the hydrogen-carbon reaction, but 630 p.p,m. if it came from N14. The experimental result was 160 =t 40 p.p.m., decisively in favor of the H-C mechanism.
this investigation and especially his help in preparing liquid butane for irradiation.
ACKNOWLEDGMENT
(1) Blaser, J. P., el al., Helv. Phys. Acta 24, 465 (1951). (2) Glickstein, S. S., Winter, R. G., Nuclear Instr. & Methods 9, 226 (1960).
I5'e have the suggestions and advice of B. A. Fries throughout
LITERATURE CITED
(3) Kreger, b ' . E., Kern, B. D., Phgs. Rev. '13, (4) Paul, E. B.,(1y5g). Clarke, R. L., Can. J. Phys. 31, 267 (1953). (5) Seagrave, J. D., Phys. Rev. 84, 1219
( 1951), ( 6 ) Khitehead, -4.B., Foster, J. S., Can. J . Phys. 36, 1276 (1953).
RECEIVED for review September 18, 1961 -4ccepted December 4, 1961.
Estimation of Manganese in Biological Material by Neutron Activation Analysis HAMILTON SMITH Departrnenf of Forensic Medicine, The University, Glasgow, Scotland, and Western Regional Hospital Board, Regional Physics Department, Glasgow, Scotland
b Neutron activation analysis combined with chemical separation is a quick, accurate method for the estimation of M n in small samples of biological materials. After nitric-sulfuric acid digestion of the activated sample, a solvent extraction separation is combined with a colorimetric yield determination. Two modified yield determination and counting techniques are examined.
Manganese-56. K h e n the only stable isotope of h l n is irradiated n i t h thermal neutrons, a n unstable isotope is produced by neutron capture. This isotope has a half life of 2.58 hours and emits @-particles and ?-rays. The capture reaction is represented by:
F
Where possible, AnalaR reagents were used. The complexing agent was tetraphenylarsonium chloride hydrochloride-3.5% in water. All absorbance measurements were made using a 1-em. optical cell in a Hilger absorptiometer with 5450-A. filter. Digestion of Samples. I t was necessary t o have M n in simple ionic form; the samples and 100-pg. carrier M n were therefore heated with a mixture of nitric and sulfuric acids until all the organic material was destroyed. The maximum sulfuric acid permissible in the extraction stage was 2 ml. (see below). A suitable digestion mixture was 2 ml. of 36N sulfuric acid and enough 16N nitric acid to destroy all the organic material. The excess nitric acid was removed by heating the mixture till fumes of sulfuric acid appeared. With normal care, no h l n was lost a t this step. Removal of Interfering Substances. After digestion, t h e sulfuric acid was allowed to cool and diluted t o 40 ml. One drop of tetraphenylarsonium chloride solution was added, and the mixture was extracted twice with chloroform. I n this way, most of the substances which would otherwise follow the M n extraction 'Ir-ere removed without removing a n y of the M n which did not form a complex in the bivalent state. Following this
OUR PROBLEMS in the quantitative determination of h l n in biological tissue were: the destruction of tissue while retaining M n in the reaction medium; the chemical separation; the yield recovery estimation; and the final determination of hln. The following outline covered these points and acted as a basis for the investigation. After neutron activation samples were placed in a beaker with some inactive carrier and digested with a mixture of nitric and sulfuric acids, a preliminary extraction using tetraphenyl arsonium chloride was carried out to remove interfering substances. Manganese was then converted to permanganate and extracted with the same reagent. A yield determination was made, and the activity was measured and compared with a standard. Preparation and Irradiation of Samples. T h e samples, preferably about 40 mg., were iveighed into polyethylene tubes which were then sealed. A sample (about 0.25 gram) of standard M n solution (100 pg. of M n per gram) was weighed into a silica tube n-hich was then sealed. The samples and standard were packed into a standard aluminum can and irradiated in a reactor at a thermal neutron flux of 1OI2 neutrons per square cm. per second for 2 hours. The unit was returned and processed as described below. The standard was diluted as necessary.
