Anal. Chem. 1984, 56,487-491
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Effect of Eluent and Sample Composition on Quantitation in UV-Visualization Liquid Chromatography F. Vincent Warren, Jr., and Brian A. Bidlingmeyer* Waters Associates, Inc., 34 Maple Street, Milford, Massachusetts 01757
The Influence of eluent and sample properties (Ionic strength, solvent strength) upon quantitative analysis by use of UVvlsuallzatlon LC is Investigated. A “high salt” eluent Is shown to provlde superior stablllty of peak area and retention tlme when uslng a UV-absorbing llpophlllc cationic reagent In the reversed-phase eluent.
UV-visualization liquid chromatography (1-5) has recently been applied to the determination of a variety of samples for which detection is otherwise difficult. By use of this technique, bile acids (6),ionic surfactants (5-B), amino acids (9), and inorganic ions (10) have been detected. However, few studies have concentrated on the use of this method for reliable, routine quantitation. In the reports that do address the issue of quantitation, some troublesome behaviors have been noted. Parris (7) reported a variation in k’with sample size for several surfactants. A similar effect is seen in data presented by Sachok, Deming, and Bidlingmeyer (5). Denkert and coworkers (9) demonstrate a dependence of the detector response on proximity of sample peaks to the ”system peak”, consistent with data recently published by our laboratory (11). These observations underscore the need for a systematic study of quantitation by the UV-visualization technique. This paper represents a first step toward that goal by investigating the influences of eluent ionic strength and sample solvent. Based on earlier work in our laboratory ( 2 1 ) we perceived the need for the consideration of ionic strength, both of the eluent and of the sample, during the development of a quantitative method using the UV-visualization technique. With increasing ionic strength of the eluent, the quantity of visualizing reagent adsorbed onto the Cls stationary phase was shown to increase according to a sigmoid dependence. Dramatic effects on the retention and chromatographic response for the alkylsulfonate samples were observed as ionic strength was varied over 3 decades of salt concentration. In addition, the solvent strength and ionic strength of the sample relative to the bulk eluent were shown to induce pairs of peaks. In this work, therefore, it was necessary to determine the optimal ionic strength for both eluent and sample. Specifically, we have considered the effect of ionic strength on retention time and on the magnitude and linearity of peak area for a test solute. Because of the sigmoid dependence mentioned above, we predicted that stable chromatographic performance should be obtained a t the extremes of ionic strength, correponding to plateau regions of the sigmoid curve (11). This prediction will be verified in this paper. We will further demonstrate trade-offs involved in applying the UV-visualizationtechnique for quantitative determinations. It is not the purpose of this paper to propose any specific LC assay, but rather to discuss the influence of several variables on the retention time, peak area, and peak height for representative solutes. Sodium valproate (NaV), the sodium salt of dipropylacetic acid, was selected as a test solute for the UV-visualizationtechnique because the chromatographic determination of aliphatic acids presents a difficult challenge, 0003-2700/84/0358-0487$0 1.50/0
For LC analyses, low detection sensitivities are often obtained due to poor UV-absorption properties of the sample. Additional problems include variable ionization during separation and strong interaction with LC supports (12). Precolumn derivatization is often used to avoid these drawbacks (13) at the expense of additional sample preparation time. By addition of the UV-absorbing ion-pairing reagent cetylpyridinium chloride (CPC) to the reversed-phase eluent, retention of free aliphatic acids is effected by a paired-ion mechanism’ and detection is facilitated at 254 nm by the coelution of reagent and sample ions. Neither precolumn nor postcolumn reactions are involved.
