Anal. Chem. 1982, 5 4 , 1129-1131
(1)high catalytic activity in luminol luminescence, (2) excellent stability of the catalytic activity, (3) simple covalent labeling of any peptides and proteins via the carboxyl group of hemin, and (4) an inexpensive labeling as compared with radioisotopes and enzyme labelings. We anticipate that the present method should be applied in general to any antigen determinations particularly including hapten determi.nationn. A possible use of hemin is under the investigation for the catalyst immunoassay of smaller molecules.
LITERATURE CITED Wisdom, G. B. Clin. Chem. (Winston-Salem, N . C . ) 1076, 22, 1243-1255. Halmann, M.; Velan, B.; Sery, T. Anal. Envlron. Mlcroblol. 1977, 3 4 , 473-477. Oisson. T.; Brunius, G.; Thore, A. J . Immunol. Methods 187% 25,127-135. Aizawa, M.; Suzuki, 8.; Kato, T.; Fujiwara, Y.; Fujita, Y. J . Appl. Blohem. 1960, 2 , 190-195. Aizawa, M.; Morioka, A,; Matsuoka, H.; Suzukl, S; Nagamura, Y.; Shinohara, R.; Ishiguro, I. J. Solld-Phase Biochem. 1976, 7, 319-328.
1129
(8) Alzawa, M.; Morioka, A.; Suzuki, S . J . Membr. Sci. 1978, 3 , 22 1-228. (7) Aizawa, M.; Morioka, A,; Suzuki, S.; Nagamura, Y. Anal. 8iOChem. 1979, 9 4 , 22-28. (8) Alzawa, M.; Morioka, A.; Suzuki, S. Anal. Chim. Acta 1060, 775, 81-88. (9) Mattiasson, B.; Borrebaeck, C.; Sanfridson, 6.; Mosbach, K. Biochim. Blophys. Acta 1977, 483, 221-227. (10) Boitieux, J. L.; Desmet, G.; Thomas, D. Clln. Chem. (Wlnsfon-Salem, N.C.)1979, 25,318-321. (11) Carrico, R. J.; Yeung, K. K.; Schroeder, H. R.; Boguslaski, R. C.; Buckler, R. T.; Christner, J. E. Anal. Blochem. 1976, 78, 95-103. (12) Schroeder, H. R.; Carrico, R. J.; Boguslaskl, R. C.; Christner. J. E. Anal. Blochem. 1976, 72, 283-290. (13) Schroeder, H. R.; Vogelhut, P. 0.; Carrico, R. J.; Boguslaski, R. C.; Buckler, R. T. Anal. Blochem. 1976, 4 8 , 1933-1938. (14) Puget, K.; Micheleon, A. M.; Avrameas, S . Anal. Blochem. 1977, 79, 447-456. (15) "Data Book of Biochemistry. I"; Biochemical Society of Japan; Tokyo Kagaku Dojin: Tokyo, 1979; pp 106-122. (16) "Data Book of Biochemistry. I"; Biochemical Society of Japan; Tokyo Kagaku Dojin: Tokyo, 1979; p 97. (17) Halfman, C. J. Anal. Chem. 1979, 57,2306-2311.
RECEIVED for review December 1, 1980. Resubmitted September 1, 1981. Accepted March 3, 1982.
Removal 01 Sodium-24 by Chromatographic Extraction with a Kieselguhr Column and a Crown Ether Solution Toshlmasa Kimura * Japan Chemical Analysis Center, Sanno, Chiba, 2 8 I, Japan
Tatsujiro Ishimorl Institute for Atomic Energy, Rikkyo University, Nagasaka, Yokosuka, 420-0 1, Japan
Tatsuji Hamada Institute of Physical and Chemical Research, Hirosawa, Wako, Saltama, 351, Japan
Sodium-24 was readily removed from aqueous solutlons by a chromatographlc process based on an Isotopic exchange reactlon. The reactlon was carrled out wlth a granular Kleselguhr column and a chloroform solutlon of crown ether-sodlum plcrate. The process took about 1 h and can be utllized In neutron actlvation analysls of many elements, CI, Br, Sc, Cr, Mn, Fe, Co, NI, Cu, Zn, As, Ru, In, Sn, Ce and W. I t cannot be used, however, for alkali and alkallne earth elements. The amount of 24Nain a treated sample was lo-' of the orlglnal amounfi. The Compton background was reduced In the range of y a y energy after the removal of sodium-24.
