plutonium-241, is only 4 X 10-5, the initial activity of the uranium is significant because of its short half-life of 6.75 days (7). Results for the alpha-gamma method showed a greater variation from the expected americium growth for two reasons. First, the measured gamma activity includes both the uranium-237 and americium-241 contribution, thus accounting for the nonlinearity shown in Figure 2. Second, because the gross gamma activity must be corrected by subtracting the non-americium activities, the errors become large when this correction is nearly equal t o the total activity, as is the case during the early period of growth shown in Figure 2. Comparison of the alpha-gamma and TOPO methods for various other samples as received are given in Table 111. I n almost all cases the alpha-gamma method yielded higher results, which might be attributed to uranium-237 present, although the differences were usually small.
Table 111. Americium-Contents of Plutonium Samples (ppm) Found by Alpha-Gamma and TOPO Methods TOPO method a,y method 5 5.6 6.3 6.3 16 10.5 36 34 38 35 43 38 44 35 49 43 65 64 103 96 218 214 228 242
The alpha-gamma method for determining americium in plutonium is rapid and sufficiently accurate for most purposes down t o 5 ppm. The TOPO extraction method is recom-
mended for extending the range to 0.2 ppm. This lower limit represents a count rate of about 100 counts/minute in 250 pg of typical plutonium, and is below the usual background of a gamma counter. Increasing the sensitivity of the method by employing much larger sample size enhances the hazard of spread of contamination during extraction and therefore is not recommended.
(7) E. K. Hyde, I. Perlman, and G. T. Seaborg, “The Nuclear Properties of the Heavy Elements,” Vol. 11, Prentice-Hall, Englewood Cliffs, N. J., 1964, pp. 737, 828.
RECEIVED for review February 13, 1967. Accepted May 18, 1967. Work performed under the auspices of the U. S. Atomic Energy Commission.
APPLICATIONS
Solvent Extraction and Determination of Magnesium in Biological Materials Kwang J. Hahn, Dean J. Turna, and Merton A. Quaife Special Laboratory of Nuclear Medicine and Biology, Veterans Administration Hospital, Omaha, Neb. 68105
PRIORPUBLICATIONS dealing with the radiochemical separation of magnesium have predominantly described a precipitation analysis (1-4). Neutron activation analysis for magnesium is limited by a low activation cross-section, low abundance of 26Mg, and a short half-life of the neutron activation product 27Mg. The separation of magnesium by solvent extraction methods has not been widely used because of poor extraction efficiency. Inadequate chemical separation may cause significant interferences in radiometric analysis. F o r example, manganese interferes with the determination of magnesium even though it is present in minute amounts. This investigation explored the efficacy of a method which employs a combination of solvent extraction and neutron activation analysis in the determination of magnesium in biological samples. A double extraction utilizing sodium diethyldithiocarbamate and thenoyltrifluoroacetone was used in isolating (1) C. K. Kim and W. W. Meinke, “Proceedings of the International Conference on Modern Trends in Activation Analysis,’’ College Station, Texas, 1965. (2) A. W. Fairhall, “The Radiochemistry of Magnesium,” Nuclear Science Series Report NAS-NS-3024, January 1961. (3) 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. (4) H. C. M. Bowen, P. A. Cawse, and M.’Dagush, Analyst, 89, 266 (1964).
magnesium from biological materials. The chelating abilities of the sodium salt of diethyldithiocarbamic acid and thenoyltrifluoroacetone (TTA) in analytical techniques are well documented as they have been utilized as reagents for colorimetric determinations for various elements (5-8). The method under study employs a two-step separation prior to radiochemical analysis. I n addition, the effect of p H variation o n the equilibrium of sodium diethyldithiocarbamate extraction was also investigated a s well as the extent of interference from the *7AI(n,p) 27Mgreaction. EXPERIMENTAL Apparatus. Samples were irradiated utilizing a Triga Mark I water-cooled reactor and the induced 27Mgactivities were measured in a RIDL 400-channel pulse height analyzer. Reagents. MAGNESIUM STANDARD.Pure magnesium metal rod (Johnson, Matthey & Co., London) was dissolved in dilute nitric acid and diluted to the desired concentrations. SODIUM DIETHYLDITHIOCARBAMATE, 5.7 %. 5.7 grams in 100 ml of demineralized water was used.
