Anal. Chem. 1994,66, 1432-1437
Optimization of Flame-Based Sulfur Chemiluminescence Detection with Microcolumn High-Performance Reversed-Phase Liquid Chromatography A. L. Howard,? C. L. 6. Thomas, and L. T. Taylor'
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 2406 1
Through a detailed optimization process, response surfaces were obtained for micro-HPLC/SCD under various reversedphase (methanol/water) elution conditions. Linear dynamic range, correlation coefficient, response factor, and limit of detection were found to vary with mobile-phase composition. The lowest sensitivity achieved was a detectivity of 600 fg of S/s at a mobile-phasecompositionof40%methanol/60%water. Application of the HPLC/SCD interface to the separation of thiocarbamates extracted from apples is presented.
Supercritical fluid chromatography coupled with flamebased, ozone-promoted sulfur chemiluminescene detection (SFC/O3-SCD) has beed demonstrated as one means of analyzing thermally labile sulfur-containing compounds because relatively mild temperatures (30-100 "C) are employed to achieve their Unfortunately, SFC/ SCD is not applicable to the analysis of many polar sulfurcontaining compounds due to the nonpolar nature of the S F C02 mobile phase. The use of organic-modified C02 in packed-column SFC has been shown to be feasible, but chromatographic integrity was poor compared to that seen when pure C02 was utilized as the mobile phases3 A logical extension of methanol-modified SFC/SCD is SCD interfaced to reversed-phase-mode HPLC.'s4 In addition to being thermally mild, HPLC can also accommodate analytes of higher molecular weight and polarity than SFC. Ultraviolet/visible absorbance is the most commonly used HPLC detection scheme due to its simple interface with the liquid mobile phase via a flow-through cell. Problems arise, however, in detecting analytes that lack a chromophore. In addition, UV/visible absorbance is not analyte specific; other absorbing species could potentially interfere with chromatographic analysis. The use of smaller inner diameter HPLC columns (i.d. CO.5 mm) (termed micro-HPLC) has led to greater compatibility between micro-HPLC and the gas-phase detectors routinely used in SFC and GC to detect sulfur-containing analytes.s,6 The reduction in column inner diameter translates into lower mobile-phase flow rates and an added advantage Current address: Merck Research Laboratory, West Point, PA. ( 1 ) Chang, H.-C. K.: Taylor, L. T. J . Chromazogr. 1990, 517, 491-501. (2) Pekay, L. A.; Olesik, S.V.J. Microcolumn Sep. 1990, 2, 27C-277. (3) Howard, A. L.; Taylor, L. T. Anal. Chem. 1993, 65, 724-729. (4) Howard, A. L.; Taylor, L. T. J . High Resolut. Chromatogr. 1991, 1 4 , 785794. (5) McGuffin, V. L.; Novotny. M. Anal. Chem. 1981, 53. 946-951. (6) Kientz, C. E.; Verweij, A.;DeJ0ng.G. J.; Brinkman, U. A. Th. J . High Resolut. Chromarogr., 1989, 12, 193-796.
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Analytical Chemistry, Vol. 66, No. 9, May 1, 1994
of decreased organic solvent consumption. Micro-HPLC/03-SCD was shown to be feasible by Chang and Tay10r.~Sample transport to the detector was found to be controlled by the micro-HPLC/SCD interface temperature. For volatile to moderately volatile analytes, an elevated detector temperature (350 "C) was necessary since vaporization was the dominant sample transport phenomenon. Conversely, nonvolatile compounds were found to require an interface temperature lower than that of the mobile-phase boiling point because aerosol formation governed sample transport. At higher detector temperatures, nonvolatile analyte deposition in the transfer line connecting the column and the interface occurred due to premature solvent evaporation. Because of the combustible nature of the micro-HPLC mobile phases employed, SCD sensitivity was shown to vary with mobile-phase composition. As the organic concentration in the mobile phase increased, the air flow rate needed for maximum sensitivity also increased. Response factors were found to be 1 for several sulfur-containing compounds under identical elution conditions. The results presented here build upon the findings obtained by Chang and Taylor' and Howarda4 A methodical optimization procedure was used to maximize the sensitivity of the SCD in the micro-HPLC mode. Mobile-phase composition, air flow rate, and hydrogen flow rate were investigated. Detector performance was then assessed under the optimized flame conditions. A separation of thiocarbamate pesticides extracted from an apple is given as an application.
