We expected and found that our data would show the effect of improvements in microwave spectroscopy instrumentation which have developed during the five-year period since the earlier work was reported. Because of differences in the amount of propene-dl in the samples analyzed and the temperature at which the measurements were made, the weakest line analyzed in the earlier work (10) (y 'v 3 X cm-l) was a factor of 6 stronger than that studied here (y = 5 X
cm-l). On the average, the standard deviations reported previously are a factor of 8 larger than we report here.
RECEIVED for review January 17, 1972. Accepted May 26, 1972. Contribution No. 1899 from the Central Research Department, E. I. du Pont de Nemours & Company, Experimental Station, Wilmington, Del.
Curcumin Method for Spectrophotometric Determination of Boron Extracted from Radiofrequency Ashed An imaI Tissues Using 2-Ethy I- 1,3-Hexa nedio1 James W. Mair, Jr., and Harry G . D a y Department of Chemistry, Indiana University, Bloomington, Ind. 47401 Work on the nutritional significance of boron in animals required the development of a sensitive and accurate method for its determination at the submicrogram level. The method developed requires gentle combustion of animal tissues in a low temperature radiofrequency excited oxygen plasma followed by extraction from a 1N HCI solution of the ash using 2-ethyl-1'3hexanediol in chloroform (10% v/v). Boron in the organic phase i s converted to the highly absorbing rosocyanin complex using glacial acetic acid (0.375% w/v) followed by concentrated sulfuric acid. The concentrate i s diluted with 95% ethanol and spectrophotometrically read at 550 nm vs. a reagent blank. Beer's law is obeyed down to 0.002 pg/ml, and the method exhibits a total error of about 10% over concentrations ranging between 0.002 and 0.020 pg/ml. Data show that 100.3*5.1% of the standard Na2B407.10H 2 0 added to the unashed tissue samples was recovered. The use of XE-243 boron-specific resin proved to be a convenient, quantitative means for concentrating as little as 1pg of boron from large volumes of solution.
SINCETHE 1950's when Spicer and Strickland ( I ) demonstrated the utility of curcumin for boron determinations, many attempts have been made to improve the use of this reagent. In the original work, boron was separated from interferences by distillation as methyl borate and retained in platinum dishes with glycerol. After destroying the glycerol during the fusion, the highly absorbing rosocyanin complex was formed in the dishes by addition and drying of an acetone and water solution of curcumin. The complex was then extracted into o-chlorophenol and its absorbance read at 550 nm. This procedure was plagued with errors due largely to the numerous manipulations. Hayes and Metcalfe ( 2 ) modified the method by forming the rosocyanin complex in a mixture of 3 ml of glacial acetic and 3 ml of concentrated sulfuric acids. The strong acid protonated the curcumin to facilitate rosocyanin formation. Excess protonated curcumin unfortunately exhibited a spectral interference, but this could be removed by dilution of the concentrate with 95% ethanol in the Hayes and Metcalfe ( 2 ) method and by an ammonium acetate-acetic acid buffer
in the method by Grinstead and Snider (3). Uppstrom ( 4 ) used propionic anhydride to eliminate water, followed by direct analysis with the acid mixture. These methods all failed to achieve the degree of sensitivity possible with the rosocyanin complex because of the suboptimal reaction conditions which were employed. In the method to be described herein, direct standardization with primary standard borax is possible without prior chemical manipulation to simulate sample treatment. Standards need not be exposed to the sample ashing conditions because the radiofrequency method of sample ashing which was used enables boron to be completely recovered from the biological matrix. Complete recovery of boron permits the maximum sensitivity of the rosocyanin complex to be realized. EXPERIMENTAL
Apparatus. Polypropylene test tubes (Falcon Plastics No. 2059 size 17 x 100 mm) were used as vessels for tissue storage, for extraction, and for rosocyanin formation, and were effective in avoiding contamination. Combustion of animal tissues was accomplished using a Trapelo (formerly Tracerlab) low temperature biological sample asher Model 600. All spectrophotometric measurements were made using a Carl Zeiss PM QII spectrophotometer. Reagents. All standard boron solutions were prepared from reagent grade NazBIOi.10 H20. Solutions of 2-ethyl1,3-hexanediol (Aldrich) in chloroform (Allied Chemical) were 10% vjv. Curcumin from Eastman Organic Chemicals was recrystallized once from ethanol and used in glacial acetic acid (0.375 w/v). Procedure. Animal tissues were freeze dried and compacted into disks inch in diameter by inches thick using a pellet press and an applied pressure of approximately 4000 pounds. This step is optional, and serves only to allow large quantities of tissue to be handled more easily. Depending on the boron concentration anticipated in the sample, as much as 4 grams of the dried tissue disks were placed on aluminum foil squares or in silica dishes and inserted into each radiofrequency combustion chamber. These samples
(1) G. S. Spicer and J. D. H. Strickland, Anal. Chim. Acta, 18, 231 (1958).