(5) J. M. Chilton, ANAL.CHEM.,25, 1274 (1953). (6) E. B. Sandell, “Colorimetric Determination of Traces o f Metals,” Interscience, New York, 1959. (7) . , A. M. Poskanzer and B. M. Foreman. J. Inora. Nucl. Chem.. 16, 323 (1961). (8) G. H. Morrison and H. Freiser, ANAL.CHEM.,30, 632 (1958). I
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Table I. Determination of Magnesium Concentration in Same Rat Liver Sample no. pg Mg/g fresh wt 1 247,6 2 245.7 3 241.2 235.2 4 5 241.4 231.6 6 237.4 7 231.2 8 Mean 238.9 6.1 Std dev,a pg 2.6 Re1 std dev, a
Std dev
=
Table 11. Recovery of Magnesium from Tissue Mg added, N-8 Mean, fig Recovery, % Std dev,a pg 20 19.8 99.0 0.81 101,5 2.40 40 40.6 2.09 60a 59.7 99.5 Mean value of seven determinations. pg
a
Re1 std dev, % 4.0 5.9 3.5
AMMONIUM ACETATE BUFFER,pH 6.1. Ammonium hydroxide, 470 ml, were mixed with 430 ml of glacial acetic acid. More acid or base was added as required t o adjust the p H to 6.1, and then the solution was diluted t o 1 liter. The buffer solution gave a p H of 5.2 to 5.4 when diluted 1 to 5. CHLOROFORM. Spectrographically analyzed grade was used. THENOYLTRIFLUOROACETONE. Ten grams were dissolved in 400 ml of tetrahydrofuran and diluted t o 1 liter with benzene. AMMONIUM ACETATE BUFFER,p H 9.0. Mix equal volumes of 3N ammonium hydroxide with 2.3N acetic acid. When necessary, p H was adjusted with acid o r base. Procedure. PRE-ACTIVATION EXTRACTION.Liver samples weighing 0.1 to 0.3 gram-when serum samples were utilized, 1 ml to 2 ml-were transferred into 125-ml Erlenmeyer flasks. Three milliliters of concentrated nitric acid were added and covered with Gooch type crucibles (Kimax, low form, with fritted disk). The samples were heated on a sand bath boiling gently so that digestion proceeded slowly. When the digest had been evaporated to dryness, 3 ml of IN nitric acid were added to the residue, then heated to boiling. The sides of the flask were then washed with 3 ml of demineralized water, The sample was transferred t o a 60-ml separatory funnel and the flask rinsed with an additional 5 ml of demineralized water. The same method was also used to obtain the reagent blank. Two drops of 0.1% brilliant yellow indicator were added to the samples and blank, and the solutions were adjusted t o approximately neutral with 1N ammonium hydroxide. At this point a modified procedure of Eckert (9) using diethyldithiocarbamate was introduced to remove heavy metals by chloroform extraction. Two milliliters of acetate buffer, p H 6.1 and 2 ml of 5.7% diethyldithiocarbamate solution were added, then shaken for 2 minutes following the addition of 10 ml of chloroform. The organic phase was discarded. The extraction was repeated adding another 2 ml of diethyldithiocarbamate and ~
~~
10 ml of chloroform. The aqueous phase was then washed with 10 ml of chloroform to ensure the removal of the interfering metals. If the chloroform layer was not clear and colorless, then further extractions were necessary. Magnesium was isolated into the organic phase by a modification of the method of Haven ( I O ) . The solution was made basic with IN ammonium hydroxide. Five milliliters of acetate buffer pH 9 were added, and the solution was shaken 3 minutes with 5 ml of TTA. The aqueous layer was drained and discarded. The magnesium complex was dissociated into an aqueous phase by shaking 3 minutes with 5 ml of 1N nitric acid. The organic layer was removed by means of a siphon and the aqueous phase was washed with 10 ml of benzene t o ensure that the chloroform and other organic matter had been completely removed. The aqueous layer was drained into a polyethylene vial and irradiated for 30 minutes. POST-ACTIVATION EXTRACTION.Four milliliters of the irradiated sample were pipetted into a separatory funnel containing 5 ml