2024
Anal. Chem. 1988, 60,2024-2027
the binding enhancement between the on and off states can be calculated from the E” values of the two observed voltammetric waves using eq 1. In this case, substoichiometric amounts of metal cations should result in two well-resolved redox couples corresponding to the free ligand and the metal complex. This response is indicative of incremental electrochemical switching. When the binding constant of the neutral ligand is low (Ks < l), can be calculated by using voltammetric data for several cation concentrations and eq 2. Only one redox couple will be observed a t all cation concentrations and the redox potential will shift anodically as the cation concentration increases. There appears to be no straightforward method to deal with cases in which 1 < Ks < lo4. In this region, the voltammetric behavior is intermediate and neither eq 1 nor 2 fully applies. Fitting the experimental cyclic voltammograms to simulated results could be a valid methodology to treat those cases and determine the binding enhancements. Thus far, among all of the crown ether systems examined, only the lariat ethers show clear, two-wave behavior that can be interpreted as the direct observation for both the bound and the unbound systems. Registry No. 1, 88548-59-8; I-, 102586-74-3; 1 Na’, 10804348-7; 1 Na, 115603-75-3; 2, 87453-20-1; 2-, 107996-01-0; 2 Na+, 115603-74-2; 2 Na, 87453-19-8; Na, 7440-23-5; C , 7440-44-0.
LITERATURE CITED (1) Kaifer, A.; Echegoyen, L.; Gustowski, D.; Goli, D.; Gokel, G. J . Am. Chem. SOC. 1983, 105, 7168.
Gustowski, D. A.; Echegoyen, L.; Goli, 0.M.; Kaifer, A,; Schultz, R. A,; Gokel, G. W. J . Am. Chem. SOC. 1984, 106, 1633. Morgan, C. R.; Gustowski, D. A.; Cleary, T. P.; Echegoyen, L.; Gokel, G. W. J . Org. Chem. 1984, 4 9 , 5008. Gustowski, D. A.; Gaeo, V. J.; Kaifer, A.; Echegoyen. L.; Godt, R . E.; Gokel, G. W. J. Chem. Soc.. Chem. Commun. 1984, 923. Delgado, M.; Echegoyen, L.; Gatto, V. J.; Gustowski, D. A.; Gokel, G. W. J . Am. Chem. SOC. 1986, 108, 4135. Maruyama, K.; Sohmiya, H.; Tsukube, H. Tetrahedron Lett. 1985, 26, 3583. Saji, T. Chem. Leff. 198S2275-276. Kaifer, A.; Gustowski, D. A.; Echegoyen, L.; Gatto, V. J.; Schultz, R. A.: Cleary, T. P.; Morgan, C. R.; Goli, D. M.; Rios, A. M.; Gokel, G. W. J . Am. Chem. SOC. 1985, 107, 1958. Wolf, R. E.; Cooper, S. R. J . Am. Chem. SOC. I964. 106, 4646. Feldberg, S.W. In Electroana&tical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1969; Vol. 3. Feldberg, S. W. Computers in Chemlstty and Instrumentation; Mattson, J. s., Mark, H. B., Jr., MacDonald, H. C., Jr.. Eds.; Marcel Dekker: New York, 1972; Vol. 2, Chapter 7. Evans, D. H.; Xie, N. J . Electroanal. Chem. Interfacial Electrochem. 1982, 136, 139. Gustowski. D. A.; Gatto, V. J.; Mallen, J.; Echegoyen, L.; Gokel, G. W. J . Org. Chem. 1987, 52, 5172-5176. Nagaoka, T.; Okazaki, S.;Fujinaga, T. Bull. Chem. SOC.Jpn. 1982, 5 5 , 1967. Nagaoka, T.; Okazaki, S.; Fujinaga, T. J. Electroanal. Chem. 1982, 133, 89. Kalinowski, A . K.; TenderendaGuminska J . Electroanal. Chem. 1974, 5 5 , 277. Peover. M. E.; Davies, J. D. J . Electroanal. Chem. 1983, 6 , 46. Galus, 2 . Fundamentals of ElectrochemicalAnalysis ; Ellis Horwood: London, 1976; Chapter 14.
RECEIVED for review November 24,1987. Accepted May 23, 1988.