EXPERIMENTAL SECTION Apparatus. The chromatographic system was a Waters Associates, Inc. (Milford,MA), Model 204 ALC which included a WISP automatic injector, a Model 6000A solvent delivery system, and a Model 440 absorbance detector operating at 254 nm. Automated runs were directed by a Model 720 system controller (Waters). The analog outputs of the UV absorbance detector were recorded with a Model 730 Data Module (printer, plotter, integrator) (Waters). For the quantitation of peaks showing negative UV absorbance at 254 nm (see text) the leads to the Data Module were reversed. A pBondapak C18 column (3.9 mm i.d. X 30 cm) (Waters) was temperature controlled at 33 “C using a circulating water bath (Haake FE2, Waters) and a column temperature control block (Waters). The time equivalent ( t o ) of the void volume (Vo)was determined by injecting 10 pL of methanol and confirmed by injecting 10 p L of water. Reagents and Mobile Phases. The salts used in this study were purchased from Fisher Scientific (Fairlawn, NJ). Cetylpyridinium chloride was obtained from Pfaltz and Bauer Chemical Co. (Stamford, CT). Purified water, obtained from a Milli-Q System (Millipore, Bedford, MA), and chromatographic grade methanol (Waters) were used for eluent preparation. Mobile phases were filtered through a 0.45-~mcellulose acetate filter (HSWP 04700) (Millipore,Bedford, MA) and degassed before use in an ultrasonic bath with the solution under vacuum. Sodium valproate was obtained from Abbott Laboratories (North Chicago, IL) and used without further purification. Decanoic acid and phenylbutyric acid (Aldrich, Milwaukee, WI) were used as received. Samples were filtered through 0.5-pm Millex SR filters (Millipore) prior to injection. RESULTS AND DISCUSSION Effect of Eluent Ionic Strength upon Sensitivity and Retention Time. In a previous report (11)we discussed the general trends in retention for alkylsulfonatesas mobile phase ionic strength is varied. Due to the observed sigmoidal dependence of the amount of CPC adsorbed onto the C18stationary phase on the logarithm of ionic strengh, we predicted that the extremes of ionic strength (corresponding to plateau regions of the sigmoid curve) would provide the most stable chromatographic performance. Two eluents were chosen with this prediciton in mind. A “no salt” mobile phase (55% methanol, 45% water, 0.16 mM CPC) and a “high salt” mobile phase (60% methanol, 40% water, 0.16 mM CPC, 5 mM KC1) were used throughout this study. For UV-visualization, a simple rule can be used to predict the type of peak (positive or negative) to expect for an injected 0 1984 American Chemical Society
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Typical chromatograms for sodium valproate analysis by UV-visualization chromatography: no salt eluent is 55 % methanol, 4 5 % water, 0.16 mM CPC; high salt eluent Is 6 0 % methanol, 4 0 % water, 0.16 mM CPC, 5 mM KCI. Flgure 1.
1412” No salt 10Peak Area [ + 1031
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Flgure 2. Calibration curves for sodium valproate standards. Chromatographic conditions are given in Figure 1.
sample species. When the sample and reagent are of opposite charge, the sample peak will have a positive UV absorbance whenever the sample elutes after the induced reagent peak (which will be negative). The opposite is also true: a sample which elutes before the reagent will yield a negative peak which precedes the positive reagent peak. Both cases of this rule are seen in the present study. For the no salt mobile phase, the positive peak for NaV elutes shortly after the negative CPC peak. Conversely, with the high salt eluent the NaV peak is negative and elutes several minutes before the positive reagent peak. Figure 1 shows typical chromatograms for NaV standards in the two eluents. The overall magnitude of the peak areas is considerably less in the high salt mobile phase when compared to no salt conditions. This is clearly reflected in Figure 2, which shows calibration lines for NaV standards in both eluents. The slope of the calibration line for the high salt eluent is approximately four times less than that for the no salt eluent. The reduced response must arise from a lack of coelution of CPC with the sample which might occur in the high salt eluent because potassium cations can coelute with NaV to some extent. Despite the decreased peak areas in the high salt eluent (HS), linearity is slightly better than for no salt (NS) (RHS= 0.999 vs. RNS = 0.994) and an acceptable limit of detection of 50 ng injected was obtained for NaV without any special effort to optimize sensitivity. Choice of eluent ionic strength affects peak shape as well. In the no salt mobile phase peaks are relatively symmetrical for both NaV and CPC. As increasing concentrations of NaV are injected, the CPC peak tails more, while the NaV peak fronts more significantly. For the high salt eluent, substantial tailing is observed for the NaV peak at all levels injected.
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Flgure 3. The effect of injected sodium valproate concentration on retention time for 50-pL injections. Chromatographic conditions are given in Figure 1.
