Determination of Copper in Hydrogenated Fats by Neutron Activation Analysis SIR: Trace amounts of copper are known to increase the oxidative rancidity of fats to a marked extent. The determination of micro amounts of copper is, therefore, an analytical task with which many laboratories are concerned. Because of the great number of determinations of this particular kind carried out in many laboratories it is highly desirable to have a rapid sensitive procedure. Neutron activation analysis is particularly well suited for the determination of copper in biological materials, and the method has been in use for some years (3, 5). More conventional methods have also been employed, in particular spectrophotometry (I, 2). Previously published procedures have, however, been rather time consuming, mostly because they include either hydrolysis or wet decomposition of the sample. Recently, a procedure was published for the determination of nickel in hydrogenated fats using neutron activation analysis ( 7 ) . The induced nickel activity was extracted with nitric acid from a toluene solution of the fat. The main advantage of this method is that hydrolysis or wet decomposition of the f a t is avoided. We decided to see if induced copper activities in hydrogenated fats could be extracted by using a similar postirradiation procedure. As the method for the radiochemical separation of copper, we used the fast procedure developed by one of the present authors (6). To test whether the data obtained represented the true concentration of copper, the data were compared with those obtained with a wet decomposition method.
The copper was extracted from the aqueousphase with l0ml.of carbon tetrachloride containing 3 mg. of Zn-dibenzyldithiocarbamate per ml. (30 seconds). The aqueous solution was discarded and the organic phase was washed once with 10 ml. of 6N hydrochloric acid. One milliliter of concentrated nitric acid and 5 drops of concentrated hydrochloric acid were added to the organic phsse. After shaking for 30 seconds, copper was stripped with 10ml. of water. The organic phase was discarded. After addition of 20 ml. of EDTA solution (50 mg. per ml.) and 5 ml. of concentrated ammonia, the solution was filtered. The copper was extracted with 5 ml. of carbon tetrachloride containing 3 mg. of ZnAibenzyldithiocarbamate per ml. (30 seconds), and the organic phase was washed once with 10 ml. of EDTA solution containing 5 drops of concentrated ammonia. Three milliliters of the organic phase was transferred to a small glass vial and the gross gamma activity was measured with a NaI(T1) well-type scintillation crystal. One milliliter of the same organic phase was diluted to 50 ml. with carbon tetrachloride, and the absorbance, A . , was measured at 435 mp using 10-mm. cells. One milliliter of the irradiated standard solution of copper was diluted to 250 ml. with water, a 250-pi. aliquot was transferred to a separatory funnel containing 20 ml. of EDTA solution (50 mg. per ml.), 1 ml. of concentrated ammonia and 0.5 mg. of copper as carrier. The copper was extracted with 5 ml. of carbon tetrachloride containing 3 mg. of Zn-dibeneyldithiocarbamate per ml. (30 seconds). Three milliliters of the organic phase was transferred to a glass vial similar to that used for the sample, and the gross gamma-activity was recorded. One milliliter of the organic phase was diluted to 50 ml. with carbon tetrachloride, and the absorbance, A . ,
EXPERIMENTAL ~
One and a half milliliters of a standard solution of copper (1 mg. Cu/ml.), prepared by dissolving electrolytic pure copper in nitric acid, was sealed into a polyethylene tube and irradiated in a reactor, together with sealed polyethylene tubes containing 1-2 grams of the hydrogenated fats, for 24 hours in a neutron flux of 2.7 X 1OI2 neutrons per sq. cm. per second. After irradiation, a known amount of the fat WBS dissolved in toluene and transferred tQa separatory funnel containing B m l . of toluene, 10ml. of 6N hydrochloric acid and 0.5 mg. of copper ss carrier. The mixture was shaken for 3 minutes. the wueous phsse was retained, &d the-organic phase was washed with 10 ml. of 6N hydrochloric acid for 1 minute. The washing was added to the first aqueous phase. The organic phase ww discarded. 1414
ANALYTICAL CHEMISTRY
1. Concentration of Copper (p.p.m.) in Mixed Hydrogenated Fats
Table
Wet decomposition 0.051 0.045 0.080 0.073 0.118
Extraction SamDle A
Extraction (filtered sample)
0.050 0.048 0.066 0.076
0.031 0.023 0.026
0.060
0.027
0.068 0.055 ~
~~~
Av. 0.070
SamDle B 0.029
0: 028
0.026 0.027 0.027 0.022 Av. 0.026
0.032 0,029 0.031 0.030
was measured at 435 mp using 10-mm cells. The absolute amount of copper in the sample was calculated by using the following equation: z = ( J , -I . - A'
I'
where : a
A
amount of copper in the 250-p1. aliquot of the diluted standard solution. I and I' = the measured activity of copper isolated from sample and standard, respectively, at the end of irradiation. A . and A.' = the measured absorbance of the diluted copperdibenzyldithiocarbamate solution containing copper isolated from sample and standard, respectively. =