190
ANALYTICAL CHEMISTRY
56RIn (n, y ) 5eRln REAGENTS AND APPARATUS
process, the aqueous layer containing the h l n was evaporated to fumes of sulfuric acid to remove any traces of chloride ion or complexing agent which would interfere in the nest step. Chromium follon ed the h l n through the above and the following oxidation step where it was oxidized to dichromate. This reacted with tetraphenylarsonium chloride and was extracted into the chloroform solution in the final step. The elimination of Cr activity is described under the activity estimation below. Oxidation to Permanganate. The cooled sulfuric acid from the last step was diluted t o 40 nil. and transferred to a centrifuge tube where the M n 1% as oxidized to permanganate by stirring with about 2 grams of sodium bismuthate. The suspension formed was centrifuged, and the supernatant liquid was transferred to a separatory funnel. If any chlorides or tetraphenylarsonium chloride were accidentally left till this stage, there were large losses of M n due to trapping of the tetraphenylarsonium permanganate in the sodium bismuthate sludge, or reduction of the permanganate by the chlorides. The Extraction. Five drops of tetraphenylarsonium chloride solution were added to the contents of the separatory funnel and niived \\-ell. The tetraphenylarsonium permanganate was then evtracted by shaking twice with 8-ml. portions of chloroform. The combined extracts were made up to 20 ml. for the next step. The extraction proved to be sensitive to varying conditions. The following experiments were made to find the n-orking limits of the extraction. Acid Concentration. It was necessary to work from a digestion residue containing about 2 ml. of concentrated sulfuric acid. Using the extraction
method above and varying the acid content, the greatest allowable concentration was 1 ml. of concentrated sulfuric acid in 20 ml. of solution (about 2W). Above this concentration, not all of the complex was extracted. If the concentration of nitric acid became even 0.1N, serious losses occurred in the extraction. It was necessary, therefore, to remove the nitric acid after the digestion by heating to fumes of sulfuric acid. Complexing Reagent. Using the method above mith the acid concentration limited as described, varying amounts of tetraphenylarsonium chloride solution rrere added. K i t h more than five drops, the extraction was not as efficient. Recovery and Activity Estimations 1. The standardized sample prepared in the extraction step was checked for recovery by estimating the optical transmission. To determine if this was a n accurate method, a set of standard tetraphenylarsonium permanganate solutions in chloroform, ranging from 1 to 10 pg. per ml., were prepared, and their transmission was measured. K h e n concentration was plotted against log transmission, a straight line graph was obtained. Using this method, it was possible to correlate the recoveries and hence the activities of the samples and standards. Recoveries were between 95 and 100%. The activity of the complev solution was estimated either by counting in a Geiger tube accepting liquid samples or in a glass or polyethylene container for a scintillation counter. Both served equally well under normal circumstances, but if a very large amount of Cr was present in the sample, it was better to use the scintillation counter with the discriminator set so that the weaker y-rays from the Cr did not register. I n most samples, no Cr activity could be detected due to its relatively long half life (2i.8 days) and the short irradiation time (2 hours). Final determination of M n content was made by comparison of count rates and recoveries u-ith a standard sample. Recovery and Activity Estimations 2. It was possible to calculate the recovery gravinietrically using 10 mg. of 1 I n carrier. After oxidation to the permanganate, the solution was transferred to a clean centrifuge tube and precipitated by a slight excess of tetraphenylarsonium chloride. The
precipitate was then centrifuged and It was washed well with water. slurried into a weighed planchet with water and dried a t 105' C. for 15 minutes. The activity of the samples was estimated by counting on a scintillation counter using a suitable discriminator setting to eliminate Crsl-activity. An end window Geiger counter was also used, but if activity due to Cr51 were present, it was necessary to allow the Mn to decay, count the residual activity, and then correct the original count rate. Thereafter, comparison of count rates and weights with a standard gave the hIn content of the samples. Recovery and Activity Estimations 3. If a known amount of long-lived activity in the form of MnS4were added to the samples with the M n carrier, the recovery was estimated by comparing count rates of the separated material after the activity due to the much shorter-lived MnSGhad decayed. This limited the sensitivity and accuracy of the method, but for samples of high 3 l n content, it was useful and convenient. The activities were measured on a scintillation counter set to ignore CrS1 activity. Normally, samples were checked for interfering long-lived isotopes, but in this method it was not possible. Manganese contents were determined by comparison of count rates and recoveries with a standard. Stability of Permanganate Complex. A solution of tetraphenylarsonium permanganate in chloroform showed the characteristic permanganate color. iifter a short time, the color changed t o brown pink, and finally all the M n precipitated. The color varied appreciably after about 30 minutes, b u t this was sufficient for accurate colorimetric measurements. The stability of solid tetraphenylarsonium permanganate a t high temperatures was investigated by keeping it a t 105' C. for some time and weighing a t intervals. Although the complex changed from magenta to light purplebrown, the weight remained unchanged for 2 hours and only lost 9% when heated for 90 hours. It was possible therefore to free the complex from mater by drying a t 105' C. Completeness of Separation. T o examine the completeness of separa-
tion of the active isotopes, the activity of a sample was observed, using a n end window Geiger counter, until it merged with the background activity some 42 hours later. On tracing the results on semilogarithmic paper, the sample gave a straight line decay until i t fell below 1 count per minute above background. This showed very good separation even though no attempt was made to remove activity from Cr5l. Reasons for the lack of interference from Crbl were given above. Sensitivity. The application of activation analysis to M n microestimations allowed amounts of the order of gram to be detected with ease, but this would be reduced somewhat if a suitable reactor was not relatively near. This is due to the rapid = 2.58 hours). decay of the MnS6 Tests on standard solutions showed reproducibility of results within 1%. Samples varying in known M n content from 0.14 pg. to 37.4 pg. were analyzed by the above method (Recovery and Activity Estimation 1); the results obtained agreed within 2%. As a further check, the method was compared with Bowen's ( 1 ) precipitation technique, in the investigation of M n in biological materials. The results again agreed within the experimental error quoted above. CONCLUSION
This method eliminated blanks other than the standard sample which gave the specific activity of Mn. Microseparations were avoided due to adding inactive carrier M n in convenient amounts, according to the recovery estimation used. Tetraphenylarsonium permanganate was shown to be relatively stable and useful for the estimation of &In by colorimetry and by activation analysis. ACKNOWLEDGMENT
The author thanks John Glaister, J. M. il. Lenihan, Edgar Rentoul, and Derek Gibbons for advice given during this investigation. LITERATURE CITED
(1) Bowen, H. J. M.> J . Nuclear Energy 3, 18 (1956).
RECEIVEDfor review RIay 8, 1961. Accepted November 17, 1961. Work performed under a grant supplied by the Medical Research Council.
VOL. 34, NO. 2, FEBRUARY 1962
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