Interestingly, the sample and reagent peaks tail toward each other in both eluents. The significance of this observation is not yet clear. Figure 3 shows the stability of the retention time (tR)for NaV as a function of its concentration. All injections were 50 pL. In both eluents, t~ varies with NaV concentration, but the overall variation is substantially less in the high salt eluent. The 1.3 min variation observed in the no salt mobile phase is reduced to less than 0.3 min for the high salt mobile phase. It is interesting to note that a linear calibration curve is obtained for the no salt eluent despite the large variation of t R with NaV concentration. The observed variation is presumably an ionic strength effect, based on the difference in ionic strength between the sample and the no salt eluent. In the presence of a high background ionic strength (high salt eluent) the effect is minimzed and t R is more stable. The exact nature of this effect is discussed more fully in the following sections. It should be noted that the observed variations in t R do not follow the behavior previously observed for a non-UV-visualization system (14) using a styrene-divinylbenzene stationary phase. In that study, t R was shown to vary inversely with the concentration of analyte injected. Effect of Sample Solvent upon Sensitivity. A previous study of quantitation by UV-visualization (5) demonstrated that equally good calibration lines can be obtained from constant volume injections of varying sample concentrations or from varying volume injections of a single sample concentration. However, the calibration line for constant volume injections more nearly passes through the origin (5). This work confirmed these observations and we chose to use constant volume injections (50 pL) for the present study. The solvent in which the sample is dissolved was also found to have a significant impact on the resulting chromatography for the UV-visualization method. Injection of a single-component sample generally leads to the production of three peaks, all of which are due to depletion or enrichment of the UV-active reagent in a particular zone of the eluting mobile phase. One of the peaks elutes at a retention time which is characteristic of the sample and is due to coelution of reagent with the sample (2). The other peaks occur as a pair of “induced vacancy” peaks (9, 15) similar to those shown in Figure 4 for the high salt eluent. Figure 4A shows a blank injection of the high salt mobile phase. In Figure 4B, 100 pL of the strong solvent methanol has been injected. This causes local desorption of CPC reagent upon injection, followed by rapid elution of an enriched band of mobile phase at the void volume and the later elution of a deficient band of mobile phase at the retention time characteristic of CPC. Injection of the weak solvent water has the opposite effect (Figure 4c)
ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
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Flgure 4. Induced palrs of peaks produced by the injection of various solvents but no sample: (A) 200 pL of high salt eluent, (B) 100 pL of methanol, (C) 200 pL of water, and (D) 200 pL of high salt eluent splked wlth additional KCi. Chromatographic conditions are given in Figure 1 (high salt eluent).
Y-'f
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as does the injection of mobile phase spiked with additional KC1 (Figure 4D). This last case is most revealing for the situation generally encountered in paired-ion chromatographic separations, in which a sample consisting of multiple ionic species is typically injected. When the sample is dissolved in mobile phase, it will necessarily have a greater ionic strength than the bulk eluent due to the presence of ionic sample species. If the sample is dissolved in the weak solvent of the eluent, the situation of Figure 4C will arise which cannot be visually distinguished from Figure 4D. In this study, we observed no difference in performance between samples dissolved in water and identical samples dissolved in the mobile phase. Effect of Sample Ionic Strength upon Sensitivity and Retention. It has been noted (5)that the injection of a mixture of alkylsulfonates results in reduction of the peak area response for either pentanesulfonic acid or hexanesulfonic acid in comparison to the responses obtained when identical quantitites of these analytes are injected alone. Since it is possible that ionic species other than NaV will be present in samples, e.g., from human plasma, several experiments were conducted to determine the influence of various mixtures of ionic species on the stability of retention time and peak area for NaV. In the first study, the influence of increasing concentration of heptane- and octanesulfonic acid (HSA and OSA) on a constant injected amount of NaV was considered. In the second experiment, the influence of added NaCl on the retention time and peak area of NaV was investigated. Finally, the effect of added CPC reagent was studied. By addition of lipophilic anions (HSA and OSA), a lipophilic cation (CPC), and inorganic ions (NaC1) a range of potential interferents could be considered in a systematic manner. The influence of added HSA and OSA is indicated in Figure 5 for the no salt eluent. In the presence of these lipophilic anions, the signal for NaV is seen to steadily decrease as the added quantity of the sulfonates increases from 0 mM to 0.16 mM. Peak area (Figure 5A) and peak height (Figure 5B) are similarly affected. The series of standards was run in duplicate, and the lines in Figure 5 pass through the average values for the two runs. The run-brunvariation is substantial for both peak area and peak height. The range of peak area variation is 12.8% of the average peak area value obtained. For peak height, a relative variation of 10.7% was found. Figure 5C indicates the variation in retention time with increasing concentration of the sulfonates. The overall variation is 0.26 min. Interestingly, the run-to-run variation in retention time was much improved in the presence of the higher concentrations of alkylsulfonates. Figure 6 presents combined data for the influence of constant concentrations of NaCl and CPC on the calibration line
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Flgure 5. The effect of increasing concentration of octane- and heptanesulfonate ions on the response for a constant injected amount of NaV (1.70 pg per 50-pL injection): (a) effect on peak area, (b) effect on peak height, and (c) effect on retention time. I
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A NaV f CPC o NaV t NaCl
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Flgure 6. Calibration curves for sodium valproate standards wtth and without added Ionic species (see legend). Upper lines are for the no salt eluent. Lower line Is for the high salt eluent.