Voltammetric and Liquid Chromatographic Identification of Organic Products of Microwave-Assisted Wet Ashing of Biological Samples Kenneth W. Pratt,* H. M. Kingston, William A. MacCrehan, a n d William F. Koch
Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
Residual organic species in nitric acid digests of freeze-dried bovine liver (NBS SRM 1577a) have been Identifled by use of voltammetry, liquid chromatography, spectrophotometry, and classical chemical tests. Data from these techniques show that malor products of microwave-assisted dissolution by nitric acld Include 0-, m-, and pnitrobenroic adds (NBA). I n addition to these compounds, other organic species present in these digests irreversibly complex copper, but not zinc, and result in low values for copper by polarography. The NBAs and these other organlc specles are all ellmlnated by refluxlng the nltrlc acld dlgest In perchlorlc acid at atmospheric pressure. Poiarographlc results obtained for copper following treatment wlth perchloric acld agree with the certifled value. The use of voltammetry In the evaluatlon of wet ashing procedures is dlscussed.
Two important factors in the evaluation of sample dissolution procedures are the time required and,the completeness of decomposition of the original sample matrix. For biological samples, microwave-assisted dissolutions in sealed pressure
vessels achieve dissolution in less than 10 min with nitric acid. Microwave dissolution of human urine in HNOB( I ) was shown to result in a 105-foldreduction in the concentration of amino acids. However, this experimental result does not indicate that the original organic sample matrix was totally converted to COz,HzO, and N2 The authors noted that the nitric acid digests contain “incomplete digestion products” along with the inorganic species of interest. Although organic decomposition products do not interfere with many instrumental techniques for trace elemental analysis, voltammetry is sensitive to interference from chelating and electroactive organic components coexisting in samples during analysis. These organic species may bias the results, making examination of their origin and mechanism of formation extremely important. Previous workers (2-5), using thermal nitric acid dissolutions of biological material a t elevated temperature and pressure, have noted that the decomposition is not complete and produces “interfering organic compounds” (2), “undefined artifacts” (3),or “organic nitro compounds” (4) that result in unwanted signals and/or errors in trace-level voltammetric determinations. Samples with high protein content are more difficult to decompose completely than other biological sam-
This article not subject to US. Copyright. Published 1988 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
ples (2, 4 ) . Fuming of the H N 0 3 digest with HC104 or decomposition using H N 0 3 in a Carius tube at a minimum of 250 "C both result in a digest that is sufficiently free of organic products to permit trace level determinations by anodic stripping voltammetry ( 4 ) . In the present work, residual organic products of microwave-assisted nitric acid dissolution of biological samples are identified. The presence of 0-,m-, and p-nitrobenzoic acid (NBA) in these digests is demonstrated by means of liquid chromatography, voltammetry, spectrophotometry, and classical chemical methods. The effects of residual organic products of microwave-assisted digestion on the polarographic determination of trace metals are also presented. It is demonstrated that these organic products of nitric acid digestion are destroyed by refluxing the digest with perchloric acid. This work extends the results of Wurfels et al. (5) who recently reported the presence of the three NBA isomers among the products of thermal nitric acid dissolution of biological samples. This latter work appeared during preparation of the present work and provides evidence for the similarity of thermal and microwave-assisted nitric acid dissolutions.
