Determination of Manganese in Bone by Neutron Activation Analysis SIR: Neutron activation analysis has been applied to the determination of manganese in tissues by many workers (1-3,7,9,11). A radiochemical separation step is often included in the analysis to simplify the counting of the induced manganese radioactivity by eliminating or greatly reducing interferences due to other activities. Usually the tissue samples are wet ashed with nitric acid or nitric acid-sulfuric acid mixtures before further oxidation and precipitation of the manganese as the dioxide or as tetraphenylarsonium permanganate. Similar methods have been used in this laboratory with limited success (5, 8). *halyses of liver, blood, and other soft tissues yielded satisfactory results, but when bone samples weighing approximately 1 gram were analyzed, the manganese values obtained were consistently lower than those that could be obtained by the colorimetric method of Gates and Ellis (6). In addition, a certain degree of difficulty was experienced in obtaining samples effectively free of sodium. Following a suggestion by Smith (10) Fve found that, if small bone samples weighing less than 50 mg. were used, they could be ashed effectively with a nitric acid-sulfuric acid mixture to yield reproducible results in near agreement with the colorimetric procedures. Khen attempts were made to ash 1gram bone samples, the required amount of sulfuric acid, plus resulting calcium, interfered with the precipitation of the manganese. Our experiences in the analysis of bone have demonstrated that, when using a wet-ashing procedure under a carefully duplicated set of conditions, one can achieve reproducible but apparently low values for manganese ( 5 , 8). The use of smaller samples can obviate this difficulty, but these small samples in turn necessitate a much longer and, hence, more expensive neutron irradiation to produce a
Table
I.
desirable count rate in the final manganese sample. I n the analysis of small pieces of bone, one is concerned whether the result obtained is truly representative of the concentration of manganese throughout the bone. -4more satisfactory method for us has been developed by using dry ashing of the bone before neutron activation. The manganese in the ash can be oxidized readily and completely. The irradiation is followed with a radiochemical purification procedure in which the manganese is precipitated, redissolved, and then extracted into an organic phase. The organic fraction is counted and analyzed colorimetrically to determine chemical yield. This has eliminated any difficulty with sodium or other activities. With this procedure we obtained consistently higher results than with the procedures involving a wet ashing with nitric acid alone. These results compare well with those obtained using the colorimetric method. EXPERIMENTAL
Samples. Samples consisting of humeri from young calves were analyzed. To avoid sampling errors, cross sections of all the bones analyzed were taken from the central diaphysis region. Each ring was fractured, and sectors of the ring were used for replicate samples. All samples were dried to constant weight a t 110' C. Irradiation. The samples were encapsulated in polyethylene vials and irradiated for 20 minutes in a thermal neutron flux of 3 X 10" neutrons/sq. cm./second. The fast neutron flux at the same position is 4 X 10" neutrons/sq. cm./second. All samples were irradiated in triplicate along with a fourth vial containing 2.5 pg. of manganese in a standard solution. After irradiation, this standard comparator sample was processed similarly to the regular bone samples. A calculation of the interference due to fast neutron reaction FeS6(n, P)Mn= was made using a method similar to that
Comparison of Manganese Analyses:
Approx. sample size, g.
Bone identification number go* 92 * 256c Method Colorimetric 1.0 0.95&0.04 0 . 9 3 f 0 . 0 6 1.37k0.07 Neutron activation 1.0 0 . 4 8 f 0 . 0 6 0.56=!=0.09 0 . 8 9 f 0 . 0 2 Wet ashing ("01) 0 . 6 6 f 0 . 0 4 0 . 7 6 k 0 . 0 3 0.91fO.06 Wet ashing ("01 HC104) 1.O 0.050 0.96 f 0.06 0.97 f 0.05 Wet ashing ("03 &Sod) 1.0 1.09 k 0 . 0 5 1.11 f 0.04 1 . 5 0 ' i ' 0 . 0 3 Dry ashing (600') a Results expressed as micrograms of manganese per gram of bone with maximum deviations from mean. Each value corresponds to the mean value of three determinations. * Bone from calves whose dams were kept on a manganese deficient diet. c Bone from calves whose dams were kept on a normal diet.
