In the catalytic mixture the manganese and chromium oxides gave excellent results for the oxidation of the sample. The tungsten oxide was added to prevent the formation of boron and silicon carbides ( I , 6) and to rule out the possibility of the formation of boron oxides which might enclose unburned sample. Tungsten trioxide was prefered to vanadium pentoxide because it is noticeably less hygroscopic and it does not spatter on heating because of its high melting point, 1473”C (12). In our equipment, the temperature of the combustion furnace was 1050”-1060” C-Le., a value about 150°-250” C higher than that commonly employed in the usual apparatus. In addition, the time of sample combustion in a static oxygen atmosphere was extended for periods varying from 70 seconds to 150 seconds. With these conditions, correct analytical results were obtained for all the silico-carborane polymers which we examined. The time of complete combustion was found to be related to the mean degree of polymerization of the sample; a longer time being required for a higher molecular weight. This is apparent from the curves in Figure 1, where the analytical data for four samples, with molecular weights between 2250 and 9500, are reported. This figure shows the per cent of carbon (found) plotted against the combustion time for these samples. It can be seen that the heat stability of these polymers is greater for higher molecular weights. Thus,
in order to obtain correct results, it is necessary to operate for at least 150 seconds on samples of mean molecular weight on the order of 10,000. Table I shows the analytical data for neocarborane, bis(chlorodimethylsilyl)neocarborane, and for polymers with different degrees of polymerization. A comparison is made between the method described here and the modified Pregl method for boron and silicon containing compounds. The combustion time for all samples in the new method was 150 seconds. It is evident that in the case of neocarborane the analyses obtained by our procedure are not very different from those obtained with Pregl’s method. On the other hand, for the polymers, the negative deviations from Pregl’s method are very large, and increase with increasing molecular weight up to an error of 100%. In Table I, the mean error and the standard deviation are also reported. It can be seen that these deviations are within the usual analytical limits. Hence, by employing the catalyst and the oxidation conditions reported above, together with suitable changes in the combustion times, it is possible to determine successfully the carbon and hydrogen content of organic polymeric compounds containing boron and silicon, to which the usual analytical methods cannot be applied, It has further advantages of requiring small amounts of sample, about 0.6-0.7 of a mg for an analysis, and rapidity of operation. RECEIVED for
(12) E. Kissa, Mirrochem. J., 1, 203-207 (1957).
review October 30, 1967. Accepted December
26,1967.
Rapid and Simple Continuous Radiochemical Separation of Copper, Magnesium, Zinc, and Manganese in Biological Materials Kwang J. Hahn, Dean J. Tuma, and John L. Sullivan Medical Research, V.A. Hospital, Omaha, Neb. 6810.5
MICROANALYTICAL TECHNIQUES for certain trace metals including copper, magnesium, zinc, and manganese are increasing in importance because these elements are constituents of numerous enzymes and they function as essential catalysts in biosynthesis. Most of the currently available data concerning determinations of Cu, Mg, Zn, and Mn in biological materials were either obtained by emission spectrographic methods ( I ) or extraction and separation procedures developed for colorimetric determinations (2). In addition, neutron activation analysis combined with radiochemical separations utilizing lengthy and multistep procedures have been reported (3-8). Various computer techniques for multielement determinations have also been reported for neutron activation analysis (9, IO); however, by applying these methods uncertainties are unavoidable. Satisfactory performance of such analysis for the elements described by the resolution of complex spectra depends on the standards provided for curve fitting and the accounting of the number of nuclides present. The major difficulties or disadvantages are due to the unaccounted-for nuclides present, a masking effect by one of the energetic nuclides present or a high concentration of one component, and especially the superimposed principal photopeaks of certain nuclides. 974
ANALYTICAL CHEMISTRY
Because of these inherent difficulties, the present study was conducted to develop a rapid and simple radiochemical separation procedure for W u , 27Mg,‘jgrnZn,and b6Mn. Determination of these elements by neutron activation analysis was made practical by this method even though the half-life of 27Mgis short and the principal photopeak is practically superimposed (1) I. H. Tipton, M. J. Cook, R. L. Steiner, C. A. Boye, H. M. Perry, Jr., and H. A. Schroeder, Healfh Physics, 9,89 (1963). (2) E. B. Sandell, “Colorimetric Determination of Traces of Metals,” Interscience, New York, 1959. (3) K. Samsahl, AE-168, Aktiebologet Atomenergi, Stockholm, 1964. (4) H. J. M. Bowen, Intern. J. Appl. Rad. and Isotopes, 4, 214 (1959). (5) D. G. Kaiser and W. W. Meinke, Anal. Biorhem., 6, 77 (1963). (6) R. M. Parr and D. M. Taylor, Biochem. J., 91,424 (1964). (7) G. W. Leddicotte, Nucl. Sci. Ser., NAS-NS-3018, October 1960. (8) W. F. Bethard, D. A. Olehy, and R. A. Schmitt, “L’Analyse
par Radioactivation et sec Applications aux Science Biologiques,” Presses Universitaires de France, Paris, France, 1964. (9) G. D. O’Kelley, Nucl. Sci. Ser., NAS-NS-3107, October 1962. (10) F. J. Kerrigan, ANAL.CHEM., 38,1677 (1966).