EXPERIMENTAL SECTION Apparatus. Microwave-assisted digestions were performed with a microwave digestion system (CEM Corp. 1705-81,Indian Trail, NC) operating at 2450 MHz with a maximum power of 574 W. Temperature and pressure measurements were recorded at 5-s intervals with a fiber-optic thermometer (Luxtron 750, Mountain View, CA) and a pressure sensor. The microwave unit was modified as described previously (1). Digestions were performed in ported, 60-mL PFA Teflon vessels (Savillex Corp., Minnetonka, MN). Spectrophotometric analyses were performed with a double-beam UV/visible recording spectrophotometer with 1-cm quartz cells. The reference cuvette was filled with pure solvent. High-pressure liquid chromatographic (HPLC) analyses were performed at a flow rate of 1.25 mL/min with a Vydac (2-18 column (25 cm, 5-wm particle size) and ultraviolet (UV) detection at a wavelength of 260 nm. The volume of the sample injection loop was 20 bL. The HPLC eluent was 5% CH30H + 15% CH3CN+ 80% H20 (proportions before mixing), which was made 0.05% in CF3COOH to ensure that organic acids were in their protonated form. Polarographic analyses were performed with a polarographic analyzer and mercury electrodes (PAR 384B and 303A, Princeton Applied Research, Princeton, NJ) in the differential pulse mode. Polarograms were recorded by using a scan increment of 4 mV and pulse height of 20 mV. Drop times of 0.5 and 1.0 s were used, with medium drop size. Solutions were deaerated for 8 min with water-saturated Ar prior to recording the polarograms. Potentials are reported with respect to the saturated Ag/AgCl reference electrode (SSCE). Reagents and Standards. Microwave-assisted digestions and elemental analyses were performed on National Bureau of Standards Standard Reference Material (SRM) 1577a, bovine liver. The SRM was vacuum-dried at room temperature for 24 h in accordance with the certificate instructions. Acids and water used in the elemental analyses for Cu and Zn were purified by subboiling distillation (6). Other reagents were analytical grade. Standard solutions, nominally 1000 pg/mL, of Cu(I1) and Zn(I1) in 1 mol/L HN03 were prepared by dissolving Cu and Zn of >99.999% purity in HN03 and diluting to 500 g with 1 mol/L HN03. All dilutions were performed on a mass basis. Procedure. Samples of bovine liver, SRM 1577a (nominally 250 mg), were weighed to an accuracy of 0.1 mg into individual PFA vessels. The vessels were covered (ports open),7.2 g of HN03 was added to each, and the samples were predigested on a hot plate overnight at 130 "C. This predigestion was required to reduce the pressure buildup in the sealed 60-mL vessels during the subsequent microwave dissolution. The predigestion step can be eliminated by using reinforced 120-mL digestion vessels, which can withstand the pressure buildup resulting from the initial reaction of HNO, with readily oxidized components of the sample. Organic samples up to 250 mg can be directly decomposed in these
2025
aw
R
a
IU R
w n
fiI-
0
2
4
6
a
TIME (min)
Figure 1.
Temperature and pressure conditions for four 0.25-9 sam-
ples of bovine liver (SRM 1577a) in 7.2 g of HNO,; 60-mL digestion
vessels. vessels without exceeding the safety limitations of the vessel. After predigestion, microwave-assisted dissolution was performed in sealed vessels using a microwave program of 30% power (172 W) for 5 min followed by 36% power (207 W) for 5 min. An additional program segment of 40% power (230 W) for 3 min was added in cases where the temperature of the solution did not reach the target value of 180 "C for a minimum period of 100 s. The maximum pressure in the sealed vessels, during the microwave irradiation, was approximately 800 kPa (8 atm). Representative temperature and pressure profiies versus time for the bovine liver sample in the sealed vessel are shown in Figure 1. After irradiation, the sealed vessels were cooled in a dry ice-ethanol bath to reduce the internal pressure to less than 100 kPa (1atm) prior to opening. This procedure prevented loss of sample from aerosol passing through the ports after opening. For the elemental determination of Cu and Zn, the digests from the vessels were heated with HC1 to volatilize Sn, extracted with methyl isobutyl ketone (MIBK) to separate Fe(II1) and Tl(III), neutralized with aqueous ammonia, and then extracted with dimethylglyoxime to separate Ni. Sodium diethyldithiocarbamate was then added and the Cu and Zn chelates were extracted into CHC13. The extracted chelates were finally decomposed in HN03 and HC104and the residues were dissolved in weighed portions (5 g nominal) of 0.5 mol/L acetic acid + 0.5 mol/L ammonium acetate buffer for the polarographic determination. For calibration, standard solutions of Cu(I1) and Zn(I1) were substituted for the digests in the above procedure and taken through the same series of separations. The MIBK extracts from the digests were evaporated in a N2 stream and redissolved in CH30H for the HPLC determinations. Portions of these extracts were also used for the classical chemical tests.