of TTA and two drops of brilliant yellow indicator. The solution was made basic with 1.5N ammonium hydroxide and shaken 2 minutes after addition of 3 ml of acetate buffer solution p H 9. Four milliliters of the organic layer were pipetted in a polyethylene vial and counted a t 8 minutes after removal from the reactor for 10 minutes live time. Calculations. An external standard calibration curve was obtained from the full preparative procedure carried o u t on known quantities of magnesium. After subtracting the average of the blanks, the area of 10 channels from either side of 0.84 MeV peak in the spectra was integrated. The values for the samples were computed from the corresponding photopeak sum of the standards. RESULTS AND DISCUSSION
Sodium diethyldithiocarbamate as a chelating reagent occupies a very important role in this method in that it forms many of the heavy metal precipitates which are soluble in various organic solvents. The successive extractions with chloroform removed the major interfering metals such as Cu(II), Mn(II), Co(II), Ni, Fe(II), and Fe(III), leaving magnesium ions as well as other ions not removed by the above extraction, especially sodium in the aqueous phase. The next extraction with thenoyltrifluoroacetone in benzene/THF removed most of the N a ; however, traces remained in the sample. The post-activation extraction with TTA removed the remaining traces of 24Na, and consistent and reproducible results were obtained. If the post-extraction was omitted, inconsistent traces of 24Naremained in the sample. The standard calibration curve for the range of 0 to 120 fig of magnesium was linear. The magnesium extracted from the samples was found to be in pure form showing a half-life of 9.6 min. Table I illustrates the results of the described extraction procedure. The analysis of eight pieces of the same rat liver gave a relative standard deviation of less than 3 %. Table I1 summarizes the results of a study of the magnesium recovery by neutron activation analysis. The recovery in the concentration tested was better than 99.0%,as compared to the 84% reported by precipitation analysis ( I ) . A magnesium level of 120 pg or higher, white Mg (TTA)2was precipitated in the organic phase. This Mg (TTA)? precipitate does not interfere with the analysis as long as a careful separation is made. A reinvestigation of the method of Eckert ( 9 ) showed calcium as well as aluminum and magnesium remained in the
~
(10) M. C . Haven, G . T. Haven, and A. L. Dunn, ANAL.CHEW,
(9) G.Eckert, 2.Anal. Chern., 153,261 (1956).
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ANALYTICAL CHEMISTRY
38, 141 (1966).
aqueous phase. The effect of the pH on the equilibrium was studied, and it was found that reproducible results were obtained between pH 5 and 6. The recovery in this p H range but recovery diminished outside this range. At p H was 95 4 or lower, manganese interference occurred. In conducting studies on interfering ions, standard solutions of magnesium containing known amounts of Cu, Zn, and Mn (a major interfering ion) in their approximate tissue concentrations were quantitatively determined. N o interference with magnesium analysis was found even when six times the normal manganese concentration was added. The competing reactions of 27Al(n,p)27Mgand 26Mg (n,-y) *'Mg were investigated. From a mean value of three determinations, the aluminum interference was calculated. Five and one-half per cent of the aluminum was converted to *?Mgby fast neutrons above 2.1 MeV. Therefore, the aluminum concentration in tissue reported by Tipton et ai. (11) which is less than 1 pg Alig tissue is much too low to interfere.
x,
~~
(11) I. H. Tipton, M. J. Cook, R. L. Steiner, C. A. Boye, H. M. Perry, Jr., and H. A. Schroeder, Healrh Pliys., 9, 89 (1963).
