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Anal. Chem. 1983, 55, 708-713
so the absolute diminution in current for iDp is even more striking. The differences in current response illustrated above serve as a guide to the behavior to be expected in practical experimental cases. In general the current AiNP gives the largest value of peak current and therefore the greatest analytical sensitivity. Correspondingly the current AiDP is by far the worst in sensitivity, and the effect is greater the slower the reaction until the limiting value is reached for total irreversibility. The effects of irreversibility on the current AiDP are more pronounced for greater values of the time tl,and the representative calculations given here are at the short end of the time range normally employed. Thus they understate the effect of slow heterogeneous charge transfer kinetics on the differential pulse response. The shape of the AiDNP voltammogram is the most sensitive to changes in kinetic parameters in the quasi-reversible region.
Auerbach, C.; Flnston, H. L.; Kissel, G.; Glickstein, J. Anal. Chem. 1961, 33, 1480-1484. Jackson, L. L.; Yarnitzky, Ch.; Osteryoung, R. A,; Osteryoung, Janet Chem. Blomsd. Environ. Instrum. 1980, IO, 175-188. Abel, R. H.; Christie, J. H.; Jackson, L. L.; Osteryoung, Janet; Osteryoung, R. A. Chem. Instrum. ( N . Y . ) 1976, 7 , 123-138. Lane, R. F.; Hubbard, A. T. Anal. Chem. 1076, 48, 1287-1293. Albery, W. John; Beck, T. W.; Brooks, Wllllam N.; Fillenz, Marianne. J . Electroanal. Chem. 1081, 125, 205-217. Anderson, J. E.; Bond, A. M. Anal. Chem. 1961, 53, 504-508. Evans, Otis M.; Hanck, Kenneth W. Anal. Chlm. Acta 1981, 129, 79-88. Aokl, Kolchi; Osteryoung, Janet; Osteryoung, R. A. J . Electroanal. Chem. 1980, 110, 1-18. Brumleve, Timothy R.; O'Dea, John; Osteryoung, R. A,; Osteryoung, Janet Anal. Chem. 1081, 53, 702-706. Brumleve, Timothy R.; Osteryoung, Janet Anal. Chem. 1081, 53, 988-99 1. Brumleve, Tlmothy R.; Osteryoung, R. A.; Osteryoung, Janet Anal. Chem. 1082. 54. 782-787. Lovri6, Milivoy; Osteryoung, Janet Electrochlm. Acta 1982, 2 7 , 983-988. Barker, G. C.; Gardner, A. W. AERE Harwell C/R 2297, 1955. Aokl, Koichi; Osteryoung, Janet J . Electroanal. Chem. 1980, 110, 19-36. Nicholson, R. S.; Olmstead, M. L. In "Electrochemistry: Calculations, Slmuiation and Intrumentatlon"; Mattson, J. S.,Mark, H. B., MacDonald, H. C., Eds.; Marcel Dekker: New York, 1972; Vol. 2. O'Dea, John: Osteryoung, Janet; Osteryoung, R. A. Anal. Chem. 1081, 53, 895-701.
ACKNOWLEDGMENT The authors express their thanks to Robert deLevie whose thoughtful questions prompted this work initially. They also thank Robert Osteryoung for helpful comments.
LITERATURE CITED (1) "Instructlon Manual. Electrochemical System Model 170"; Prlnceton Applied Research Corp.: Prlnceton, NJ, 1970.
RECEIVED for review September 8,1981. Resubmitted August 3,1982. Accepted January 10,1983. This work was supported by the National Science Foundation under Grant No. CHE 7917543.