( 2 ) M. R.Hayes and J. Metcalfe, Analyst (London),87,956 (1962).
R.Grinstead and Sigrid Snider, ibid., 92, 532 (1967). (4) Leif'R. Uppstrom, Anal. Chim.Acta, 43,475 (1968). (3) Robert
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
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Table I. Data for the Recovery of 2-3 pg of Boron &gig (dry) Recovery, Rat liver 0.708 94.1 Rat kidney
0.608 0.218 0.835 1.172 1.442 1.490 1.769 2.569 5.716
101.9 106.8 100.5 99.9 93.5 96.2 109.4 101.3 99.4
RESULTS AND DISCUSSION
100.3 =k 5.1z
were ashed for 40-50 hours at 100-150 W forward power, an oxygen flow of 80 standard cc/minute, and a pressure of 1.0 mm Hg. Less ashing time was required if the samples were not compacted. Flue gases which included nitrogen oxides, carbon dioxide, and water were trapped at a liquid nitrogen cold finger. After combustion, the ashed disks were quantitatively transferred to polypropylene test tubes, and dissolved with 2 ml of 1N HC1. Extraction of boron with an equal volume of Z-ethy1-1,3-hexanediol solution in chloroform (10 v/v) was complete after 1 minute. A 0.50-ml aliquot of the organic phase was treated for 15 minutes, first with 1 ml of curcumin in glacial acetic acid followed by 0.30 ml of concentrated sulfuric acid. This order of addition gave the best results. The concentrate was diluted to 50 ml with 9 5 z ethanol, and its absorbance read us. a reagent blank at 550 nm. This procedure was modified slightly to provide a greater applicability to samples of small size and low boron content. The modification requires that as much as a 1.5-ml aliquot of the organic phase be taken for treatment with curcumin and sulfuric acid. Prior to the addition of reagents, the sample is placed under a mild vacuum provided by a water aspirator to evaporate most of the CHC13 (bp 62 "C) while leaving the diol-borate complex (bp 244 "C) behind in a total volume not to exceed 0.50 ml. Using this modification, three times as much sample can be taken for analysis, thus giving a wider range of applicability. Use of XE-243. XE-243 is a boron-specific resin produced by the Rohm and Haas Company by polymerization of a chloromethylated styrene-divinylbenzene copolymer with N-methylglucamine. Its general utility as a boron-specific ion exchange resin was described by Kunin and Preuss in 1964 (5). Mair and Day (6) showed that the resin could be utilized successfully in the diet of rats to remove either trace quantities of boron or toxic levels of ingested boric acid. Carlson and Paul (7) demonstrated its utility as an analytical tool. We have confirmed that XE-243 provides a quantitative method for concentrating as little as 1 pg of boron from large volumes of solution. Boron from samples of rat urine and large quantities of ashed rat diet, low in boron, was successfully concentrated on an XE-243 resin bed 4 cm tall by 0.50 cm in diameter. After flushing any interfering substances through the column with water, the boron was eluted with 1 ml of 2M H2SO4followed by 7-8 ml of water. Using one 2-ml portion of the organic phase previously described, the entire 8-9 ml of effluent was repeatedly extracted in 2-ml aliquots and the analysis continued
z
( 5 ) Robert Kunin and Albert F. Preuss, Znd. Eng. Chem., Prod. Res. Deaelop., 3, 304 (1964). (6) James W. Mair, Jr., and Harry G. Day, Department of Chem-
istry, Indiana University, Bloomington, Ind., unpublished data,
1972. (7) R. M. Carlson and J. L. Paul, ANAL.CHEM., 40, 1292 (1968). 2016
as outlined. Better than 96z recovery of added boron has been obtained using the resin in this manner. The resin in the column can then be regenerated for future use by rinsing with 2 M N H 4 0 H followed by water until the effluent is neutral.