Most environmental and biological materials contain a large amount of natural sodium, 23Na. The cross section of 23Na for thermal neutron capture is relatively large and its neutron activation product, %Na (15.0 h), emits two high-energy y-rays. The two y-rays produce a large Compton background which often interferes with y-ray spectrometry of other products during the instrumental neutron activation analysis. Various methods for avoiding the interference of 24Nahave been reported resonance activation methods with sodium (I, 2) and cadmium (3), and a chromatographic method with hydrated antimony pentaoxide, HAP ( 4 , 5 ) . This paper describes a method for removing the sodium-24 from irradiated aqueous solutions by a chromatographic process based on an 0003-2700/82/0354-1129$0 1.2510
isotopic exchange reaction. The reaction was carried out by using a "Extrelut" prepacked column filled with granular Kieselguhr, also called "diatomaceous e a r t h .
EXPERIMENTAL SECTION Radionuclidesand Reagents. Carrier-free radioactive nuclide solutions of 22Na,"Mn, 59Fe,C O @ ,' 'OeRu, W n , 133Ba,13"Cs,and 144Cewere purchased from Japan Radioisotope Association. Radionuclide solutions of 46Sc,'%, 65Ni,16As,114mIn,and 18"W were prepared by neutron activation of the stable elements. The "Extrelut" prepacked column was purchased from Merck Japan Co. Kieselguhr in the column can support 20 mL of aqueous solution. The crown ether used was dicyclohexyll8-crown-6made by Nippon Soda Co. It was a mixture of two isomers and was used without purification. All chemicals used were of analytical grade. Instruments. Irradiations were carried out at a thermal neutron flux of 1.5 X 10l2n-cm-2d in a Triga Mark I1 Reactor at the Institute for Atomic Energy, Rikkyo University, Japan. Radioactivity was measured with a 50 cm3Ge(Li) detector having 2.1 keV resolution at 1.33 MeV, in conjunctionwith a 4096 channel pulse height analyzer, or a NaI(T1) detector in conjunction with a single-channel scaler. Preparation of a Chloroform Solution of the Crown Ether-Sodium Picrate for Chromatographic Extraction. A 7.5-g sample of dicyclohexyl 18-crown-6was dissolved in 750 mL of chloroform, after which the solution was transferred to a 1-L separatory funnel containing 225 mL of 1.3 X 10-1 M sodium picrate solution. After the funnel had been shaken for 1min and allowed to stand for 10 min, the organic phase was transferred to a different 1-L separatory funnel containing 225 mL of 1.3 X 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
Table I. Relationship between the Removal of Sodium-22 and the Amount of Sodium Ion Carrier in Chromatographic Extractiona amt of Na+,mg
removal, %
0.5 4 10 60
97.3i 97.9 f 98.9 i 95.3 i
0.2 0.1 0.2 0.1
a Aqueous sample; 20 mL of 5 mM acetic acid-5 mM lithium acetate solution containing 22Naand Na+. ExtracM [(C-l8)Na]+[picrate]tant: 150 mL of 2.7 X chloroform solution.
lo-' M sodium picrate solution. After this funnel had been shaken and allowed to stand as above, the organic phase was transferred to a red bottle. The chloroform solution could be used for extraction up to 1 month after preparation. Procedure for Chromatographic Extraction. An aqueous sample solution of less than 20 mL was adjusted to about pH 4 with 1 mL of 0.05 M acetic acid-0.05 M lithium acetate buffer, if necessary with 1M hydrochloric acid or 1M lithium hydroxide added, and its volume was adjusted to 22 mL with sodium ion carrier solution and water. Lithium salt or lithium hdyroxide was used in order to prevent the increase of sodium ion concentration more than a predetermined one. A large amount of sodium ion in the aqueous solution results in an increase of eluent amount. For lithium ion the sodium crown ether was scarcely consumed because of its low affinity toward the crown ether. After 20 mL of the sample solution had been applied to the top of the "Extrelut" prepacked column and allowed to be absorbed for 15 min, the chloroform solution of the crown ether-sodium picrate was passed through the column at a flow rate of 3.7 mlsrnin-'. Aliquots of effluent were taken in counting vials 10 mL by 10 mL or 50 mL by 50 mL. The Kieselguhr which supported the aqueous solution in the column was also transferred to a counting vial after chromatography had been carried out. Extractions of various ions were examined by y-ray measurement of the effluent or the Kieselguhr.