Determination of Pertechnetate by Liquid Chromatography with Reductive Electrochemical Detection Jane Y. Lewis, Julius P. Zodda, Edward Deutsch, and Wllliam R. Heineman" Department of Chemistry, Universiv of Cincinnati, Cincinnati, Ohio 4522 1
A method utlllzlng llquld chromatography with electrochemlcal detectlon has been developed for the determlnation of total Tc04- (gg"rcO[ and %O.,-) in " M o ~ generator c eluents. Pertechnetate, which Is the startlng material for the preparation of many diagnostic radlopharmaceutlcals, Is generally present In these eluents in the concentratlon range of 5 X IO-' M to 5 X I O d M. No sample pretreatment Is necessary since lmpurlties and other components are separated by the HPLC ",-bonded column. By use of both static mercury drop (SMDE) and solid electrode detectors, in conjunction with rigorous deoxygenatlon procedures, total Tc04- In generator eluents is readily determined. A severe electrode foullng phenomenon lhits the use of solid electrode detectors to Tc04- concentrations less than lo-' M, the working range for a carbon electrode belng 8.5 X I O 4 to 1.0 X IO-' M. The working range for the SMDE Is 2.1 X IO-' to 1.0 X M Tc04-.
In diagnostic nuclear medicine, a particular chemical form of a y-ray-emitting isotope is introduced into the body so that the isotope localizes in the organ of interest. A scan obtained with a y-ray camera then provides valuable diagnostic information about the organ ( I ) . In this essentially noninvasive technique, nearly 80% of the clinically used radiopharmaceuticals incorporate w m Tbecause ~ it is an isotope that has 0003-2700/83/0355-0708$01.50/0
ideal nuclear properties, has diverse chemistry, and is readily available. 9 9 m T in~the form of 99mT~04is supplied by a ggMo/99mTcgenerator which utilizes the following decay scheme: 9
9
- a~
66 h
99mTc ~
ggTc
6h
99Moin the form of 99M0042-is adsorbed on alumina and packed in a column at a reactor site. It is then shipped to the hospital packaged in lead. Each morning an aliquot of saline is drawn through the generator eluting the -1 charged Tc04- (both q c 0 ; and &Tc04) and leaving the -2 charged 99M0042-on the column. Tc04-is then mixed with a reductant, a ligand which will confer some desirable biological properties on the final complex, and a stabilizer to form the fiial radiopharmaceuticalmixture ( I ) . In order to understand the chemistry and kinetics of this preparation scheme, the total pertechnetate concentration (i.e., both 99T~04and gSmT~04-) in the original generator eluent must be known (2). The current method for determining total Tc04- is to measure the &Tc activity with a y counter. Given certain assumptions about the history of the generator, [99T~04-] can then be calculated. However, these assumptions have never been validated, and the calculated results have never been experimentally verified. Recently, a liquid chromatography method utilizing UV detection (LCUV) at 254 nm has been developed in this 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55,
laboratory for determining total Tc04- in ggMo/wmT~ generator eluents ( 3 , 4 ) . This HPLC procedure involves a simple NH2-bonded phase separation in acetate buffer and has a detection limit of 6.4 X lowsM Tc04-. It is convenient, inexpensive, and readily applicable to generator samples as interferences are easily overcome by the HPLC separation. It was found that wMo/99mT~ generators typically yield eluents containing pertechnetate in the concentration range of 5 X M to 5 X lo4 M. The LCUV method can be used to monitor the majority of such eluents, the major exceptions being the second of two consecutive elutions performed within a short time period or samples drawn late in the generator life. Thus, in order to monitor all generator eluents a more sensitive detection method is needed. In addition, it is of interest to monitor radiopharmaceutical mixtures for any remaining, w e a c t e d Tc04-, since the presence of this species is almost always detrimental to the quality of the y-ray image. Because the major fraction of radiopharmaceutical mixtures contains Tc in a reduced form, the concentration of Tc04- in these mixtures is very low, and again a very sensitive detection system is required. In this work, liquid chromatography/electrochemistry (LCEC) is applied t o the determination of total Tc04- in generator eluents as LCEC is known to be one of the most sensitive HPLC methods (5). Tc04- contains the most oxidized form of Tc (i.e., the VI1 oxidation state), and therefore reductive LCEC is necessary. There have been several papers in the literature recently that report the use of LCEC in the reductive mode. Though reductive LCEC is not a new technique (6-9),this trend reflects development of the method into a highly sensitive analytical tool with a wide range of applications. These include the determination of organics (10-15),organometals (16), and metal ions (14,17, 18). State-of-the-art developments have been reviewed (11,19). Many of the typical problems associated with reductive LCEC were encountered while developing the method for the determination of TcO,. These include difficulties associated with electrode fouling, the necessity of detecting at somewhat large negative potentials, and interferences from dissolved O2 However these problems were overcome to the extent that generator eluent levels of Tc04- could be readily detected.