for NaV. As was the case for OSA and HSA, both CPC and NaCl cause a reduced response for N a V in the no salt eluent. The effect of added NaCl at a constant level of 36 mM was less dramatic than that of the added CPC at 0.55 mM, indicating that ionic strength of the sample alone does not suffice to explain the observed variability of peak area and retention time. It appears that the lipophilic nature of the added ions also contributes significantly to reduction of the peak area response for NaV. The net result is a significant uncertainty in the NaV signal for the no salt eluent depending on what ionic species are present in the sample. Since this is not a
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984 A
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Flgure 7. Chromatograms in the no salt eluent illustrating the effect of increasing concentration of NaV on the peak area for a constant injected amount (0.74 p g per 50 pL Injection) of the internal standard phenylbutyric acid (PhBAj. Amount of NaV injected increases from left to right: 1.38 pg, 4.14 pg, 8.27 pg.
variable that can generally be controlled in real world samples, it appears that UV-visualization will not be an acceptable method of quantitation for the no salt eluent. This is reflected in the calibration "wedge" seen in Figure 6 for the no salt eluent. Fortunately, the variations in the NaV peak area response are substantially reduced when the high salt eluent is used. The lower calibration line of Figure 6 reflects this, as one line passes through all three sets of points with a correlation coefficient of 0.988 and a y intercept very near the origin. The comparable line for the combined data in the no salt eluent has a correlation coefficient of 0.975 and intercepts they axis at a point which is 18 times as far from the origin. Interestingly, it appears to be possible to trade sensitivity for stability in the present system. By selecting the high salt eluent, t R becomes nearly independent of sample concentration (Figure 3) and of competing ionic sample components (Figure 6). The slope of the calibration curve is substantially reduced, but the sensitivity which remains would be adequate for the analysis of the NaV at parts-per-million levels. Choice of Internal Standard. A further test was conducted to verify the utility of the high salt eluent for reliable quantitation by UV-visualization. For each eluent, an appropriate carboxylic acid internal standard was chosen on the basis of retention time from compounds which were judged appropriate and which were available in the lab. For the no salt eluent, a compound was desired which would elute before NaV or slightly after, to keep the analysis time near 10 min. Since sample peaks are positive UV signals in the no salt eluent, the sample could be a UV-absorbing carboxylic acid, and phenylbutyric acid was chosen. For the high salt eluent, however, sample peaks are due to a diminished UV absorbance (compared to the background absorbance of CPC) as the sample elutes. Thus, a non-UV-absorbing internal standard was required and decanoic acid was chosen. For each internal standard, a series of six samples was prepared for which the concentration of the internal standard was held constant while the concentration of NaV was varied. Figure 7 presents three representative chromatograms for the no salt eluent. A serious problem is immediately apparent. As the injected amount of NaV increases, the peak for phenylbutyric acid (PhBA) gradually disappears into the base line, despite the fact that a constant quantity of PhBA is injected in each case. This confirms the previous conclusion that UV-visualization with a no salt eluent is entirely unacceptable for quantitation. Figure 8 emphasizes this conclusion. In Figure 8A, the NaV linearity is quite good, but the response for PhBA deviates significantly from the expected horizontal line. The internal standard calibration plot (Figure 8B) is decidedly nonlinear and useless for calibration or quantitation. For the high salt eluent no comparable problem was found, and the calibration plot showed excellent linearity (R = 0.999). Retention times for both NaV and decanoic acid showed good stability as well, thus indicating further that the high salt
25
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Flgure 8. Calibration curves in the no salt eluent for NaV standards
containing a phenylbutyric acM (PhBA) Internal standard (A) calibration curves for NaV and PhBA, (B) callbration curve according to the internal standard method.