on 5"n and also despite the fact the thermal neutron capture reaction of e*Zn,(n, 7)6gmZnis not the most sensitive. The method finally developed on the basis of the present results for routine analysis are the extractions of copper dithizonate, zinc dithizonate, magnesium thenoyltrifluoroacetone (possibly as butylammonium Mg(TTA),), and manganese diethyldithiocarbamate. Copper was extracted first, then zinc, manganese, and magnesium, respectively. The y-activities were measured in the order of 64Cu, 27Mg, 6gmZn,and @Mn. An optional procedure was developed for those who wish to determine only magnesium.
Table I. Analysis of Solution of Known Chemical Composition Re1 std Std dev dev Added Found Yield, Solution No. 1, N = 5
cu,
Mg, Zn, pg
Mn,pg
0.796 36.2 36.1 50.7 0.531
99.6 90.9 90.7 86.1 99.8
Solution No. 2, N
cu, Pg
EXPERIMENTAL
Mg, pga Mg, c(g
Apparatus. Samples were irradiated utilizing a Triga Mark I water-cooled reactor and the induced gamma-activities were measured in a RIDL 400-channel pulse height analyzer. Reagents. STANDARDS. Pure metal rod or oxide (Johnson, Matthey, and Co., London) was dissolved in dilute nitric acid and diluted to the desired concentration. ACETATE BUFFER,pH 4.75. Equal volumes of 4N sodium acetate and 4N glacial acetic acid were mixed. AMMONIUM ACETATEBUFFER,pH 6.1. Ammonium hydroxide (470 ml) was mixed with 430 ml of glacial acetic acid. More acid or base was added as required to adjust the pH to 6.1, and then the solution was diluted to 1 liter. The buffer solution gave a pH of 5.2 to 5.4 when diluted 1 to 5. AMMONIUM ACETATE BUFFER,pH 9.0. Equal volumes of 3N ammonium hydroxide and 2.3N acetic acid were mixed. When necessary, pH was adjusted with acid or base. SAMPLEPREPARATION. Liver tissues weighing 0.3 gram were transferred into 125-ml Erlenmeyer flasks and digested in 3 ml of concentrated nitric acid according to the method of Hahn, et al. (11). The residue was dissolved in 1N nitric acid with heating and the volume was adjusted to a total of 4 ml. The aqueous samples then were irradiated for 1 hour in a thermal neutron flux of 1.1 x n/cm2/second. The same method was used to obtain the nitric acid blanks. Procedures. RADIOCHEMICAL SEPARATION OF COPPER. Three milliliters of the irradiated samples were transferred into a separatory funnel containing 2 ml of 0.1N HCl. At this point a modified procedure of Cholak, et al. (12), using dithizone, was introduced to extract copper. Five milliliters of 0.01% dithizone in CC14 were added, then shaken for 2 minutes. Four milliliters of the organic layer were pipetted in a polyethylene vial and counted at 10 minutes after removal from the reactor for 10 minutes' live time. RADIOCHEMICAL SEPARATION OF ZINC. The aqueous layer from the copper extraction was washed with 10 ml of carbon tetrachloride to ensure the removal of any copper dithizonate that may have remained in the separatory funnel. Two drops of 0.1% brilliant yellow indicator were added to the sample and the solution was adjusted to approximately neutral with 5N ammonium hydroxide. A modified procedure of Fischer and Leopoldi (13) was employed for this extraction. Three milliliters of acetate buffer, pH 4.75, 2 ml of 25% sodium thiosulfate, and 5 ml of 0.01 dithizone in CCla solution were added, then shaken for 2 minutes. Four milliliters of the organic layer was counted for 10 minutes' live time at 40 minutes after removal from the reactor. RADIOCHEMICAL SEPARATION OF MANGANESE. The resulting aqueous phase from the previous extracts was washed with 10 ml of carbon tetrachloride to remove any remaining zinc dithizonate. The solutions were adjusted to neutral
Zn,pg Mn,pg
(11) K. J. Hahn, D. J. Tuma, and M. A. Quaife, ANAL.CHEM., 39, 1169 (1967). (12) J. Cholak, D. M. Hubbard, and R. E. Burkey, IND. ENG. CHEM., ANAL.ED., 15,754 (1943). (13) H. Fisher and G. Leopoldi, 2.Anal. Chem., 107,241 (1937).