EXPERIMENTAL SECTION Apparatus. Voltammograms were obtained with a Bioanalytical Systems, Inc. (BAS), CV-1A or CV-1B potentiostat, a HewletbPackard 7015-B x-y recorder, and a Keithley 178 digital multimeter. The standard bulk solution voltammetry cell with glassy carbon working (area = 9.1 mm2),Ag/AgCl (3 M NaCl) reference, and Pt auxiliary electrodes was from BAS. Differential pulse polarography was performed at a EG&G Princeton Applied Research (PAR) Model 303 static mercury drop electrode (SMDE), which incorporates a Ag/AgC1(3 M NaC1) reference electrode and a Pt auxiliary electrode. Large drops (surface area of 2.7 mm2)were used. A PAR 174A polarographic analyzer controlled the SMDE. Chromatograms were obtained with a liquid chromatograph that could be connected to either a thin-layer detector with a glassy carbon electrode or a mercury drop detector. The BAS liquid chromatography system, depicted in Figure 1(A-E), is a LC-304 apparatus which includesa LC-22 temperature controller,a mobile phase deoxygenationsystem as recommended by BAS, a PM-30 dual piston pump, a TL-5 electrochemical cell, an LC-4 amperometric controller (time constant = 0.5 s), and a Houston Instruments Omniscribe strip-chart recorder. The TL-5 thin-layer cell contains a glassy carbon working electrode (area = 8.0 mm2) and a 5-mil Teflon spacer. The Ag/AgC1(3 M NaC1) reference and stainless steel auxiliary electrodes are located downstream. No pulse dampener was used since it proved to be unnecessary. Detection at a mercury:drop electrode was accomplished by replacing the BAS detection system with an EG&G PAR Model 310/174A SMDE system (time constant in the sampled DC mode = 16.7 ms). This is depicted in Figure 1; again park A-D are used,
NO.4,
APRIL 1983
709
/
A
G
F
Flgure 1. LCEC apparatus: (A) temperature controller: (6) solvent reservoir: (C) pump; (D) BAS LC component rack which holds
(clockwisefrom left) anaiytlcal column, injection valve, pressure gauge, and TL-5 thin-layer Celt, left of the rack are two vials for deoxygenating sample with Ar (lower and upper contain water and sample, respectively): (E) BAS amperometric controller: (F) EG&G PAR SMDE; (G) EG&G PAR polarographic analyzer. except that the TL-5 thin-layer cell in D is replaced by F, and detector E is replaced by detector G. The SMDE system utilizes a mercury drop assembly that is coupled to the column outlet tubing by an LC adapter body. The adapter body as well as the Ag/AgCl reference electrode, and Pt auxiliary electrode are suspended in a 50-mL bulk flow cell. Medium drops with a surface area of 1.7 mm2 were used. The HPLC Knauer column (25 cm X 4.6 mm i.d.) from Unimetrics was packed in our laboratory with 5-pm NH2-bonded Spherisorb from Jones Chromatography, Inc. A guard column of 10-pm Amino Spheri-5 material from Brownlee Labs was installed between the injection valve and the analytical column; this column is located behind the component rack and thus is not depicted in Figure 1D. Deoxygenation Requirements. Detection in the reductive mode at potentials in excess of -1.0 V required rigorous deoxygenation procedures. These include adaptations recommended by BAS: a reflux apparatus for deoxygenating the mobile phase; lines from the Ar tank that are 1/8 in. copper tubing with brass fittings (no further purification of the Ar proved necessary);and exclusive use of 316 stainless steel tubing and fittings between the reflux apparatus and the electrochemical cell. Additionally, an Ar line was extended to the SMDE electrochemical cell and fitted to a f i e porosity glass dispersion tube. This tube was passed through the spiking hole in the side of the SMDE electrode support block as shown in Figure 1. The deoxygenation procedures were as follows: (a) The appropriate aqueous mobile phase was placed in a 2-L flask and refluxed at 100 "C for 1h (using the LC-22 temperature controller and probe) while being purged with Ar. The temperature was then allowed to drop to 45 OC, and, with Ar flowing over the solution, the mobile phase was pumped directly from this vessel through stainless steel tubing. If the LC had been exposed to 0 2 in the meantime,the deoxygenatedmobile phase was pumped through it overnight to purge all O2 from the porous packing material. Our system incorporated two such apparatuses so that shubdowntime for deoxygenating was eliminated. This procedure is essentially that