Table I. Day to Day Reproducibility: No Salt Eluent ( % RSD, N = 4) p g of NaV area (NaV) 5.82 7.3 4.85 9.3 3.88 11.6 2.91 3.4 1.94 16.2 0.97 30.8 13.1 av Table 11. Day to Day Reproducibility: High Salt Eluent (% RSD, N = 5 ) Pi2 of area area NaV (NaV) (decanoic) ratio 5.47 8.8 7.8 1.6 4.69 8.2 10.6 6.2 3.91 5.6 7.3 4.9 3.13 7.8 8.6 6.0 2.34 7.4 10.2 7.2 1.56 7.3 9.2 5.0 av 7.5 9.0 5.2 eluent provides the stability needed for good quantitation. Precision of Analysis. Day to day reproducibility is an important characteristic for a reliable quantitative method. To investigate the reproducibility of analysis available with the no salt and high salt eluents, two sets of standards were prepared, one for each eluent. The standards for the high salt eluent contained a decanoic acid internal standard, but no internal standard was used in the standards intended for the no salt eluent for the reasons mentioned previously. Each set of six standards was analyzed repeatedly over a period of 3 days. The reproducibility statistics are presented in Table I for the no salt eluent and Table I1 for the high salt eluent. The relative standard deviation (RSD) of the area measurements is seen to be more consistent for the high salt eluent, despite the fact that much larger peaks are obtained with the no salt eluent. In addition, the average RSD for NaV peak area is much improved for the high salt eluent although these values are still poor compared to the precision which can often be achieved in LC analyses. Use of the internal standard ratio adds further improvement to the average precision, as expected. Overall precision for the retention time of sodium valproate was 4.3% in the no salt eluent and 2.9% in the high salt eluent.
Anal. Chem. 1984, 56, 491-497
CONCLUSION For the UV-visualization analysis of sodium valproate, an eluent containing 5 mM KC1 has been shown to provide chromatographic performance which is superior to what is obtained by using an eluent containing no added salt. Stability of retention time and peak area, freedom from interference by ionic sample components or sample solvent, compatibility with the internal standard method, and precision of analysis are all benefits of using the high salt eluent. The implications of these findings for paired-ion chromatography in general are intriguing. Similar effects of ionic strength may be present for the usual paired-ion systems but might not ordinarily be noticed because the ion-pairing reagents are detector-transparent. Further research in this area is ongoing in our laboratory. ACKNOWLEDGMENT The authors wish to thank K. Bergeron and J. Newman for assistance in preparing the manuscript and P. Watson for preparation of the figures. LITERATURE CITED (1) Bidiingmeyer. B. A.; Deming, S. N.; Price, W. P.; Sachck, B.; Petrusek, M. J. Paper presented at Advances in Chromatography, 14th Interna-
49 1
tionai Symposium, Lausanne, Switzerland, Sept 24-28, 1979. Bidlingmeyer, 8. A,; Deming, S. N.; Price, W. P., Jr.; Sachok, B.; Petrusek, M. J . Chromatogr. 1079, 186, 419. Bdilngmeyer, B. A. J . Chromatogr. Scl. 1080, 78, 525. Bidiingmeyer, 6. A.; Deming, S. N.; Sachok, B. Poster presented at 13th International Symposium on Chromatography, Cannes, France, Julv 2. 1980. Sachok, B.; Deming. S. N.; Bdilngmeyer, B. A. J . Liq. Chromatogr. 1082.5.389. Paris,’ N.’ Anal. Blochem. 1070, 100. 250. Paris, N. J . Liq. Chromatogr. 3 , 1743. Helboe, P. J . Chromatogr. 1083, 261, 117. Denkert, M.; Hackzeii, L.; Schiii, G.; Sjogren, E. J . Chromatogr. 1081, 218, 31. Barber, W. E.; Carr, P. W. J . Chromatogr. 1083, 260, 89. Bidiingmeyer, B. A.; Warren, F. V., ;r. Anal. Chem. 1082, 54, 2351. Snyder, L. R.; Kirkiand, J. J. Introduction to Modern Liquid Chromatography”; Wiiey-Interscience: New York, 1974. Durst, H. D.; Miiano, M.; Kitka, E. J., Jr.; Connelly, S. A.; Grushka, E. Anal. Chem. 1075, 4 7 , 1797. Iskandarani, Z.; Pietrzyk, D. J. Anal. Chem. 1082, 5 4 , 1085. Stranahan, J. J.; Deming, S. N. Anal. Chem. 1882, 54, 1540.