Serum which contains a large amount of calcium was also analyzed. The spectrum from serum is unlike the spectrum obtained from the tissue samples and consists of a complex spectrum of Ca, Mg, and a trace of Na. This spectrum was solved by the simplex method of linear programming (12). The mean values of magnesium (20.6 pg/ml) and calcium (104.1 pg/ml) agree with the reported values; however, there are various more rapid methods available for magnesium and calcium determinations in serum. ACKNOWLEDGMENT
The authors wish to thank F. J. Kerrigan for the computations, and those who gave their technical assistance. The review and critique of the manuscript by A. J. Barak, is gratefully acknowledged. RECEIVED for review February 6, 1967. Accepted May 17, 1967. (12) F. J. Kerrigan, ANAL.CHEM., 38, 1677 (1966).
Structural and Bonding Characteristics in the Molybdenum (VI)-lminodiacetate System Determined by Infrared and Proton Nuclear Magnetic Resonance Techniques Richard J. Kula Department of Chemistry, Unicersity of Wisconsin, Madison, Wis. 53706
PREVIOUS INVESTIGATIONS of ethylenediaminetetraacetic acid (EDTA) and methyliminodiacetic acid (MIDA) molybdenum (VI) complexes in aqueous solution provided evidence for the chemistry and certain structural and bonding features of Mo(V1) aminopolycarboxylic acid chelates (I, 2). A similar ligand, which enables a n even more explicit evaluation of these features, is iminodiacetic acid (IDA). IDA differs from MIDA only by the replacement of an amine proton for the N-methyl group, and therefore these two ligands should have nearly identical structural and equilibrium characteristics. The equilibrium expectations were verified by the potentiometric study of these complexes in which the formation constant of Mo-IDA was determined to be only about 0.4 pK unit smaller than that for Mo-MIDA (3). In the present investigation the basic structural features for MoMIDA and Mo-IDA are found to be similar, but the absence of the N-methyl group does change certain chelate features, particularly those involving kinetic processes. Also, proton nuclear magnetic resonance (NMR) spectra of the MoIDA complex enable an absolute configurational assignment and an indication of the lability of individual metal ligand bonds for the complex. EXPERIMENTAL
N M R spectra were obtained on a Varian A60A (probe temperature = 36" C) and on a Varian HA-100 (probe temperature = 28" C), and chemical shifts were measured in (1) R. J. Kula, ANAL.CHEM., 38, 1581 (1966). (2) Ibid.,p. 1382. (3) R. J . Kula and D. L. Rahenstein, Ibid., 38, 1934 (1966).
cps relative to internal 0.008M tetramethylammonium ion (TMA). Solutions were prepared in a manner similar t o that described previously (2). The iminodiacetic acid was obtained from K and K Laboratories and was twice recrystallized from water. Infrared spectra were obtained on a Perkin-Elmer Model 421 using KBr pellets for crystalline samples and 0.1M solutions in D20 with a BaF2 cell (0.025 rnm spacing). RESULTS
Titration of an equimolar solution of MOO,-? and I D A v 2 with H N 0 3 results in a p H titration curve nearly identical with that obtained for the MIDA system, two breaks being observed in the titration curve: the first a t 2H+/Mo and the second a t 3Hf/Mo (2). On going to the first break, the reaction can be considered as Mood-'
+ IDA-? + 2H+ S MOOJDA-' + H20
and a t 2H+/Mo (pH = 5) the formation of MoOJDA-? is virtually complete. The N M R spectra a t 2H+/Mo in water and D 2 0are given in Figure 1 along with the line assignments. The spectrum of Mo031DA-?, like that for Mo03MIDA-?, is a basic AB pattern for the -CH2C02- protons, with further splitting caused by the N-H proton (Le., an ABX pattern). The A protons are more strongly coupled (JAx = 7.3 cps) with the N-H proton than are the B protons (Jsx = 1.0 cps). In D 2 0 solution, the amine proton is exchanged with deuterium and the ABX pattern collapses t o the simple AB case (with the A resonances being somewhat broader than the B resonances because of weak coupling with deuterium). In solutions containing both H 2 0 and D20, superimposed VOL. 39, NO. 10, AUGUST 1967
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