described by Bratin and Kissinger (11). (b) The SMDE flow cell wm rinsed with distilled, deionized H20each day. The trap was then filled with Hg and the cell was filled with nondeoxygenatedbuffer that was being used as the mobile phase. Deoxygenation was accomplishedby purging with Ar through the porous glass frit for 45-60 min. The 310 is equipped with an Ar inlet so that Ar continuously flows over the solution to prevent 0 2 from reentering the cell. Thus, once purged, the cell may be operated with no further deoxygenation. If this cell is not deoxygenated sufficiently,not only does the magnitude of the base line current rise but the peak-to-peak base line noise increases. (c) If the BAS solid electrode system was used, only the mobile phase was deoxygenated; there are no special requirements for the flow cell. (d) The sample solution was deoxygenated by purging with an Ar stream (that had been saturated with H20) for 2-5 min (Figure 1D). Immersing the inlet of the injection valve
710
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
v
- 0 0V
w -A
Cycllc voltammogram of M KTcO, In 0.1 M, pH 5 acetate buffer,glassy carbon electrode, scan rate = 25 mV/s. Flgure 2.
in the sample solution, a syringe was used to pull an aliquot into the 20-pL loop with no exposure to air ( 1 1 ) . Available sample volumes were large enough (0.5 to 2.0 mL) that this deoxygenation procedure was adequate. Reagents and Materials. Instrument mercury (A.C.S. grade, Bethlehem Apparatus Co., Inc.) was used without further purification. Acetate buffer was prepared from reagent grade acetic acid and concentrated NaOH. Phosphate buffer was prepared from HPLC grade KHzP04 and reagent grade concentrated NaOH. All water had a resistivity of at least 17 Ma cm and was obtained from a Sybron/Barnstead system. Technetium-99, obtained from Oak Ridge National Laboratory in the form of NH49eTcO4, was converted to K9eTc04by metathesis with KOH, and this material was used for preparing standard solutions. Pertechnetate generator samples were eluted from a commercial ggMo/mTcgenerator;these samples were the same as those used in previous studies (3).All solvents were filtered through a 0.2-pm Gelman Metricel GA-8 membrane filter.
RESULTS AND DISCUSSION Voltammetry. A cyclic voltammogram of M geTc04in pH 5 acetate buffer a t a glassy carbon electrode is shown in Figure 2. The voltammogram (initiated at 0.0 V in the negative direction) exhibits highly irreversible behavior with a reduction wave at -0.68 V and an oxidation wave at 0.25 V. The symmetry of the latter indicates that it could be a stripping wave. Further experiments employing the technique of anodic stripping voltammetry confirm this; i.e., the height of this wave is linearly dependent on TcO, concentration and on the time spent in the deposition step (during which stirring occurred). One further notable feature of Figure 2 is the appearance of a poorly defined cathodic wave at -0.57 V. This occurs while scanning in the positive direction and indicates that there is some alteration of the electrode surface following reduction of Tc04-. Figure 3 shows the results of further experiments designed to elucidate this phenomenon. With the same conditions as before, scans were initiated at 0.0 V in the negative direction. Voltammogram A exhibits the normal behavior that is also seen in Figure 2. If the positive scan is then reversed a t 0.0 V instead of scanning through the stripping wave, voltammogram B (Figure 3) is obtained. There is an obvious distortion in the voltammogram exemplified by a shift toward positive potentials of both the Tc04- reduction wave and the Hzevolution background wave. Voltammogram C is obtained after holding the potential at -0.9 V for approximately 2 min. Clearly the electrode characteristics have changed; most likely the electrode no longer has just a carbon surface but consists of a technetium f i on the carbon surface. This same phenomenon was observed on gold, platinum, wax-impregnated graphite, hanging mercury drop, and Au amalgam electrodes. A detailed analysis of this phenomenon is complicated by the proximity of the Tc04- reduction wave
-0.4V
ia
Flgure 3. Cycllc voltammograms of M KTcO, in 0.1 M, pH 5 acetate buffer, glassy carbon electrode, scan rate = 25 mV/s: (A) initial scan, (B) second scan (Inner tracing), (C) third scan (following the application of E = -0.9 V for 2 min).