RECEIVED for review January 25,1983. Resubmitted July 28, 1983. Accepted October 31,1983. Portions of this work were presented at the Fall 1982 ACS Meeting (KansasCity, MO), at the 1982 FACSS Meeting (Philadelphia, PA), and at the 1983 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Atlantic City, NJ).
Post-Column Reaction Detector for Platinum(I I) Antineoplastic Agents Kennan C. Marsh,l Larry A. Sternson,* and Arnold J. Repta Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045
The development and evaluation of a post-column reaction detector sensitive to piathum( 11) complexes Is presented In which sodlum bisulfite Is used as the derivatlzlng agent wlth potassium dlchromate as an activating agent. The Influences of mobile phase changes (Le., pH, organic modlflers, electrolytes), oxygen, metal ions, and order of reagent addition on reactlon klnetlcs and product yield are defined and used 290 In optlmizatlon of detector response. Detectlon at A, nm results In an on-ilne post-column sensltivlty of 40-60 ng/mL for selected cls-dichloroplatinum complexes and a sensltlvity of 300-1200 ng/mL for four (substituted)malonato-piatlnum complexes. The reactlon detector Is used to monitor the klnetlcs of aquation ol clsplatln (CDDP) and to quantitate CDDP degradation In plasma. As the sensHlvity for CDDP in plasma is comparable to that achleved from HPLC effluent fractlonatlon/off-line flameless atomlc absorption (AAS) quantltatlon, slgnlficant utillty for this time-efflclent post-column reactor in cilnlcal analysis is suggested.
Currently the most widely accepted analytical procedure for quantitation of cisplatin (CDDP) (1-4) utilizes initial HPLC separation of the platinum species followed by off-line quantitation of the platinum in each fraction with flameless atomic absorption (AAS) (5-8). For time-efficient application in clinical analysis, the need for a selective direct on-line detector system was indicated. Recently, on-line electrochemical detection of HPLC effluent was shown to be useful Present address: Abbott Labs, D-493,North Chicago, IL 60064. 0003-2700/84/0356-0491$01.50/0
in the sensitive quantitation of both CDDP and CHIP (Table I) in urine samples but was not responsive to the other platinum antineoplastic agents investigated (9, 10). Spectrophotometric monitoring of column effluent is probably the preferred detection system in HPLC, but due to the poor molar extinction coefficients of the platinum(I1) compounds, a derivatization reaction was required to provide the sensitivity needed for clinical application. Nucleophiles which react rapidly with platinum(I1) would convert a variety of platinum compounds (differing in ligand composition) to a common final product (11). Therefore, to maintain the selectivity of the system, chromatographic separation of the underivatized compounds (12,13) must precede derivatization. In earlier studies of the reaction between CDDP and N,N’-bis (3-dimethylaminopropyl)dithiooxamide (DTO) (14 ) , a dramatic rate enhancing effect was observed when CDDP was exposed to (bi)sulfite prior to reacting with DTO. Hussain et al. (15) found that (bi)sulfite rapidly degrades cisplatin, resulting in product(s) exhibiting increased absorbance a t 290-300 nm. The rapid rate of reaction to form chromophoric product(s) indicated potential applicability of bisulfite as a derivatizing reagent in a post-column reactor. Variables affecting the CDDP/HS03- reaction were optimized and considered in the design of the post-column reactor. The response of the reactor is maximized, using CDDP as the model platinum compound, and then evaluated for a series of substituted platinum(I1) antineoplastic agents.
EXPERIMENTAL SECTION Apparatus. Spectrophotometrickinetic studies and spectral characterization of platinum compounds were obtained with a Perkin-Elmer (Norwalk,CT) 555 UV/vis spectrophotometer with 0 1984 American Chemical Society