Biological Ashing. One of the most difficult aspects of trace elemental analyses in biological samples is that of finding a suitable way to destroy large quantities of organic material without loss of the trace element. Wet ashing techniques using alkali metal nitrates (8,9), hot oxidizing acids, or digestions in strong base generally are incomplete, dangerous, turbulent, and subject to contamination from the large quantities of reagents used. Boron analyses of tissues ashed in this manner always require a tedious separation step such as the distillation of boron as methyl borate ( I ) . Dry ashing has also been employed using high temperature muffle furnaces at about 500 OC. In these procedures, contamination from oven walls and evaporative losses of trace elements are sources of error which are too significant to be tolerated in boron analyses at the submicrogram level. The technique which shows the most promise in the area of biological ashing is the plasma technique first described by Gleit and Holland (IO). Later Evans and Morrison (11) used this method in their trace element survey of biological materials. In this technique, the sample is ashed in a radiofrequency induced oxygen plasma which reacts very readily with the organic sample. The combustion process is nonturbulent, free from contamination by the chamber walls or oxygen, and is relatively cool at 100-150 "C. Ashing conditions can be reproducibly controlled over a wide range for use with samples of different combustion properties. Internal Standard. The internal standard method was used to find the percentage recovery of boron which could be expected when using the plasma ashing system. The recovery procedure required that to half of a given tissue, 2-3 pg of boron be oven dried to.unashed tissue disks at 110 O C as a basic borate-mannitol complex while the other half of the same tissue was not treated with boron. Both halves of the tissue were then simultaneously ashed in separate chambers using the plasma ashing system. By analyzing each half, the percentage of boron recovered was determined. Most of the simple organoborane compounds probably were metabolized or otherwise air oxidized to borate prior to sample preparation, or subsequently oxidized to borate during ashing. However, a check for possible loss of organoborane compounds not so affected was conducted. Flue gases were collected in a liquid nitrogen-cooled trap during combustion of two liver samples from rats fed high amounts of boric acid. In 5.55 grams of dry sample, a total of 84.7 kg of boron was found while the trapped combustion products showed 2.2 pg of boron. Three other samples treated similarly showed even lower percentages of boron in trapped combustion products. All values were well within the allowable range of recovery indicated by data in Table I. These data establish radiofrequency ashing of biological samples as the method of choice when boron is the trace element to be determined. Caution. It is desirable to report some of the experiences encountered in trapping flue gases. To minimize contam(8) H. J. Bowen, ANAL.CHEM., 40, 969 (1972). (9) Thomas Greweling, ibid., 41, 540 (1969). (10) C . E. Gleit and W. D. Holland. ibid., 34, 1454 (1962). (11) C. A. Evans, Jr., and G. H. Morrison, ibid., 40, 869 (1968).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
ination of flue gases with grease necessarily used on the ground glass joints, attempts were made to trap flue gases with liquid nitrogen as soon after combustion as was possible. Thus the glass traps were attached directly to the exhaust port of two ashing chambers. When combustion products were trapped in this manner, the vessels were shattered from the force of violent explosions occurring within one minute from the time the traps had been removed from the cold system, and were warmed by the surroundings. When the flue gases were trapped several feet from the exhaust ports of the ashing chambers, no such incidents occurred during four years of operation. During tissue combustion, appreciable quantities of blue, crystalline N203and of C 0 2 and H 2 0 accumulated in the traps irrespective of where the traps were located. However, only those traps located immediately behind the exhaust ports exploded. No detailed study of the cause for the explosions was undertaken. However, it is recommended that combustion products be trapped at a point sufficiently removed from the exhaust ports of the ashing chambers to allow the decomposition of any thermally unstable compounds (e.g., peroxides) which