RESULTS AND DISCUSSION Initial evaluation of the applicability of microwave-assisted dissolution to voltammetric trace element analysis revealed the existence of systematic errors and spurious peaks in polarographic determinations. ,These observations prompted investigation into the nature of the residual organic products of microwave-assisted dissolution. For this work, bovine liver, SRM 1577a, was digested in HNOs using microwave-assisted dissolution. Evaporation of the microwave digests yielded a residue containing organic components that were relatively insoluble in cold H 2 0 and HN03 but soluble at 100-130 "C. These organic species were insoluble in hexane but readily soluble in MIBK. Polarograms obtained for these digests following separation displayed one or more peaks in addition to those expected from the Cu and Zn present in the original sample. A typical example is shown in Figure 2. The peaks at -0.22, -0.39, and -0.72 V do not result from trace metals present in the sample. Refluxing the digest with HC104 to destroy residual organic products prior to the MIBK extraction yielded a polarogram that contained only the Cu and Zn peaks at -0.01 and -1.00 V, respectively. In addition to the spurious peaks noted above, analytical results for Cu in those digests that were not refluxed with HC104 ("HN03 digests") were
2026
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
i\i 1
H C I 0 4 Digest
1
I
n
1
I1
-77
I
11 Ub 4 u
1
, 0.1
-~--,
~
(1
-
-0.1
-0.3 -0.5 -0.7 E (volts vs. SSCE)
-0.9
variable and well below the certified value for SRM 1577a. Results for Cu in the digests refluxed with HC104 ("HC104 digests") agreed well with the certified value. Actual results for Cu were 142.4 ppm (standard deviation 4.0 ppm, n = 8) without HClO, treatment and 159.8 ppm (range 4.6 ppm, n = 2) with HClO, treatment. The certified value for Cu is 158 f 7 ppm. In contrast to the results obtained for Cu, those for Zn showed little change when the digests were refluxed with HClO,. Actual results for Zn were 123.5 ppm (standard deviation 1.6 ppm, n = 8) without HC104 treatment and 119.2 ppm (range 3.1 ppm, n = 2) with HC104 treatment. The certified value for Zn is 123 f 8 ppm. The variability noted for Cu in the HN03 digests was not observed for Zn in the same samples. The apparent decrease noted for Zn in the samples refluxed with HClO, likely results from elimination of an electroactive organic digestion product that is reduced a t -1.0 V. This decrease is noticeable polarographically but is not significant in comparison with the uncertainty in the certified value. Laboratory experience and literature indications ( 3 , 4 )both point toward the protein in biological samples as the source of these interferences in polarographic determinations. The aromatic amino acids of this protein are most resistant to complete decomposition, due to the large energy of stabilization of the aromatic rings. Likely digestion products of these compounds would result from nitration of the aromatic nucleus and f or oxidative degradation of the amino acid moiety. The most obvious compounds resulting from such processes would be the nitrobenzoic acids (NBA). Spectrophotometric Measurements. UV absorption spectra of the MIBK extracts of the HN03 digests and of solutions of 0- and m-NBAs in MIBK both exhibited a shoulder, extending to approximately 300 nm, which was absent in the HClO, digests. The solutions all displayed a strong, broad absorbance peak a t 241 1 nm. Spectra obtained by using hexane for the extraction in place of MIBK showed no significant absorbance a t 280 nm. These data are consistent with the known insolubility of NBAs in hexane (7) and observed solubility in MIBK. HPLC Identification of Digestion Products. The results of the HPLC analyses of the MIBK extracts are shown in Figure 3. Chromatographic peaks for the three NBA isomers are evident in the H N 0 3 digest, with p-NBA as the largest
*
1
10
15
Lllnutes
-Ll
Flgure 2. Differential pulse polarogram of bovine liver sample after separation, showing peaks due to organic products of dissolution: 1, Cu; 2, Zn; lU, mixed NBAs; 2U and 3U, unknown.
. .1 ~ -7--r---
2
chromatograms of HCIO, digest of SRM 1577a, top; HNO, digest of SRM 1577a, middle; and standard mixture of NBAs, bottom. Off-scale peak on HCIO, digest is from MIBK. Flgure 3. Liquid
Table I. Estimation of Phenylalanine (Phe) in SRM 1577a from Determined Concentrations of Nitrobenzoic Acid WBA)
sample no.