-
EXPERIMENTAL SECTION Instrumentation. A schematic of the micro-HPLC/SCD system is given in Figure 1. The 15 cm X 320 wm (Ld.) CB column (LC Packings, Amsterdam, Holland; d , = 5 pm)was connected directly to a helium-actuated injection valve (Valco, Houston, TX) which contained a 60-nL injection rotor. The column effluent was fed into a flame ionization detector (FID) via a 20 cm X 50 pm (i.d.) segment of deactivated fused silica (SGE, Inc., Austin, TX) which was placed approximately 8.6 cm into the FID jet. A Waters MS-600 quaternary gradient reciprocating HPLC pump (Milford, MA) was utilized to deliver the mobile phase at conventional HPLC flow rates (5-0.5 mL/min). A preinjector split where two analytical (7) Chang, H.-C. K.; Taylor, I.. T. Anal. Chem. 1991, 63, 486-490.
0003-2700/94/0366-1432$04.50/0
0 1994 American Chemlcal Society
To VMum pump
~~
~
Table 1. Mlcro-HPLC/SCD Optlmlratlon Study Condttloni
parameters mobile-phase composition air flow rate hydrogen flow rate mobile-phase flow rate probe molecqlb/solvent amount injected injection volume column probe height
anprt Flgure 1. Micro-HPLC/SCD system schematlc: (A) pump controller, (e)host pump, (C) spllttlng apparatus, (D) injection valve, (E) packed capillary column, (F) oven, (0)fused silica transfer Ilne, (H) ceramic probe, (I) FID jet, (J)ozone generator, (K) chemiluminescencereaction cell, (L) photomultiplier tube, (M) SCD, (N) SCD transfer line, and (0) FID block.
scale columns are utilized to maintain a constant back pressure was used to generate microflow rates (1-20 pL/min.). The split microflow rate was determined to be directly proportional to the conventional-scale (host) pump pressure. When premixed mobile phases were employed, the unused split from the host pump was recycled back into the mobile-phase reservoir rather than to waste. The operation of the O&CD (Sievers Research, Inc., Boulder, CO) was similar to that previously except that the FID block temperature was set at 50 OC. In actuality, however, block temperature varied from 50 to 80 OC as a result of heat generated by the combustion process. Approximately 10-12 psigrade 4.3 oxygen (Airco, Inc., Radford, VA) served as the ozone precursor. SCD output was recorded with a Model SP4290 Spectra Physics integrator (San Jose, CA). Air and hydrogen flow rates were measured with Top Trak (Sierra, CA) mass flow meters in standard cubic centimeters per minute [cm3(STP) m i d ] . An Edwards Model 1.5 vacuum pump was used to maintain subambient pressure conditions in the SCD reaction cell. Since the SCD exhaust contains unreacted ozone, a trap was placed prior to the vacuum pump. The pump exhaust was vented to a fume hood in order to prevent ozone from pervading the laboratory. Chemicals. All mobile-phase components (HPLC grade methanol, acetonitrile, and water; Fisher, Pittsburgh, PA) were vacuum filtered prior to use with 0.22-pm pore size Millipore hydrophilic membrane filters (Milford, MA) and degassed with helium. Analyte solutions were also prepared with HPLC grade solvents and filtered with 0.45-pm Teflon filters (Fisher). The thiocarbamate pesticides and carbon disulfide were obtained from Chem Services (West Chester, PA) and Matheson, Coleman and Bell (Norwood, OH), respectively. RESULTS AND DISCUSSION Detector Optimization. There are many physical parameters which must be carefully monitored in order to take full advantage of the flame-based ozone-promoted SCD's sensi-
conditions 0-100% methanol in water, 10%incrementa 200-400 cma(STP) min-' in 25 cma(STP) min-l 100-400 cma(STP) min-1 in 50 cm*(STP)min-1 incrementa 7 pllmin CSZin methanol 30 ng of CS2 (25 ng of S) 60 nL
15 cm X 320 pm i.d. Cla
4 mm above FID jet