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ANALYTICAL CHEMISTRY
reported by Borg et al. (1). The results, based upon a concentration of approximately 120 pg. of iron per gram of marrow-free dry bone, indicate a correction factor of less than 0.01 pg. of manganese per gram of bone. If the iron were to form a significant portion of the matrix, then it would constitute an important interference in a mixed neutron flux such as this. Counting. A gamma-ray spectrometer assembly consisting of a 3-inch X 3-inch N a I (Tl) scintillation crystal detector connected to a Nuclear Data Model 130-1 512 channel pulse height analyzer was used in these experiments to count the manganese activity and determine the purity of the final samples. A simple scintillation detector could be used for routine analyses. CHEMICAL PROCEDURES
Neutron Activation Method with Dry Ashing. The unirradiated bone samples were heated in a furnace at 600' C. for 18 hours in a covered porcelain dish. They were then dissolved in a minimum volume of 8M HKOz and irradiated. To determine that appreciable amounts of manganese were not picked up during the dry ashing and subsequent ash dissolving steps, blanks were run concurrently with the bone samples. The amounts of manganese found in the blanks were less than 0.005 One and one-half milligrams of carrier manganese were added to each irradiated solution along with 20 ml. of 90% fuming nitric acid. Four milliliters of 50y0 sodium chlorate solution were then added to precipitate manganese dioxide. The manganese precipitate was washed with distilled water and redissolved in 1 drop of 6-11HN03, 1 drop of 30% H202, and 1.5 ml. of distilled water. It was then complexed with 2-thenoyltrifluoroacetone and extracted into a 1 : l mixture of benzene and acetone according to the procedure developed by De and Rahaman (4). Each solution was counted with the scintillation detector. After counting, the absorbance of each solution was measured a t 530 mp to determine the chemical yield of the manganese. Colorimetric Analysis Method. The samples were prepared and ashed in the manner described above and were analyzed by the colorimetric procedure of Gates and Ellis (6). RESULTS A N D DISCUSSION
Analyses were performed on aliquot fractions of three different bone samples using neutron activation and colorimetric methods, The data presented in Table I show that the results obtained on the 1-gram samples by the activation procedures deviate from each
other more than can be accounted for by experimental error. One possible explanation is that the wet-ashing procedures did not result in complete osidation of the manganese in the larger bone samples. Other biological samples have been analyzed in this laboratory by the neutron activation analysis procedures outlined above. For samples other than bone, the wet-ashing procedure using nitric acid gives results comparable to the dry-ashing method (8). ACKNOWLEDGMENT
The authors express their appreciation to R. G. Yount of the Department
of Agricultural Chemistry for his willing advice and assistance and to the WSU reactor operating staff for sample irradiations. LITERATURE CITED
(1) Borg, D. C., Segel, R. E., Kienle, P., Campbell, L., Intern. J . Appl. Radiation Isotopes 11, 10 (1961). (2) Bowen, H. J. M., J . Nucl. Energy 3, 18 (1956). (3) Bowen, H. J. M., Cowse, P. A., Atomic
Energy Research Establishment, Harwell, Berkshire, England, Report No.
AERE-R-2925 (1959). (4) De, Ani1 K., Rahaman, Md. Syedur ANAL.CHEM.35, 159 (1963). (5) Dyer, I. A., Cassatt, W. A., Jr., Rao, R. R., Bioscience 14, 31 (1964). (6) Gates, M. E., Ellis, H. G., J. Bid. Chem. 168, 537 (1957).
(7) Meinke, W. W., Science 121, 177 (1955). (8) Rao, R. R., “Manganese Deficiency
and Reproductive Phenomena in Beef Cattle and Rats,” Washington State University, h1.S. thesis (1963). (9) Smith, H., AXAL. CHEM. 34, 190 (1962). (10) Smith, Hamilton, private communi-
cation.
(11) Testa, C., Anal. Chim. Acta 25, 525 (1961).
MIGTJEL A. ROJAS IRWINA. DYER Department of Animal Sciences Washington State University Pullman, Wash. WAYNE A. CASSATT Department of Chemistry Washington State University Pullman, Wash.