The equation presented above is only valid if the same amount of carrier solution is used for both sample and standard. Note that it is not necessary to know the true concentration of the carrier solution. Beer's law must, of course, be valid in the concentration range used. RESULTS
The results of the analysis of two samples of mixed hydrogenated fats are shown in Table I. For comparison, the data obtained by using a wet decomposition method are also shown. The data show that both methods gave identical results. Sample A was obviously inhomogeneous with respect to copper. A single preirradiation filtration step (filterpaper No. 589, blue ribbon, from Schleicher and Schull, Germany) removed about 50% of the copper, and, apparently, rendered the sample homogeneously with respect to copper. The time required for the separation of copper and the measurement of the absorbance of the two diluted copperdibenzyldithiocarbamate solutions containing copper from sample and standard, respectively, is of the order of 15-20 minutes. The validity of Beer's law was confinned working with 10-mm. cells at the wavelength of maximum absorption, 435 mp, in the concentration region 0.2-2.5 pg. Cu/ml. CCI,. With a chemical yield of 50%, the sensitivity is about l o + p.p.m. using 2 grams of fat, 24 hours irradiation time in a thermal neutron flux of 2.7 X 10" neutrons per sq. cm. per second, and a well-type NaI(T1-scintillation crystal
(2' x 11/2") for recording the gross gamma-activity of T u (12.8 hours). The method is fast enough to permit the use of 6 C u (5.1 minutes). Based on earlier work (6),the sensitivity using T u is estimated to 0.1 p.p.m. using 2 grams of fat and a Sminute irradiation a t a thermal neutron flux of 2-3 X IO1* neutrons per sq. cm. per second. The reproducibility, using the data for sample B, was found to be *7% (standard deviation). The radiochemical separation resulted in a sufficient high purity of the isolated copper. All decay curves showed that the initial activity of the impurities amounted to 1% or less of the initial activity of copper. DISCUSSION
In view of the work of Souliotis on the determination of nickel in hydrogenated fats (7) and that of Brune (4) on the application of the Szilard-Chalmers effect in the neutron activation analysis of biological samples, it was not surprising that it was possible to exQact the induced copper activity with 6N hydrochloric acid from a toluene solution of the irradiated fat. Although this most probably can be fully ascribed to copper existing in the nonirradiated fat as simple ions or organic complexes which are readily extractable in 6 N hydro-
chloric acid, it is possible that part of the radioactive copper is extracted due to a Szilard-Chalmers effect on copper (which is nonextractable in nonirradiated fat), or that radioactive copper, still bound owing to recombination, is extracted through isotope exchange with the copper carrier (4). Although the extraction procedure was checked on only two samples of mixed hydrogenated fats, the induced copper activity will probably also be extracted with 6N hydrochloric acid from all types of fats which can be dissolved in organic solvents having a low solubility in 6N hydrochloric acid. The apparent inhomogeneity of sample A with respect to copper prior to the filtration step might be due to copper associated with particulate matter inhomogeneously distributed in the fat or copper dissolved in a possible water phase also inhomogeneously distributed. Both particulate matter and water can be retained on the filter used. Obviously, one should be aware of the possibility of having also other trace elements inhomogeneously distributed in similar samples. The usefulness of the recommended method is, of course, not restricted to the 24 hours irradiation time, nor the flux and instrumentation used. Although several days were required to get the
results of the analysis in our work, a reduction of the required time could easily have been achieved by reducing the irradiation time and using a multichannel analyzer for recording the activity and checking the purity of the isolated activity. However, the most important point is that the time required for the radiochemical separation and the measurement of the absorbance of the two diluted copper-dibenzyldithiocarbamate solutions in only 15-20 minutes. The procedure is, therefore, most suitable for analyzing series of samples. LITERATURE CITED
(1) Abott, D. C., Pohill, R. D. A,, Analyst 79, 547 (1954). (2) Borchardt, L. G., Butler, J. P., ANAL.CHEM.29, 414 (1957). (3) Bowen, H. J. M., I n i a . J . Appl. Radialia and Isotopes 4, 214 (1959). (4) Brune, Dag, Anal. Chzm. Acta 34, 447 (1966). (5) Fritze, K., Aspin, N., Holmes, T. H., Radiochim. Acta 3 , 204 (1964). (6) H#gdahl, 0. T., Meinke, W. W., U. S. At. Energy Comm. Rept. TID17272, 62 (1962). (7) Souliotk, A. G., ANAL.CHEM.36, 1385 (1964).