I
-0.2
I
I
-0.6
I
I
-1.0
I
I
-1.L
E , V vs AgiAgCl Dlfferentlal pulse polarogram of M KTcO, in 0.05 M, pH 6 phosphate buffer: SMDE, scan rate = 10 mV/s, 1 drop/s, moduiatlon amplitude = 25 mV. Flgure 4.
to the H2evolution wave; this creates difficulties in the LCEC application. A differential pulse polarogram of Tc04- using the static mercury drop electrode is shown in Figure 4. It has been reported in the literature that Tc04- has a well-behaved, diffusion-controlled, polarographic wave corresponding to three electrons at approximately -0.7 V in pH 7 phosphate buffer (20). Figure 4 shows that this same wave occurs in pH 6 phosphate buffer. At pH 5.3, it is still present, along with waves at -1.05 V and -1.35 V. Thus the potential of this peak is independent of pH over this range. More importantly, this peak is well removed from the H2 evolution wave, a fact that simplifies the LCEC analysis. Liquid Chromatography/Electrochemistry. LCEC generally employs either solid electrodes or a dropping mercury electrode. Solid electrodes used for reductive processes commonly are either carbon or a Au amalgam, and in the absence of adsorption problems these electrodes can be used for routine analyses with minimal daily preparation; they are simple to use and inexpensive. Solid electrodes, however, are subject to passivation, and considerable effort has been expended to circumvent this limitation (21-23). These effects can be avoided by the use of a dropping mercury electrode which presents a renewable surface to the electroactive solution. The dropping mercury electrode also has a large negative potential range and a uniform surface that is not subject to inhomogeneities associated with the surfaces of solid electrodes. On the other hand, dropping Hg electrodes can be somewhat inconvenient because they require more operator attention. Since it is evident from the data in Figure 3 that reduction of M Tc04- on a carbon surface involves a serious adsorption problem, two approaches have been taken
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
Table I. Linear Least-Squares Analysis for Kg9Tc0, Standard Curves' slope SMDE detector glassy carbon electrode detector SMDE detector under generator working conditions
12.80
Y intercept
* 0.04
0.16
corr coeff 0.9989 0.9996 0.9995
0.01 -0.0069 i 0.0002 0.18 0.01
1.47 i 0.01 11.68 * 0.02
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f:
*
a At least three sample injections were made at each concentration; the mean value of i in nA was plotted vs. concentration Tc0,- in pM. Y values are weighted in the least-squares analysis as l / u m 2 where urn = the standard deviation of the mean.
c
1.0
T
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I
T
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0.0
-0.2
-0.8
-1.4
Eapplied, V vs Ag/AgCI Hydrodynamic voltammogram of lo-' M KTcO,: i* = normalized current, mobile phase = 0.01 M, pH 5.7 phosphate, SMDE, sampled DC mode, 1 drop/s, flow rate = 1.0 mL/min.