sample wt, g
total NBA by HPLC, mg
1 2 3
255.9 234.8 274.6
7.32 8.24 7.60
%
Phe calcd 2.83 3.47 2.74
Table 11. Typical Levels of Individual Amino Acids in Rat Liver Protein (Calculated from Data in Reference 7 and 8 as a Percentage of the Total Liver Sample) weight percent amino acidn
glutamic acid leucine
glycine serine
lysine aspartic acid arginine **phenylalanine valine threonine isoleucine alanine **tyrosine
methionine **histidine **tryptophan cystine aromatic amino acids
wet (69.0% H20)
dry
1.91 1.51 1.44 1.31 1.26 1.24 1.19 1.10 1.08 0.95 0.86 0.85 0.70 0.58 0.45 0.27 0.25 2.52
6.15 4.87 4.64 4.23 4.06 4.00 3.83 3.54 3.48 3.07 2.78 2.73 2.26 1.86 1.45 0.87 0.81 8.12
Double asterisk denotes aromatic compound. peak. Retention times for these three peaks agree to within 1.1% of the values for the standard solution, which contained 0-,m-, and p-NBA a t nominal levels of 20 ppm. The large peak noted in the chromatograms of the extracts of the HN03 and HCIO, digests a t a retention time of 8.5 min results from residual MIBK from the extraction procedure. The chromatograms illustrated in Figure 3 were also used to estimate the concentrations of the three NBA isomers based on the response factors obtained for 0-,m-,and p-NBA standards. These results are summarized in Table I. In addition, the corresponding concentration of phenylalanine
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
is calculated from the total concentration of NBA, assuming that the NBA results solely from phenylalanine originally present in the sample. The resulting values compare to a typical value of 3.54% estimated for phenylalanine in bovine liver (see Table 11) calculated using data from the literature for the overall composition of rat liver (8) and the amino acid composition of animal liver (9). The HCIOl digests showed no detectable HPLC peaks a t the retention times of the 0-, m-, or p-NBA standards. Analysis of a HN03 blank, preheated and irradiated concurrently with the bovine liver samples, yielded a chromatogram with small peaks at retention times of 6.42 and 12.05 min, which were also present in the HCIOl digest (the 6.42-min peak is present but not visible in Figure 3). These two peaks therefore result from an impurity in the reagents used in the procedure. Polarographic Identification of Digestion Products. Polarographic analysis was performed on an acetate-buffered NaOH extract of the MIBK extracts of the HNO, digest. The buffered extract yielded a polarographic peak, at -0.23 V, which was also observed in the final solution used earlier in the determination of Cu and Zn. This test confirmed that at least one of the unwanted products of incomplete digestion displayed acid-base and solubility properties characteristic of the NBAs, Le., preferential extraction of the protonated NBA into the organic (MIBK) phase at low pH and into the aqueous phase, as the nitrobenzoate anion, at high pH. Further polarographic investigations demonstrated that addition of 0-,m-, and p-NBAs to acetate-buffered standard solutions of Cu(I1) and Zn(I1) at room temperature did not result in a reduction of either the Cu or the Zn peak height beyond the decrease attributable to dilution of the original solution by the spike. Thus, the NBAs are not responsible for the decrease in Cu peak height, and consequent low values for Cu, noted previously for the HNO, digests of bovine liver. The polarograms for the spiked solutions also displayed peaks for the reduction of the NBAs in addition to the Cu and Zn reduction peaks. Peak potentials for the NBA reduction in the acetate buffer were -0.288, -0.249, and -0.233 V for the ortho, meta, and para isomers, respectively. Signals for mixtures of the three isomers were not polarographically resolved but yielded a single, slightly broadened peak at an intermediate E, value, determined by the relative proportions of the three isomers. Polarograms of the acetate-buffered NaOH extract discussed above yielded an E, value of -0.233 V (vs -0.230 V for the HNO, extracts) at a similar pH value, thus providing additional evidence for the presence of the NBAs as digestion products of microwave-assisted HNO, digestion. Correlation with the Literature. The above results extend the results of the