OVET. H ~ G D A H L MEISOM SIGURD Central Institute for Industrial Research Blindern, Oslo 3, Norway
Fast Quantitative Separation of Iron(l1) and lron(ll1) by Pa per Chromatography SIR: The importance of the separation of Fe+*and Fe+3has been reviewed (6) and some qualitative separations have also been developed (1,5,6,9, IO). Stevens (9) made a few quantitative studies on the separation of these ions and Pollard et al. (5) carried out a comprehensive and detailed study of the materials and conditions of their F e + L Fe+3 separation on paper chromatograms. A qualitative separation of these ions by paper chromatography was reported from these laboratories earlier (6). Because time is an important factor in the separation of the different valence states of a metal (79, it was considered worthwhile to study the quantitative and other aspects of this separation in detail. The present communication summarizes the results of such a study. EXPERIMENTAL
Apparatus. Development was performed in 20- by 5-cm. glass jars using the ascending technique. The dimension of the paper strips was 14 by 3.5 cm. &timation was done on a Hilger Spekker absorptiometer.
Reagents. Whatman No. 3 M M paper was used in quantitative work only. For qualitative studies, Whatman No. 1 was used. Solutions, 0.1M of nitrates, chlorides, or sulfates of the metal ions, containing a little acid to prevent hydrolysis, were used. Solutions, 0.1M of the sodium, potassium, or ammonium salts were taken for the study of anions as impurities. The stock solutions of ferrous ammonium sulfate (British Drug Houses Analar grade) and ferric ammonium sulfate (Riedel) containing 15,000 and 20,OOO p.p.m. of iron, respectively, were prepared in 1% sulfuric acid and standardized according to the usual procedure (S, 4 ) . A 10% solution of hydroxylamine hydrochloride (8) and a 0.3% aqueous solution of l,l0-phenanthroline were used. The phthalate buffer of pH 3.98 was prepared according to the procedure of Harvey, Smart, and Amis (9). The developer was a mixture of 4M HC1, n-butanol, acetic acid, and acetone by volume in a ratio of 1 : l : l : l . Fe+* and Fe+' were detected usually by exposure to ammonia gas and occasionally with ferro- and ferricyanides and 1,10phenanthroline. The detection with ammonia makes the use of pilot papers
unnecessary in quantitative work. Other cations were detected by the usual, specific color reagents. Procedure for Quantitative Studies. SEPAFLATIONAND DETERMINATION OF Fe+l AND Fe+3 IN ABSENCEOF IMPURITIES. The solution containing ferrous and ferric ions was streaked on the line of application by means of a micropipet. The paper was then conditioned for 15 minutes and the developer was allowed to ascend 10 cm. Blank paper was streaked with 1% sulfuric acid and treated similarly. The paper strips were taken out of the jars and treated with ammonia gas. Zones were cut and eluted successively with 30 ml. of 1% hydrochloric acid and 30 ml. of distilled water. The extract was reduced to 1 ml. by evaporation, cooled, and diluted to 10 ml. with distilled water. To 1 ml. of this solution, 2 ml. of hydroxylamine hydrochloride solution, 2 ml. of buffer solution, and 4 ml. of I ,10-phenanthroline solution were added. The volume was made up to 10 ml. with distilled water. The absorption was measured after 5 minutes and the amount of iron present was determined with the help of the calibration curve drawn previously. VOL 38, NO. 10, SEPTEMBER 1966
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