0.800 39.8 39.8 58.8 0.532
Mg, pga
1.610 59.7 59.7 49.2 0.122
1.614 54.2 54.4 48.8 0.117
2.410 79.6 79.6 24.6 0.266
2.468 72.3 72.9 23.5 0.261
0.028 1.0 2.0 0.5 0.002
1.8 1.9 3.7 0.9 1.8
0.042 2.0 1.7 0.4 0.005
1.7 2.7 2.3 1.8 1.9
= 5
102.4 90.8 91.4 95.5 98.1
Mg, pg" Mg, pg Zn, pg Mn, pg Mg optional procedure.
2.3 2.2 4.2 3.3 1.5
= 5
100.3 90.8 91.0 99.2 95.6
Solution No. 3, N
cu, Pg
0.018 0.8 1.5 1.7 0.008
0
~
Table 11. Tissue Analysis and Recoveries from Tissue cu Mg Zn Mn Normal liver value, N = 5 pg/g fresh wt
Std dev Re1 std dev
4.35 0.11 2.5
Added
239.3 9.8 4.1
Found
Yield,
30.9
1.5 4.7
2.89 0.02 0.8
Std dev
Re1 std dev
0.03 2.3 0.4 0.006
1.6 2.1 1.8 1.5
Recoveries
c u , pg
Mg, 1.18 Zn, pg Mn, pg
1.90 53.3 22.1 0.41
1.88 56.0 22.2 0.41
98.9 101.3 100.5 100.0
with 5N ammonium hydroxide. Two milliliters of acetate buffer, pH 6.1, 2 ml of 5.7% sodium diethyldithiocarbamate, and 5 ml of chloroform were added, then shaken for 3 minutes. Four milliliters of the .organic layer were counted for 10 minutes' live time at 55 minutes after removal from the reactor. RADIOCHEMICAL SEPARATION OF MAGNESIUM. The aqueous phase from the above extraction was washed with 10 ml of chloroform to ensure complete removal of manganese diethyldithiocarbamate. The solution was adjusted to the brilliantyellow indicator end-point with 5N ammonium hydroxide. Five milliliters of acetate buffer, pH 9.0, 1 ml of 40% n-butylamine, and 5 ml of 1% TTA in chloroform were added, then shaken for 2 minutes. Four milliliters of the chloroform layer were counted for 10 minutes' live time at 25 minutes after removal from the reactor. The optional procedure for those who wish to isolate magnesium only is as follows. The major interfering ions were removed as described by Hahn, et af. ( I I ) , and the magnesium extraction procedure described above was used, Four milliliters of the organic layer were counted for 10 minutes' live time at 10 minutes after removal from the reactor. CALCULATIONS. The values for the samples were computed from the corresponding photopeak sum of the standards. After subtracting the blank, the area of 5 channels from either side of the principal photopeak was integrated. In the case of e4Cuthe annihilation photopeak was used. VOL. 40, NO. 6, M A Y 1968
975
RESULTS
The results of the described procedures on the analysis of known concentrations of chemically mixed standards are illustrated in Table I. It is seen that the average chemical yield for Cu, Zn, and Mn was better than 95 %. The yield of Cu is nearly 100% for the three concentrations tested. The chemical yield of zinc at the lowest concentration tested (24.6 pg) was 95.5%; however, at twice this concentration a yield of 99.0 % was obtained. A constant yield of about 87 was observed for concentrations of zinc above 55 pg. Concentrations of zinc up to 80 pg were tested and this yield was still observed. The overall yield of magnesium from three concentrations tested was 91 %. This is true for both Mg procedures. Thus, in the actual analysis of biological tissues, the Mg values were corrected for 91 % extraction. The chemical yield of manganese varied from 95.6% at the lowest concentration tested (0.122 pg) to almost a 100% at the highest concentration (0.532 pg). Table I1 summarizes the results of a study of the recovery from tissue, An excellent recovery was obtained for all procedures. The results of tissue analysis showed a good correlation with reported values but they are not intended for normal tissue values. The Cu, Mn, and Zn recoveries were nearly 100%. The mean recovery of Mg after correction for 91 % extraction was 101.3%. The spectra of extracted elements demonstrated the high purity of the isolated elements as well as the effectiveness of the extraction procedures. The following half-lives- W u , 12.8 hours; 27Mg,9.6 minutes; 69mZn,14 hours; and 56Mn, 2.60 hours-were observed for these extracted elements. The time required for the entire radiochemical separations of these elements was about 18 minutes. DISCUSSION