recent work of Wurfels, et al. (5) to microwave-assisted nitric acid dissolutions. The similarities in the ratios of the three NBA isomers for thermal- and microwave-assisted dissolutions suggest that the mechanism is similar in the two cases. The classical literature provides additional information as to the mechanism of decomposition. Morner (10-15) showed that oxalic, benzoic, picric, terephthalic, succinic, and m- and p-NBAs are present in digests obtained by conventionally heating proteins in HNO,. Tests of individual amino acids indicated that only phenylalanine yields p-NBA on digestion with HNO, (10, 12). Sufficient p-NBA was formed to result in the formation of crystals in the digest after cooling. Oxalic acid was decomposed on ex-
2027
tended digestion in 98% HNOp The preponderance of p-NBA over the meta and ortho isomers provides information on the mechanism of attack of HNO, on phenylalanine. Oxidation of the side chain to -COOH followed by nitration would result in the formation of m-NBA as the primary product, due to the meta-directive effect of the carboxylic group. Nitration of the phenylalanine followed by oxidation of the amino acid side chain would result in o- and p-NBA tu the major products. With phenylalanine, the para product is favored over the ortho isomer for steric reasons. Additional evidence for the mechanism of nitration followed by oxidation is provided by the results of Erlenmeyer and Lipp (16) and of Takayama and Tsubuku (17),both of whom observed the formation of p-nitrophenylalanine as a product of mild nitration. In conclusion, microwave-assisted digestion of bovine liver yields, 0-,m-, and p-nitrobenzoic acid (NBA) as products of dissolution. Additional organic products are also produced that interfere with the polarographic determination of Cu by complexing the free metal. The components of the bovine liver sample that are most resistant to attack are the constituent aromatic amino acids of the original protein, of which phenylalanine yields p-NBA. The relative yields of the three isomers indicate that the dominant mechanism of attack both in thermal and in microwave-assisted nitric acid dissolutions is nitration of the aromatic ring followed by oxidation of the amino acid side chain. The extreme resistance of p-NBA to decomposition suggests its use, together with subsequent polarographic determination, in the evaluation of the completeness of other digestion techniques using nitric acid. Further work using closed-vessel microwave heating, other reagent combinations, and other reaction conditions to achieve complete decomposition of organic matrices is in progress.
LITERATURE CITED (1) Klngston, H. M.;Jassle, L. B. Anal. Chem. 1988, 58, 2534. (2) Stoeppler, M.; Muller, K. P.; Backhaus, F. Fresenlus’ Z . Anal. Chem. 1979, 297. 107. (3) Jackwerth, E.; GomI&k, S. Pure Appl. Chem. 1984. 56, 479. (4) Kotz, L.; Henze, 0.;Kaiser, G.; Pahlke, S.; Veber, M.; Tolg, G. Takrnta 1979, 26, 681. (5) Wurfels, M.; Jackwerth, E.; Stoeppler, M. Fresenlus’ 2.Anal. Chem. 1988, 330, 160. (6) Kuehner, E. C.; Atvarez, R.; Paulsen, P. J.; Murphy, T. J. Anal. Chem. 1972, 4 4 , 2050. (7) The Merck Index. 8th ed.;Stecher, P. G., Ed.; Merck 8. Co.,Inc.: Rahway, NJ, 1968; p 737. (6) Biochemist’s Handbook; Long, C., Ed.; Van Nostrand Reinhold Co.: New York, 1961; p 677. (9) Block, R. J. The Amino AcM Composnion of Protelns and Foods, 2nd ed.; C. T. Thomas: Springfield, IL, 1951; p 489. (10) Morner, C. T. Hoppe-Seyler’s 2.fhysiol. Chem. 1915, 95, 263. (11) Miirner, C. T. Hoppe-Seyler’s Z . fhysiol. Chem. 1918. 98, 89. (12) Morner, C. T. Hoppe-Seyk’s 2.fhysiol. C t ” . 1918, 98, 93. (13) Miirner, C. T. Hoppe-Seyler’s 2.fhysiol. Chem. 1918, 98, 98. (14) Morner, C. T. Hoppe-Seyler’s Z . Physiol. Chem. 1917. 101, 15. (15) Miirner, C. T. Hoppe-Seyler’s Z . fhysiol. Chem. 1918, 707, 210. (16) Takayama, Y.; Tsubuku. Y. Bull. Chem. SOC.Jpn. 1942, 17, 109. (17) Erlenmeyer, E.; Lipp, A. Justus Liebigs Ann. Chem. 1883, 279, 179.
RECEIVED for review March 23,1988. Accepted June 23,1988. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best for the purpose. This work was presented at the 193rd National Meeting of the American Chemical Society, Denver, CO, April 5-10, 1987, paper no. ANYL-118.