tivity and selectivity. For example, optimal probe height as well as probe concentricity with respect to the flame must be considered in addition to the flame composition. Many of these parameters could not be adjusted by other a ~ t h o r s l * ~ due to a poor one-piece probe holder design. For this earlier probe system, concentricity was assessed visually and then the probe was placed in the FID tower. Placement of this one-piece assembly inside the FID without bumping the probe against the FID housing or collector electrode was impossible. Currently, concentricity of the probe is maintained by systematically changing the depth of three set screws on the base of a two-piece probe holder assembly. After each adjustment of the set screws, an injection of methomyl was made and the SCD response was monitored. This procedure was continued until the optimum SCD response was obtained. Once centered, this base could be permanently fixed to the FID housing. The goal of this detailed optimization study was to map the overall response of the SCD in reversed-phase microHPLC mode. Through this mapping procedure, it should be possible to determine at which mobile phases and flame compositions the SCD is most sensitive, linear, and selective. Conditions for the mapping study are given in Table 1. Carbon disulfide was used as the probe molecule in order to shorten analysis times since it was unretained on the C18 column under all elution conditions. Total gas flow rates varied from 300 to 800 cm3(STP) min-l, which in most cases met or exceeded the capacity of the vacuum pump. A minimum of 54 experiments were conducted to construct each surface. Peak area reproducibility was not assessed for each individual set of conditions, but was assessed at the beginning and end of each day to be 3 4 % (n = 5). Microsoft Excel was used to generate the 3-D surface plots. Overall, the SCD response under all elution conditions was shown to be equally dependent on both hydrogen and air flow rates, contrary to the results of Chang.' Many of the surfaces, despite differences in mobile-phase composition, appear to have the same general shape (Figures 2 and 3). In addition, when mobile phases with 30% or greater water content were utilized, the surfaces appear flatter with a single maximum per surface, whereas compositions with higher methanol content have steeper, multiple maxima (Figures 4 and 5 ) . The presence of increasing amounts of methanol in the mobile phase may afford sufficient fuel to the flame, thereby enhancing SO production with the result that other response maxima are created. On the other hand, since the flame conditions used often produce incomplete combustion, Ana/yticai Chemistty, Voi. 66, No. 9, May 1, 1994
1433
Table 2. Mkro-HPLC/SCD Opthnal Flame Gas CondltlaMI mobile-phase comp optimal optimal total (MeOH/water) air f l o e hydrogen flow gas flow
.
.
. -..
..-.
4.-.
150
0' _. -_,100 200 225 250 275 300 325 350 375 4W 425 450 ~
~
A i r Plow Rata (SCCN)
Flgure 2. Micro-HPLC/SCD response surface for 60 % methanol/ 40% water mobile phase. Conditions as in Table 1.
ow Rate
0 1 200
--, YE.
'
. 25.0
275
ux) 325
Air Flow Rata
3m 375
I
-200 150 loo
400
36W260
4261300
9
H:"~~?;?E~5z~3;x ?low Rate (BCC)()
Flgure 4. Micro-HPLCISCD response surface for 90 % methanol/ 10% water mobile phase. Conditions as in Table 1.
100000FO
m 0
3000006
ayarogon ? l o r Rat0
2000000
(Sen)
0
Air P l o w Xata (BCQO
Flgure 5. Micro-HPLC/SCD response surface for 100% methanol mobile phase. Conditions as in Table 1.
the presence of uncombusted organic species may selectively enhance chemiluminescence quenching. Similar response surfaces were determined by Pekay and Olesik2 for open tubular column SFC/SCD and GC/SCD where carbon disulfide was the probe molecule. These surfaces, however, were generally flatter (contain broader maxima) than those obtained here for micro-HPLC/SCD. Sharp maxima on the 1434
27W200
AWH, MI6 1.42
(aCCX)
Peak A r m 5UXkXxl
I
A l r m a g . n F l m Rat"
Flgure 3. Micro-HPLC/SCD response surface for 80% methanol/ 20 YO water mobile phase. Conditions as in Table 1.
Atr
500 600 600 600 550 525 550 550 550 975 725
In cm3(STP) min-l.