The TTA extraction of Mn, Cu, Mg, and Zn in biological samples was reported ( 1 4 ) ; however, the spectrum obtained by this manner was dominated by 5"n. A small copper peak was observed and the zinc photopeak was completely masked. In addition, the principal photopeaks of j6Mn and 27Mgwere almost superimposed and the secondary photopeak of 27Mg at 1.02 MeV was indistinguishable. These factors made it practically impossible to resolve this complex spectrum for the desired metals; hence it was necessary to perform radiochemical separations of these metals to isolate them in radiochemically pure form in order to obtain quantitative data for these metals. The chelating agents used in this procedure extract the divalent ions; ions such as Na, K, and other monovalent ionsalthough present in large quantities in biolcgical materialsdid not interfere, but remained in the aqueous layer throughout the separation procedures. Dithizone is known to react or chelate with at least 19 elements. The extraction specificity of one element can be achieved by the different strengths of acidity, by extraction conditions such as various buffer systems, as well as by complex forming agents. Thus copper was extracted specifically at pH 0 to 1. Zinc was also extracted as zinc dithizonate at pH 4.75. The concentrations of Na2S203,acetate buffer, and dithizone were (14) M. C . Haven, G. T. Haven, and A. L. Dunn, ANAL.CHEM., 38, 141 (1966).
976
ANALYTICAL CHEMISTRY
greatly increased from that of the reported procedures (13). This and other modifications did not lower the rate of extraction. If copper, however, was not separated before the zinc extraction and 2 pg or more was present, it did interfere with the zinc extraction. If the concentration of Ni(1I) and Co(I1) were present in large amounts, it was necessary to use 1 ml of 10% potassium cyanide as an additional complex forming agent; but in the tissue analysis this was not the case. In a previous article ( I I ) , sodium diethyldithiocarbamate was utilized to remove any interfering ions, especially Mn, in the magnesium extraction procedure. This method required pre- and post-activation extractions under carefully controlled conditions. In the present procedure, sodium diethyldithiocarbamate was employed for the quantitative determination of Mn by extraction of the manganese diethyldithiocarbamate with chloroform. The extraction of Cu prior to Zn, the separation of Cu and Zn before Mn, and the removal of Cu, Zn, and Mn prior to Mg extraction conveniently removed the interfering ion or ions for the respective subsequent extractions. Anionic chelate-extraction systems were well reviewed and summarized by Morrison (15). Out of several anionic chelate-formers, only one-that developed by Umland and Hoffman (16)-resembles the described magnesium procedure. According t o Umland and Hoffman, magnesium appears to form a complex with three molecules of 8-hydroxyquinoline (oxine) which pairs with butylammonium ion to form ( R . N H J MgOx3. Similar studies were conducted with the described magnesium procedures and found that the behavior of Mg using TTA in chloroform with n-butylamine differs slightly to that of Umland and Hoffman. Furthermore, Mg(TTA)* was prepared (IZ) and the solubility was compared with the cloroform-extractable Mg compound of this procedure. Mg (TTA)2 was insoluble both in benzene and chloroform, but it became soluble as soon as n-butylamine was added. This indicated that a complex different from that of Mg(TTA)2was formed. Although only a 91 % extraction was observed for this procedure, the TTA-CHCI3 and n-butylamine extraction yield was found to be about 100%. The 10% loss of Mg was due to the previous NaDEDTC CHC&extraction. This did not affect the Mn determination because almost 6 half-lives of Mg had elapsed before the Mn was counted and also the high activity of Mn was hardly effected by the small activity represented by the 10% loss of magnesium. Although Bode (17) reported that magnesium was not extracted by NaDEDTC in CCI, at pH 4 to 11, the present procedure showed a constant 10% extraction loss of magnesium with the NaDEDTC in CHCI3 (or CCI, when used in lieu of chloroform) at a pH of 5.2-5.4. ACKNOWLEDGMENT
The authors thank F. J. Kerrigan for the computation, L. J. Arsenault, and those who gave their technical assistance. RECEIVED for review January 8, 1968. Accepted February 9, 1968. (15) G. H.Morrison, Zbid., 30, 632 (1958). (16) G. Umland and W. Hoffman, Anal. Chim. Acta, 17, 234 (1957). (17) H. Bode, 2.Anal. Chem., 143,182 (1954).