Colorimetric Determination of Micro Amounts of Manganese as Manganese Dioxide on Millipore Filters SIR: ;\lost of the commonly used methods for the trace determination of manganese depend on the oxidation of manganese(I1) t o the violet permanganate followed by a colorimetric measurement. Recently, in our laboratory, a method was developed for the determination of selenium by precipitation as the element and filtering it on white Millipore filters ( 5 ) . The selenium was estimated by comparison of the red coloration of the filter with standards prepared in the same manner. I t seemed possible that a similar method could be developed for the determination of micro-amounts of manganese by precipitating it as manganese dioxide. Advantages would include sensitivity, rapidity, no instrumentation needed, ease of operation, and no need to minimize solution volumes. The present paper concerns the study of the optimum conditions for the precipitation of the manganese dioxide. Rlanganese(VI1) is precipitated via the Volhard reaction (6) which results in a multiplication of the manganese in the sample. The amount of manganese recovered on filters has been determined and methods for oxidizing manganese (11) to manganese(VI1) prior to precipitation were studied. The effects of some common metal ions on the recovery of manganese mere investigated as well as methods for increasing the sensitivity. EXPERIMENTAL
All chemicals used were reagent grade. Potassium permanganate was standardized coulometrically with electrogenerated molybdenum(V) (3, 4) and was stored in the dark. Manganese (11) was standardized by titrating with (ethylenedinitrilo) tetraacetic acid ( I ) . Potassium persulfate solutions were prepared fresh daily. The borate buffer
solution was prepared by dissolving 38.1 grams of sodium tetraborate decahydrate in 1 liter of water, and adding 72% perchloric acid to ,adjust the pH to 7.0. Millipore filters (type AAWP of 0.45-micron pore size) 25 mm. in diameter, were supported by a Millipore pyrex microanalysis filter holder type XX-(10-025-00) for collection of precipitates. For the determination of less than 1 pg. manganese, a smaller holder was constructed to provide a filter with an exposed diameter of 6.5 mm. (6). All solutions were passed through Millipore filters before use. Atomic absorption measurements were made with a Jarrel-Ash atomic absorption spectrophotometer and coulometric titrations were performed with a ChrisFeld Microcoulometric Quantalyeer. Samples were heated on a ThomasLabconco high temperature Kjeldahl digestion apparatus. Preparation of Standards. Manganese standards in the range of 1 to 50 fig. and 0.1 to 1 pg. were prepared on 25-mm. filters and 6.5-mm. filters, respectively. Standards for the determination of manganese(VI1) were prepared by adding known amounts of potassium permanganate to a 50-ml. beaker and diluting with water to 10-15 ml. The solution was cooled in an ice bath to 5’ C. and 4 ml. of the pH 7.0 borate buffer solution were added. One ml. of a solution of manganous sulfate (50 pg. Mn/ml.) was added and the mixture was cooled in the ice bath for an additional 5-10 minutes before filtering. The filtered precipitate was washed with cold distilled water. Preparation of standards from manganese(I1) differed in that it was necessary to oxidize the manganese to manganese(VI1). More satisfactory results were obtained if the manganese was in the permanganate form prior to precipitation of manganese dioxide (see below). The standard permanganate solution employed above could also be used for preparing these
standards. The pipetted sample was first reduced to manganese(I1) with 1 ml. of 301, hydrogen peroxide after acidifying with nitric acid. The pH was then adjusted to 7.0 with a saturated sodium tetraborate decahydrate solution (pH 9.0) to give a final volume of 10-30 ml. One ml. each of 1% silver nitrate and saturated potassium persulfate was added to the neutralized solution which was then heated until the manganese (11) was oxidized to the violet permanganate. The solution was then boiled for a few minutes to decompose excess persulfate. Precipitation of manganese dioxide in the cooled solution was accomplished as described. Preparation of Samples. Acid digests were neutralized with a saturated solution of sodium tetraborate decahydrate and the appropriate procedure for precipitating manganese dioxide, depending upon the oxidation state of the manganese, was followed. If iron was present-e.g., steel-the solution was acidified with nitric acid following precipitation and before filtering. The intensity of the coloration of the filter was compared with that of the standards. RESULTS AND DISCUSSION
The requirement of the proposed procedure is that the amount of manganese dioxide retained on the filter must be stoichiometric or it must be sufficiently reproducible t o allow accurate comparison of samples and standards, The Volhard reaction, 2Mn04-
+ 3;1ln+2 + 2Hz0 = 5Mn02
+ 4H+
(1)
predicts a multiplication of the manganese on the filter by a factor of 2.5 over the starting manganese(VI1). This was confirmed by determining the actual amounts of manganese recovered on the filters. The filters were washed VOL. 38, NO. 6, M A Y 1966
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