I"
t
250 400 400 400 350 200 350 350 350 400 300
200 200 200 200 200 325 200 200 200 575 425
0/100 10190 20180 30170 40160 50150 60140 70130 80120 90110 100/0
AnalyticalChemistry, Vol. 66,No. 9, May 7, 1994
Flgure 6. Effect of total air plus hydrogen flow at fixed ratio on MicraHPLC/SCD sensitivity. SCD responses were obtained from the 100% methanol response surface.
micro-HPLC/SCD surfaces indicate that finer control over flame gas flow rates is necessary for optimal SCD operation. Table 2 summarizes the air and hydrogen flow rates at the absolute SCD sensitivitymaximum for each response surface. For most of the mobile-phase compositions, the sensitivity maximum was obtained at high hydrogen (-400 cm3(STP) min-l)/low air flow rates (-200 cm3(STP)min-l) and a total gas flow rate of 500-600cm3(STP)min-l. The mobile-phase compositions of 50/50,90/ 10, and 100/0 (methanol/water) required flame composition ratios of 600 cm3(STP) min-l for maximal sensitivity. According to Pekay and Olesik,2 the absolute maxima for GC/SCD and SFC/SCD occurred at lower hydrogen (- 150 mL/min) and air (-250 mL/min) flow rates and at total gas flows of -400 mL/min. Theoretically, the reducing character of the flame should be identical at thesame fuel/oxidant ratio and therefore should produce the same amount of SO which is crucial to the chemiluminescentreaction. In constructingthe micro-HPLC/ SCD response surfaces, the contrary was found to be true. Various SCD responses under constant elution and flame composition ratio are given in Figure 6. Obviously, from this comparison, the fuel/oxidant ratio is not the only factor governing SO production. As the total gas flow (e.g., constant gas ratio) to the flame increases so does the SCD response, which indicates that transport of the flame products to the SCD is also important. For example, the increased hydrogen flow rate (added concentrically to the micro-HPLC mobilephase flow) may serve to improve sample transport to the flame as a result of better eluent nebulization. A similar trend was observed for the 90/ 10 mobile-phase composition.
Table 3. Micro-HPLC/SCD Calibratlon Curve Comparison
mobile-phase comp (MeOHIwater)
slope (m)
corr coeff (r)
LDRa
O( 100 10190 20180 30170 40160 50150 60140 70130 80120 90110 100/0
4682 3023 2779 1725 1231 1047 1429 1197 813 1627 668
0.999 63 0.991 6 0.999 66 0.998 65 0.999 88 0.998 9 0.999 3 1.000 0 0.999 91 0.998 23 0.999 85
1.4 1.4 1.4 1.4 2.4 2.7 2.7 3.2 2.7 2.4 2.4
8cD BaQkgrOUPa
8iqnal
(mv)
Ryarogen FlOU Rat. (SCCM) 01
--
475 450
I 425
400
375 350 325
Air Flow Rate
3w 275 250 225 200 (SCCN)
a
4CCl
375
3M
325
3W
275
2M
225
Linear dynamic range.
2w
M r Plow Rata (scat)
Figure 7. SCD backgroundsignal vs flame gas composition response surfaces for (A) 100% methanol and (B)40 % methanoV60% water mobile phases.
Investigation of these trends was not possible with the other mobile-phase compositions due to gas flow rate constraints. In addition to monitoring the CS2 response, the SCD background signal and SCD reaction cell pressure were monitored for every set of conditions examined. In general, these surfaces were of two general shapes. For mobile-phase compositions of 100/0, 90/ 10, and 50/50, the background signal surfaces contained an absolute maximum centered around the optimal flame gas compositions (Figure 7A). The other SCD background signal surfaces (Figure 7B) showed little variation with flame composition. Background signal surfaces obtained by Pekay and Olesik2 for GC and SFC/ SCD have shapes similar to those in Figure 7A. There appears to be little correlation between reaction cell pressure (17 f 5 Torr) and the gas flow rates used under optimal SCD conditions. Detector Performance. The micro-HPLC/SCD system was evaluated by determining linear dynamic range (LDR), response factors, selectivity, and detection limits under each of the mobile-phaseand flame gas compositionsgiven in Table 2. Calibration curves (peak area vs mass S injected) were based on an average