1
1
1
1
1
1
1
1
1
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Figure 5.
to circumvent the associated difficulties. First, a static mercury drop electrode has been employed for use with higher concentrationsof TcO,, and, second, a glassy carbon electrode has been employed to detect those levels of Tc04- (Le. less than 1 X lo-' M) that do not give extensive adsorption effects. SMDE Detection. Figure 5 shows a hydrodynamic voltammogram constructed from chromatographicdata using 0.01 M phosphate buffer (pH 5.7) as a mobile phase. This was done in the sampled DC mode by making successive TcOC injections at increasingly negative potentials. Current was measured at each potential and normalized by dividing all values by the largest current value (at E = -1.50 V). A plot of E vs. inormalized yields EIl2= -0.86 V. Any potential 2-1.0 V results in a current equal to the limiting current, thus producing maximum sensitivity. All further analyses are conducted at E = -1.10 V. Three different modes of detection were employed to determine the optimum type of analysis, i.e., differential pulse, normal pulse, and sampled DC detection. On the basis of the peak heights, normal pulse gives the maximum current response (Le., a factor of 1.5 greater than that observed in DC detection), followed by sampled DC, and then differential pulse. However, the signal to noise ratio is essentially the same for all three modes; thus, sampled DC is used for the analysis since the regular base line associated with this mode facilitates peak height measurements. Peak response is directly dependent on Hg drop area; however, if the drop is too large, the surface can be distorted by the eluent flow. Thus, a medium drop was used which provided both sensitivity and reproducibility. A 1-sdrop time proved advantageous for two reasons: fiit, electrode alteration problems late in the drop life are avoided and, second, sufficient data points during a peak (i.e., 30 drops/peak) are obtained. Figure 6 shows a typical chromatographic peak obtained with these parameters and a mobile phase of 0.01 M phosphate (pH 6). Thoroughly deoxygenating the sample ensures the absence of an O2 peak. Table I shows the calibration data obtained under these optimal conditions. With 14 different pertechnetate concentrations, linearity extends over 4 orders of magnitude from 2.1 X M to 1.0 X
1
0
Figure 6. Chromatogram of 2.6 X
IO-' M standard KTcO, solution: mobile phase = 0.01 M, pH 6.0phosphate, SMDE, sampled DC mode, ,Eepplled = -1.1 V, 1 dropls, flow rate = 1.0 mL/min.
I10nA A I
B
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0
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l
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l
l
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8
t (mins) Figure 7. Chromatographic background: (A) flow cell not deoxygen-
ated, (B) flow cell deoxygenated; mobile phase = 0.01 M, pH 5.3 phosphate, SMDE, sampled DC mode, E,,,, = -1.1 V, 1 drop/s, flow rate = 1.0 mL/min.
M Tc04-. Response may continue to be linear above 1.0 X M Tc04-, but these levels were hot investigated. The detection limit, defined as a peak height that is twice base line peak-to-peak noise, is 2.1 X M 2~8.3%. Three repetitive injections were made at each concentration, and precision is reported as the percent relative standard deviation (% rsd) between the three measured current values. Precision varied over the range from 8.3% at the detection limit to 0.2% a t the high end of the range. As mentioned in the Experimental Section, the presence of O2 is a significant problem in reductive LCEC. This has been solved in many cases by simple purging of the mobile phase and sample; in other cases it has been solved by more novel approaches (17,18,24,25). Nonetheless, detection limits have generally been dependent on the magnitude of the base line peak-to-peak noise which in turn has been found to be
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
I
I
l-
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8
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2L
t (minsi Chromatogram of lo-' M KTcO,, moblle phase = 0.1 M, pH 5 acetate buffer, glassy carbon electrode, Eapplled = -0.9 V, flow rate = 1.0 mL/mln. Flgure 8.
I
I
0
2
I
I
L 6 8 t (mins)
I
I
1
0
Flgure 8. Chromatogram of a KTcO, sample from a gsMo/ggmTc generator: mobile phase = 0.1 M, pH 5.3 phosphate, SMDE, sampled DC mode, Eeppllsd = -1.1 V, 1 dropls, flow rate = 1.0 mL/mln.
beginning a t approximately 17 min. This results from alteration of the electrode surface following TcO; reduction and makes subsequent detection on the particular electrode imdirectly dependent on the amount of dissolved O2both in the possible without a lengthy cleanup procedure. However, if mobile phase and in the flow cell. Deoxygenation of the latter the TcO,- concentration is kept at levels