might form and thus prevent their accumulation. Extraction. The use of 2-ethyl-1,3-hexanediol (10 viv) in chloroform as an extracting medium was reported by Agazzi (12) in which the optimum composition of the organic mixture and the pH of the aqueous phase for quantitative extraction was determined. The extraction of aqueous boron by this method eliminates two ions, fluoride and nitrate, which are known to interfere with the curcumin method of boron analysis. Fluoride, in amounts normally encountered in most animal tissues, does not enter the organic phase, and boron can be extracted in the presence of nitrate at a level of 100 ppm without significant interference arising in the subsequent treatment with curcumin. The organic phase is quite compatible with polypropylene. Thus the extraction can be conducted directly in the polypropylene test tubes. Deprotonation of Excess Curcumin. While the Hayes and Metcalfe ( 2 ) modification improved the reproducibility of the original work by Spicer and Strickland (I), the use of large quantities of concentrated sulfuric acid to protonate curcumin during rosocyanin formation necessitated a final dilution of the concentrate to 100 ml in order to deprotonate the excess curcumin and remove it completely as a spectral interference. The present method requires only 0.30 ml of concentrated sulfuric acid in one sixth of the reaction volume used by Hayes and Metcalfe ( 2 ) . Thus the spectral interference from excess protonated curcumin is eliminated by dilution to only 50 ml. With the use of radiofrequency combustion techniques, evaporative losses of boron during ashing were completely eliminated. Thus Beer's law curves could be based directly on extracted standard borate solutions without further chemical manipulation, and exposure of dried standards to the radiofrequency plasma under simulated ashing conditions prior to extraction was unnecessary. For the first time, the (12) E. J. Agazzi, ANAL.CHEM., 39, 233 (1967).
4 m l
Table 11. Beer's Law Data Least Mean (10) absorbance square i S* (Re1 s*)= data
0.002 0.157 Se 0.006(3.77) 0.004 0.319 i 0.004(2.32) 0.008 0.574 i 0.013(2.21) 0.016 1.156 i 0.027(2.37) 0.020 1.449 i 0.062(4.30) S* represents standard deviation.
Deviation,
0.1452 0.2904 0.5808 1.1616 1.4520
z
8.36 9.80 1.03 0.52 0.14
full sensitivity of curcumin was realized by complete boron retention. Table I1 presents absorbance values and standard deviations for given boron concentrations. The percentage deviation from the least square data is also presented. The molar absorptivity of the rosocyanin complex was found to be 1.96 X 106 1. mole-' cm-'. All analytical work was done using a 4-cm path length for which the lowest instrumentally valid absorbance value of 0.140 was used to establish the lower limit of analytical utility at 0.002 pg/ml or 0.10 pg of boron per total tissue sample following the procedure as previously outlined. Using a 9-cm pathlength, the lowest instrumentally valid absorbance value of 0.140 could be obtained at a boron concentration as low as 0.0009 pg/ml or 0.045 pg of boron per total sample in 50 ml of solution. For a 4-gram tissue sample, this would represent a lower limit of applicability of about 0.010 pg of boron per gram of tissue, and would provide a powerful tool for analyzing boron in animal tissues at the submicrogram level. The ultimate sensitivity is limited by practical path length considerations and complex formation. Using the criterion developed by McFarren, Lishka, and Parker ( 1 3 ) and the mean observed and least square absorbance values, the average total error over the entire concentration range is about 10 %. This total error is well within the 25 value required to classify it as an excellent method according to their criterion. ACKNOWLEDGMENT
The authors wish to thank Ginger Cooper for her assistance in the laboratory and Gary M. Hieftje for valuable suggestions. A special thanks is extended to Joyce Ann Lowe for her diligent care of the laboratory animals and sample preparation. RECEIVED for review December 28, 1971. Accepted June 15, 1972. This work was presented in part September 16, 1971, at the 162nd National Meeting of the American Chemical Society, Washington, D.C., Division of Inorganic Chemistry, Symposium on Boron in the Aquatic Environment. The work was supported by a U S . Public Health Service Research Grant AM 08209 which is gratefully acknowledged. (13) Earl F. McFarren, Raymond J. Lishka, and John H. Parker, ANAL.CHEM., 42, 358 (1970).
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