SCD response (n = 3-4 injections/ concentration level) in the concentration range 50 ng of S to 50 pg of S. At least three concentration levels were used for each curve. A general decreasing trend (Table 3) in response factor as measured by the slope, m,with increasing methanol concentration in the mobile phase was observed. Since organic species have been shown to be better quenching agents than water,* this trend was expected . This trend also illustrates that the SCD is not equimolar when different elution conditions are compared. The correlation coefficient was determined to be at least 0.99 for all mobile phases. The LDR was at its highest value with compositions from 50/50 to 80/20. One of the most important reasons for investigating the micro-HPLC/SCD detection system was sulfur selectivity, which is not available with any conventional HPLC detection technique. Detector selectivity was assessed to be as high as lo6 (SCS2 to methanol) for mobile-phase compositions with ~
70% or greater methanol content and as low as lo2for mobile phases with 60%or less methanol. The selectivity decrease is due to the fact that a response was obtained from the injection solvent (methanol). Two reasons for this injection solvent response under these mobile-phaseconditionsappear plausible. First, there may be a low-level sulfur contaminant in the methanol which was only detected at these mobile-phase compositions due to increased sensitivity. Second, the methanol (or a contaminant dissolved in it) could be forming a chemiluminescent species upon combustion and reaction with ozone which is detected within the SCD bandpass. Response of injection solvent has been previously noted by Chang and Taylor’ in SFC/SCD analysis as well. In addition to being sulfur selective, the SCD must also be very sensitive (low detection limits) in order to compensate for the small injection volumes (60 nL) employed in microHPLC. In order to estimate LOD values at a signal/noise (S/N) ratio of 3, most researchers usually assess height to noise and then adjust the amount of analyte injected so that its peak height is 3 times the height of that baseline noise. With the micro-HPLC/SCD system, this method is invalid since peak area is typically used for quantification. There are, however, statisticallybased means of determining LOD that were developed for spectroscopic measurements9
where SB is the standard deviation of the blank measurement, m is the slope of the calibration curve, and the constant k equals 3 for LOD measurements. Proper determination of detection limits for chromatographic analyses using this formula is not straightforward since a SB measurement is impossible to obtain in many cases. In the case of the microHPLC/SCD system discussedhere, the data collection system (integrator) does not recognize responses under a set peak threshold value (1000-3000 area units). Therefore, SB in this case was assumed to be zero since the area responses obtained are never at a “blank” level as they are in a purely spectroscopic experiment. Because the above equation is not valid for SB = 0, the expanded propagation of error equation for LOD determination9 was used to obtain the values given in Table 4. After the mass LOD was calculated, this mass of sulfur (CS2) was injected into the micro-HPLC/SCD system for
~~~
(8) Karnicky, J. F.; Zitelli, L. T.; van der Wal, S. J. Anal. Chem. 1987,59, 327333.
(9) Winefordner, J. D.; Long, G. L. Anal. Chem. 1983, 55, 712-724A.
AnaIyticalChemistry, Vol. 66, No. 9, May 1, 1994
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Table 4. Mlcro-HPLC/SCD D.t.ctMty Gomparkon
mobile- hasecomp (MeO%/water)
(pg of S)
LOP
peak width (8)
detectivit at LOD (pg ofS1s)
0/100 10190 20180 30170 40160 50150 60140 70130 80120 90110 100/0
76 114 73 145 43 112 70 48 37 145 28
84 84 78 72 72 60 48 42 30 30 25
0.9 1.4 0.9 2.0 0.6 1.9 1.5 1.1 1.2 4.8 1.1
I 3
,n,
I
0
Retention Time
1
21 min.
11
Calculated bv the propagation of error method? k = 3,$B = 0.