RESULTS AND DISCUSSION Two methods of removing 24Nafrom an aqueous solution using a "Extrelut" prepacked column were initially examined. The first method was to allow the aqueous solution containing sodium picrate to be absorbed into the Kieselguhr and to perform liquid-liquid extraction of sodium picrate in the aqueous phase of the Kieselguhr with a chloroform solution of crown ether. The other method was to allow the aqueous solution to be absorbed into the Kieselguhr and to extract the
sodium-24 with a chloroform solution of crown ether-sodium picrate. The latter method was much more effective in removing 24Nathan the former. The latter method is thought to be based on a rapid isotopic exchange between sodium ions in the aqueous phase of the Kieselguhr and those in the crown ether-sodium picrate in chloroform. The latter method employed the procedure described in the Experimental Section. Sodium-22 in an aqueous solution (4 mg of Na+, 22Na,the buffer reagent; 20 mL) was removed by the extractant (2.7 X M [ (C-l8)Na]+[picrate]- chloroform solution). The chromatographic extraction diagram is shown in Figure 1. The hydrogen ion concentration in the aqueous solution was adjusted to pH 4 with the buffer solution to prevent formation of picric acid (6) and precipitation of metal hydroxides. In Figure 1the horizontal axis represents the volume of effluent and the vertical axis represents the percent of sodium-22 extracted in each effluent fraction and in the integral effluent. After 150 mL of the chloroform solution had passed through the Kieselguhr, about 98% of the sodium-22 was extracted. Table I shows the relationship between the percent of sodium-22 removed and the amount of the sodium ion carrier in the aqueous solution. With 60 mg of Na+ carrier, a 150 mL elution was not sufficiently effective in removing the sodium-22. With 0.5, 4, and 10 mg Na+ carriers, the sodium-22 was almost all extracted by a 150-mL elution and was not found in the effluent above 150 mL. However, when values of the extraction percentage were examined carefully, they did increase slightly with increasing content of the carrier sodium ion. This tendency may be caused by the adsorption of a very small amount of sodium ions in the Kieselguhr which then do not exchange with sodium ions in the chloroform solution. In practice, a range of sodium ions of from 4 to 10 mg in a sample solution is suitable for efficient removal of sodium-24. Table I1 shows the percentage of retention and extraction of various inorganic ions in the Kieselguhr during chromatographic extraction. The extraction percentage was taken to be zero for net counting rate less than zero. Retention was calculated with the original net counting rate of each fraction and that of the total tracer used; a negative original net counting rate was not rounded to zero when calculating the percentage of retention. When the percentage of retention was calculated to be more than 100, it is shown as 100 in the table. For inorganic species examined, an aqueous solution (4 mg of Na+, the buffer reagent, radioactive tracer concerned, 1mg of its carrier; 20 mL)was prepared and chromatographic
Table 11. Extraction and Retention of Inorganic Species in the Kieselguhra % extraction 100-150 mL 50-100 mL 0-50 mL species K+
cs+
Ba2+ SCJ+b
37.2 i 0.6 53.3 f 3.3 0 i 0.1 0.01
cr3+
oi
Cr0,'Mn2+ Fe3+
0.08 i 0.12 0 F 0.01 0.06 5 0.11 0.01 i 0.02 o i 5.9 0.05 li. 0.01 0 F 2.4 O i 0.03 0.07 f 0.04 O F 0.2 0 i 1.9
coz+ Ni" Ru3+orland Ru4+
1 ~ 3 +
Sn2+orland Sn4+ Ce3+orland Ce4+ wo42-
As3' 6r/and As5+
9.2 f 0.2 19.4 i 1.5 0.1 f 0.1 0
i
0.01
0.17i 0.09 0 f 0.01 0.02 i 0.12 0.02 f 0.02 4.3 t 4.4 0.05 f 0.01 0.2 i 0.8 O i 0.04 0.01 f 0.03 0.1 f 0.2 0 i 0.4
150-200 m L
2.4 i 0.1 5.8 k 1.1 0 i 0.1
0.8 f 0.1 4.1 i 0.7 0 F 0.1
0 0
0
0.01 b 0.1 0 i 0.01 0.03 i 0.11 0.01 i 0.01 0 i 1.3 0.01 i 0.01 i i
0 f 0.7 0 + 0.04 0 i 0.03
o + 0.2
0.2 f 0.1
*
0.01
0i 0i 0i 0i
0.1 0.01
0.10 0.01 0 i 1.6 0 i 0.01 1.7 i 0.8 0.03 i 0.04 0.07 ?: 0.03 0.4 i 0.3 0 i 0.1
ClBr' a
Error is calculated on the basis of counting errors ( l a ) .
Contained 20 mg of sodium tartarate.
retention, % 11.1 k 2.7 50.4 i 0.6 17.4 i 1.1 100 i1.1 100 t 0.1 100 c 1.4 100 i 0.3 99.9k 1.0 99.9 i 0.1 98.4 3 16.9 99.9 i 0.1 98.1 f 9.1 100 r 0.1 99.9 * 0.1 99.8 F 5.6 100 i 4 . 5 98.5 i 3.3 97.6 i: 8.1
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
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Table 111. Determination of Mn, Cu, and Zn in Bovine Liver by This Methoda element MTl
by this method 1 0 . 8 f 0.3 196 f 28 118r 21
56Mn
cu
a
content, Mg/g dry
nuclide used for analysis 6 4 c u
Zn ’mZn Error is calculated o n the basis of counting errors ( l o ) .
