pressure and at 2250 K and with a diameter of 18 mm. The laser power was 40 mW, the bandwidth 0.005 nm, and the beam diameter about 1 mm. In curves b and c, the rims of the flame were screened off and only the visible Na colored part of the flame was focused on the photocathode. Curve c was obtained with a Na concentration of 160 ppm and shows roughly the same dip as the authors showed in their Figure 8b (1). Use of 300-mW laser power and the same bandwidth did not change the shape of our curves. Since the waveANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
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