verification. The detectivity was then obtained by dividing the resulting peak area by its peak width. Since the propagation of error LOD methodg is based on a comparison between the experimental detector response and the calculated one based on the slope and intercept, one would expect that the lowest LOD values were obtained for the curves with the beat correlation coefficient. No other trends such as the quenching ability of the mobile phase can be drawn from these results. The lowest SCD detectivity (600fg of S/s) was obtained with a mobile-phase composition of 40% methanol/ 60% water whereas the highest (4.8pg of S/s) was obtained at 90110. The detectivities obtained here for the 40/60mobile phase rival those obtained by Shearer et al.1° for GC/SCD (400fg of S/s), while those for the 50/50 and 70/30 mobile phases are lower than those previously obtained by Chang and Taylor' for micro-HPLC/SCD (3 or 4 of pg S/s at 50/50 and 75/25 mobile-phase compositions, respectively). However, direct comparisons to these or other researchers' LOD results are invalid since most researchers do not specify how LOD values are obtained and/or calculated. In evaluating the calculated propagation of error LOD values as amount of CS2 injected, it was observed that lower concentrations than this level actually gave a response that looked to be at S/N = 3 if visual height assessment is used. This would indicate that with thevisual assessment thedetectivities would be lower than those given in Table 4. Propagation of error detection limits are typically higher than LODs calculated from other methods because both the error in the slope and the intercept of the working curve are considered. On the other hand, the RSD values based on peak area for these lowest injected concentrations were very poor (100-200%) compared to those for thecalculatedvalues(5-1096). It is felt that thecalculated and verified LOD values are more valid given the better RSD values. Even though detection limits expressed as concentration are not technically valid for the mass-sensitive SCD, they must be examined for the sake of practicality (Table V). These values are based on the LOD values in Table 4 and an injection volume of 60 nL. Most of these values fall within the 1-3 ppm concentration range. Application. The selectivity of the micro-HPLC/SCD system was demonstrated by the analysis of thiocarbamate pesticides in a red delicious apple. The pesticides (Figure 8) were spiked onto the apple (83 and 2 ppm level) and extracted (10)Shearer, R.L.;O"ea1, D.L.;Rim, R.;Bakn, M. D.J . Chromcrtogr. Sci. 1990,28,24-28.
1436 Analytical C h " S t t y , Vol. 66,No. 9, May 1, 1994
t
0
I
20 min Flgure 8. (A) HPLCIUV separation of a spiked (83 ppm) apple COe Retention Time
supercritical fluid extract. Peak identity Is as follows: (1) methomyl, (2) methiocarb, and (3) eptam, IS Thianaphthene. HPLC/UV conditions: 50% acetonitrlle/50% water mobile phase, 250 mm X 4.6 mm 1.d. Hypersil Cle column, 1GpL Injection volume, 1 mL/mln flow rate, UV detection at 235 nm. (B) Micro-HPLCISCD conditions: 70% methanol/30% watermobilephase, 150" X 320pm1.d.C18cdumn, 60 nL injection volume, 7 pL/min flow rate, 200 cmS(STP) m1n-l air flow rate, and 400 cm3(STP) mln-' hydrogen flow rate.
Table 5. Mlcro-HPLC/SCD Concmtratbn Detection umllr
mobile- hase comp (MeOkwater)
amount CS2 injected (pg)
concn LOD (pg of CSdnL or ppm)
01100 10190 20180 30170 40160 50150 60140 70130 80120 90110 100/0
90 134 86 171 51 132 83 57 44 171 33
1.5 2.2 1.4 2.9 0.85 2.2 1.4 0.94 0.73 2.9 0.56
with supercritical 2% methanol (w/w) modified carbon dioxide at 50 OC and 350 atm. The extracted thiocarbamates were deposited in one of two tandem traps (solid phase or liquid) after the decompression of the supercritical fluid. The solid phase trap was then rinsed with methanol (10 mL) in order to remove the analytes, followed by a dilution (water) to mobile-phase solvent strength. The resulting extracts were chromatographed by both analytical scale HPLC/UV and micro-HPLC/SCD. Figure 8 is the separation of the solidphase trap rinse. Eptam could not be detected in this extract by micro-HPLC/SCD due to its low concentration. When the SCD and UV chromatograms are compared, the selectivity of the SCD is apparent. The difference in sensitivity is a result of the minute injection volume (60 nL) used in the micro-HPLC/SCD system compared to that of the analyticalscale HPLC/UV system (10 pL). In addition, for example, methiocarb is only 16% sulfur by weight whereas the probe molecule utilized in the optimization studies (CS2) is 84% sulfur. The corresponding difference in sulfur content translates into inherently higher detection limits for analytes with more realistic sulfur levels such as methiocarb. Despite the lower sensitivity, micro-HPLC/SCD can be a valuable
analytical tool even for samples with low sulfur content if concentration enrichment is used.
ACKNOWLEDGMENT We thank Mario Ursem and J. P. Salzmann, LC Packings, Inc., for their donation of packed capillary columns; and Michael Balough, Waters Chromatography, for their donation
of the MS-600 micropumping station. The following peopleaided with their technical support: Brian Clay of Sievers Research and Randy Shearer of Shell Development Co. Received for review August 4, 1993. Accepted February 14, 1994.' *Abstract published in Aduonce ACS Akrrucrs, March 15, 1994.
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