50
100
unprocessed 9.5 It 0.5 181 f 124 1 2 6 t 71
0
by NBS 10.3 f 1.0 193 i 10 130* 13
2 Energy ( M e V )
1
Amount of outflow (rnt)
Flgure 1. Chromatographic extraction diagram of sodium-22: aqueous sample, 20 mL of 5 mM acetic acid-5 mM lithium acetate solution containing 22Naarid 4 mg of Na+; extractant, 2.7 X lo-* M [(C-18)Na]+[picrate]-chloroform solution.
extraction was carried out with the extractant (2.7 X M [ (C-l8)Na]+[picrate]- chloroform solution; 200 mL). Twenty milligrams of sodium tartarate was added to an aqueous solution of tin or Bcandium to prevent hydrolysis of the metal ions. Tracers were not used to obtain the retention values of K+, C1-, and Br-. Their retention values were obtained from the ratios of activities of the respective ion before and after the removal of sodium-24 in activation analysis of these ions in a sample of liquid effluent from a research institute. Potassium, cesium, and barium ions were partially extracted, but all the other ions were retained in the column. The partial extraction of alkali and alkaline earth ions must be caused by the formation of alkali and alkaline earth ion stable complexs with the crown ether (7). Chromatographic extraction was applied to neutron activation analysis of the liquid effluent sample. The Kieselguhr was subjected to half of an irradiated liquid effluent sample with an appropriate amount of water and then was measured by y-ray spectrometry and compared with a sample treated by chromatographic extraction. The other half of the irradiated liquid effluent sample was added to sodium ion carrier, the buffer reagent, and water, and the solution was transferred to the column. After chromatographic extraction was carried out, the Kieselguhr in the column was measured by y-ray spectrometry. The results are shown in Figure 2. From the two spectra, the amount of 24Nain the treated Kieselguhr of the original amount. The sample was found to be 1 X Compton background was reduced throughout the range of y-ray energy after the removal of sodium-24. Figure 3 shows y-ray spectra of‘ 82Br (776 keV) and 56Mn (844 keV) in the irradiated liquid effluent sample. In the measurement of the original sample, 24Nainterfered so that neither peak can be clearly identified above the background. On the other hand, both peaks could be clearly seen in the measurement of the treated Kieselguhr sample. An additional example of use of this method was activation analysis of bovine liver, NBS standard reference material no. 1577. After irradiation, the sample was decomposed by heating it with nitric acid containing a small amount of hydrochloric acid. To the solution obtained, this method was applied. As shown in Table 111, the results agreed well with the values certified by WBS, and also, statistical errors on the basis of counting error were reduced by use of this method,
3
4
Flgure 2. y-ray spectra of irradiated liquid effluent: (A) original sample; (B) Kieselguhr sample after the removal of sodium-24.
>-c,
I
770
780
840
850
Channel (1keV c h - )
Flgure 3. y-ray spectra of 82Br (776 keV) and 56Mn (846 keV) in irradiated drainage: (0)original sample; (0)Kieselguhr sample after
the removal of sodium-24. especially for Cu and Zn which gave low counting rates in this sample. Chromatographic extraction is suitable for neutron activation analysis of environmental and biological materials which contain a large amount of sodium ions. The removal of sodium-24is especially effective for analyses of radionuclides such as 56Mn,64Cu,65Ni69mZn,76As,and lo5Ru,which have half-lives of the same order as that of 24Na. The present chromatographic extraction took about 1h and can be applied to analyses of many radionuclides as shown in Table 11. Hydrated antimony pentaoxide has been useful to selectively remove Na+ ions from other alkali and alkaline earth ions (4, 5). However, an “Extrelut” prepacked column can be also useful to remove sodium-24 without chemical procedure. Its operation is simple and the removal of sodium-24 is reproducible.
LITERATURE CITED ( I ) AI-Shahristanl, H.; Abbass, K. J. Radloanal. Chem. 1975, 2 7 , 105-1 13. ( 2 ) Hanna, A. G.; AI-Shahrlstani, H. J. Radioanal. Chem. 1977, 3 7 , 581-589. (3) Cesana, A.; Rossitto, F.; Terrani, M. J. Radioanal. Chem. 1978, 4 5 , 199-202. (4) Girardl, F.; Sabbionl, E. J. Radioanal. Chem. 1968, 1 , 169-178. (5) Glrardl, F.; Pietra, R.; Sabbioni, E. J. Radioanal. Chem. 1970, 5 , 141-171. (6) Klmura, T.; Iwashlma, K.; Ishimori, T.; Hamada, T. Anal. Chem. 1979, 51, 1113-1116. (7) Kolthoff, I.M. Anal. Chem. 1979, 51, IR.
RECEIVED for review August 21, 1981. Resubmitted and Accepted February 23,1982. This work was partially supported by the Science Research Promotion Fund of the Japan Private School Promotion Foundation.
